Created: 1st December 2000, last updated: 30th December 2000, © 2000 ABRF
Technologies for Resolving Macromolecular Communications
An International Symposium Sponsored by The Association of Biomolecular Resource Facilities
Town & Country Resort
San Diego, California
February 24-27, 2001
P1-S
Compatibility of the RapXtract Dye Terminator Removal Kit with purification of sequencing reactions for the applied biosystems Prism 3100 Capillary Sequencer.
A.L. Springer, L.R. Booth, K.A. Hughes, R.J. Kaiser, D.A. Spicer; Prolinx, Inc., 22322 20th Avenue SE, Bothell, WA 98021
Successful automated DNA sequence analysis requires that sequencing reactions be purified of excess unincorporated dye terminators. Common methods for purifying sequencing reactions involve several steps and require processes that are not easily automated. Prolinx,® Inc. has developed the unique RapXtract™ Dye Terminator Removal Kit for this application that is rapid, easy to perform, yields high quality product and does not require modified primers. The RapXtract kit uses a magnetic particle format to remove dye terminator contaminants by mass action. The protocol has three steps: 1. Remove storage buffer from particles, 2. Add sequencing reactions and mix, and 3. Remove purified extension products. The ABI PRISM 3100 Genetic Analyzer is a 16-capillary instrument that utilizes technology from other ABI capillary sequencers in a medium-throughput format. RapXtract-purified samples analyzed on the ABI PRISM 3100 yielded sequences with good Phred quality scores and signal strength. The RapXtract kit has been optimized to minimize the use of expensive sequencing reagents and can be performed on reactions containing as little as 1 µl of Big Dye Reaction Mix and as little as 5 µl total volume. The RapXtract kit has been used successfully with double-stranded plasmid, PCR product or single-stranded DNA templates. RapXtract purification can also be used for dye primer reactions. The ease of use and versatility of the RapXtract kit make it a good choice for manual or automated purification of sequencing reactions.
P2-M
Serial analysis of gene expression using the Applied Biosystems capillary electrophoresis platforms.
K.M. Gunning, B. Nutter, A. Swei, J. Zon; Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404
In today's genomic environment, the draft completion of the human genome has allowed researchers access to a wealth of information with respect to gene identity and gene expression profiles. Scientists have developed novel ways in which to elucidate this information and SAGE is deemed one of the more comprehensive methods available for rapid, detailed analysis of large numbers of cellular transcripts. To further enhance the rapidity at which one can determine this global gene expression profile, one needs more efficient and precise methods for analyzing these transcripts and for assessing their abundance. Automated capillary electrophoresis provides a platform whereby the sequence of these many transcripts can be determined with speed and precision. The ABI PRISM.® 3700 and the ABI PRISM.® 3100, the most recent addition to the CE family of platforms, provide the SAGE researcher with a means by which they can determine quickly and reliably, the transcript information necessary to build these gene expression profiles. These platforms partnered with the uniformity of signal strength and length of read of BigDye™ Terminators, are the labor saving answer to large scale transcript analysis using the SAGE method. We will demonstrate the ease of use and flexibility of these platforms as a preferred method for the sequence analysis of ditags in the SAGE process.
P3-T
Improved signal strength on ABI Prism 377 DNA Sequencer and 310 Genetic Analyzer by using a new sequencing run module.
S-M. Chen, H. Zielke, G. Amparo, S. Spurgeon; Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404
In many sequencing labs DNA sample template quality varies greatly due to the diverse sources of these samples and the different DNA preparation methods used. This variable DNA template quality results in variable sequencing data quality. Many low quality data are associated with low quality or quantity of DNA templates. This frequently results in low signal strength and low signal to noise ratio. A new sequencing module was developed for the ABI PRISM 377 and 310 systems that will generate greater signal strength during DATA collection. With the use of this module in data collection we were able to improve signal to noise ratio and were able to improve success rate in data analysis.
P4-S
Two steps cycle-sequencing improves base ambiguities and signal dropouts in certain BigDye DNA sequencing reactions.
L. Wen; San Diego State Univ., 5500 Campanile Drive, San Diego, CA 92182-4614
The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. While dideoxy-terminators labeled with energy-transfer dyes (BigDye; PE/ABI) provide the most versatile method of automated DNA sequencing, premature terminations results in a substantially reduced reading length of the DNA sequence. Premature terminations are usually evidenced by base ambiguities and are often accompanied by diminished signal intensity after that point. I studied a two-step protocol for Taq cycle sequencing using the ABI BigDye terminator for reducing premature terminations in DNA sequences. I demonstrated that combining the annealing step with the extension step at one temperature (60°C) reduces premature terminations in DNA sequences that regularly contain premature terminations when the three temperature steps is used. This procedure can be significantly improved sequence quality in our core facility. Sequence results from initial studies are still forthcoming and will be presented.
P5-M
Using high speed data collection on the ABI Prism 377XL to improve core facility DNA sequencing throughput.
F. Lach, J. Medalle, M. Randesi, B. Imai; Rockefeller Univ., 1230 York Avenue, Box 105, New York, NY 10021
The variety of samples subjected to a core facility presents a challenge when it comes to achieving high throughput DNA sequencing. Any given run can exhibit different degrees of success based on template type and size, template base content and arrangement, primer design, and overall sample quality. Although the ABI Prism 3700 DNA Analyzer excels in high throughput capabilities, the instrument still requires stricter sample management that is harder to achieve in the core facility environment. The ABI Prism 377XL continues to be an important resource since it requires less sample stringency than the 3700. Modification of the 377XL module parameters and new polyacrylamide premixes give the potential for increased sample throughput with minimal loss of resolution. This study will investigate the conditions required to obtain quality data using the 4X high speed data collection module on the 377XL. A data comparison will be made between the 2X standard speed and 4X high speed modules using various samples and polyacrylamide premixes.
P6-T
Update on the continuation of the DNA Sequencing Research Group 2000 Study: an evaluation of methods used to sequence and isolate bacterial artificial chromosomes.
T. Thannhauser1, L.S. Hall2, J. Hawes3, T. Hunter4, E. Jackson-Machelski5, K. Knudtson6, D. Leviten7, M.A. Robertson8; 1Cornell Univ., Rm 149 Biotechnology Bldg, Ithaca, New York 14853-2703, 2Albert Einstein Col. of Med., 1695 Poplar Street, NewYork, NY 10464, 3Indiana Univ. Sch. of Med., 4Univ. of Vermont, 5Washington Univ. Sch. of Med., 6Univ. of Iowa, 7ICOS Corp., 8Univ. of Utah
Recent emphasis on BACs as a primary source of template has created significant challenges for those who operate DNA sequencing facilities. Due to their large size (80-350 KB) BACs behave differently in sequencing reactions than do plasmid clones. To achieve optimal performance, standard sequencing reactions have to be modified. Moreover, due to their low copy number it is difficult to isolate enough BAC template using standard miniprep protocols. Using data submitted to part1 of the original DSRG 2000 BAC study, the DSRG extracted a recommended protocol for BAC sequencing and performed carefully controlled experiments in an internal study to test the efficiency and reliability of the protocol. This protocol was made available along with a standard BAC template and primer for testing in the community.
Part 1 of the study examines the effect of a DSRG recommended sequencing protocol on the quality of the results obtained on a standard BAC template sent to participating laboratories. The sequencing results are analyzed for quality and length of read on the wide variety of instrumention used by the DNA sequencing community. The success rate of the standard protocol is compared to the internal study results and to the original results where applicable.
Part 2 of the study continues to assess and evaluate the quality of BAC isolation methods commonly used in DNA sequencing facilities with the goal of providing a recommended isolation protocol. The isolated BAC DNA is sequenced using the DSRG recommended protocol. Results will be analyzed for quality and length of read on standard instrumentation to determine the success of the different BAC isolation procedures.
P7-S
Automated transfer of DNA sequencing files to an ftp site.
K.M. Ivanetich, R. Taylor, W. Yan, F. Sandifer, D. Wolber; UCSF, Box 0541, San Francisco, CA 94143
The Organize Samples program has been developed to automate the renaming, consolidation, and transfer of sequencing data from Applied Biosystems 373, 377 and 3700 DNA sequencers directly onto the facility's ftp site. The program accesses (1) information in the Applied Biosystems sequencer's analysis file, (2) the Applied Biosystems Tool Kit, (3) a file containing a list of user's names paired with their laboratory's principal investigator's names, and (4) the compression program DropStuffit. Using the above, the program compresses all sequence files for one user into one document, names the document with the user's name and the date, and places the compressed file into the appropriate laboratory's folder on the facility's ftp server. The laboratory's folders are password protected. Users can access their laboratory's folder, download the file, and uncompress it with a single click. The result is a set of uncompressed files, with one file for each sequence analysis file. The uncompressed files are named with the run and lane numbers or coordinate and capillary numbers plus the user's template and primer names. Separate sequence text files are handled equivalently by Organize Samples, and are differentiated by a ".seq" suffix. Users can access the ftp site and download their DNA sequencing data from their own computer 24 hours/day-7 days/week.
P8-M
Sequencing performance of the ABI Prism 3100 Genetic Analyzer.
P.A. Baybayan, B. Johnson, K. Roy, R. Pingue, Q. Liwei; Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404
The new ABI Prism 3100 Genetic Analyzer is the latest addition to the suite of capillary electrophoresis platforms offered by Applied Biosystems. This 16-capillary electrophoresis platform employs complete automation designed to meet the demands of mid to high-throughput sequencing and genotyping laboratories.
The ABI Prism 3100 Genetic Analyzer offers flexibility by providing options for a variety of applications. In de novo sequencing where throughput, consistency and efficiency are important, several arrays (including 36 cm, 50 cm, and 80 cm) are available. These arrays require different run parameters and therefore, perform differently. The user will have the option to an array depending on their requirements (readlength and throughput). In comparative sequencing where accuracy and sensitivity are required for the detection and identification of polymorphisms, a 36-cm array is sufficient. Here, we will present the performance (readlength, accuracy, consistency) of the ABI Prism 3100 Genetic Analyzer in the area of de novo and comparative sequencing and demonstrate how this instrument is the platform to best accommodate to your research needs.
P9-T
A comparison of alternative methods for sequencing through difficult templates.
D.A. Bintzler, Y. Song; Univ. of Cincinnati, 231 Bethesda Avenue, Cincinnati, OH 45267-0524
Template DNA that has a seventy percent or greater content of guanosine and cytosine bases (GC rich) has been a potential problem for automated fluorescent DNA sequencing. Two well-known strategies for improving the base read length of the GC rich template include the addition of dimethyl sulfoxide (DMSO) or betaine to the PCR amplification of the template. Applied Biosystems Incorporated (ABI) has offered a third method with dGTP big dye chemistry. The dGTP chemistry has been significantly effective for extending through templates that contain a chain of ten or more guanosine bases. The dRhodamine chemistry (ABI) has also been effective for some problematic templates. However, no single method has been effective for all problematic templates in this facility. Therefore, research has continued to find an alternative method. A PCR additive available from Qiagen, Q buffer, was introduced as a potential method for improving the base read length of the GC rich template. Preliminary studies showed that Q buffer had improved PCR extension through difficult templates that were not successfully sequenced with the other methods previously mentioned. However, a thorough investigation of the Q buffer compared to DMSO, betaine, dRhodamine and dGTP big dye chemistry was required. The results of the investigation are reported here.
P10-S
Fragment and sequence analysis in Portugal: a survey of facilities and applications.
L.M. Souto; Univ. de Aveiro, Portugal, Departamento de Biologia, Universidade de Aveiro, Aveiro, Aveiro 3810 Portugal
Since 1992, with the first unit of Automated Sequencers starting to work in Portugal, a significant improvement in the number of equipments and institutions dealing with sequencing and fragment analisys has been registered.
The leading groups are the forensic institutes which deal with a heavy routine work, namely in paternity cases. These groups are using very standardised techniques and markers, in accordance to the EDNAP (European DNA Profiling Group) and the Portuguese and Spanish Working Group of the ISFH (GEPISFH).
Recently, the universities as well as some private research and diagnostic labs began to use DNA Sequencers, opening the spectrum of applications.
We present in this poster the state of the art of Automated Sequence and Fragment Analysis in Portugal and its relationship with other iberian and latin-american countries, with a special emphasis onto the forensic genetics field.
P11-M
Integration of new sequencing technologies into the W.M. Keck Biotechnology/HHMI Biopolymer Lab. at Yale.
K.M. Hager; Yale Univ., 295 Congress Ave., New Haven, CT 06536
In the past year, we have successfully integrated the Applied Biosystems 3700 DNA Sequencer into our DNA sequencing service. This machine utilizes 96 capillaries and a proprietary polymer rather than polyacrylamide slab gels to separate fluorescently-labeled DNA termination fragments. Because of 3700's unattended robotic sample loading from as many as four sample plates per operator interaction, we have increased the lab's overall sequencing throughput by 25% with no increase in staff and lowered the average sample turnaround time. In addition, the 3700's automation has allowed us to initiate Genescan and SNAPSHOT single-nucleotide polymorphism (SNP) detection assays as new services. At present, about 75% of our total DNA sequencing samples are run on the 3700 with the remainder run on one 377 (slab gel). About 2-5% of the samples run on the 3700 give a poor separation and these samples are rerun on the 377. Such poor separations are due to either improper loading of the separation polymer into the capillary or overloading of the capillary with excessive amounts of DNA. On the vast majority of runs, our pgem standards yield 650-750 bases of sequence data at 99% or greater accuracy (POP-6 polymer).
Recently, we have begun an evaluation of Pyrosequencing, a non-electrophoretic DNA sequencing technology using an enzyme coupled assay where light is ultimately generated by luciferase from PPI released following nucleotide incorporation (Science 281, pp. 363-364 (1998)). Our initial applications for Pyrosequencing will include sequence tag (20-40 base) determination and SNP detection.
P12-T
Investigation of the entire spectrum of genes induced by staphylococcal enterotoxin B in human lymphoid cells using differential display-PCR.
C.A. Mendis, C. Sanchez, R. Das, M. Jett; Walter Reed Army Inst. of Res., 503 Forney Drive, Silver Spring, MD 20910
Since its introduction in 1991, Differential Display has evolved into a powerful and efficient technique in analyzing differentially expressed genes in various cells under altered conditions. Here we used differential display not only to screen the entire gene population in human lymphoid cells induced by SEB but also to understand the specificity of the expression pattern of altered genes by comparing it to the expression by Cholera Toxin (CT). In order to approach our goal in a systematic manner as well as to increase the efficiency of cDNA product recovery we used a set of arbitrary and anchored primers to further subdivide the gene population. Out of the 750 altered genes, at least 200 genes showed a SEB specific expression pattern when compared to CT exposure. The altered genes were then subjected to a high throughput cloning procedure and sequenced. Out of the 250 genes that were sequenced, 150 genes matched with available sequences in the Gene Bank and EMBL databases. Unique to SEB are genes involved in ancillary functions as adhesion molecules, regulators of vascular tone, wound healing, inflammation, heat shock, cell death and T-cell proliferation. Altered genes that matched with known sequences were verified by northern blots, RT-PCR or real time PCR. Genes that were specific for SEB were then placed on glass chips for use with other cell lines or to understand the gene expression profile of other toxins. In the quest for finding a set of genes that can be used as surrogate markers, differential display has shown to be a proven technique as it has the capability to look at the whole gene spectrum irrespective of wide use or availability of individual genes.
P13-S
Microsatellite analysis using fluorescent PCR primers synthesized in tandem.
S.L. Wegener, G.J. Wiebe, S. Yu, R.T. Pon; Univ. of Calgary, 3330 Hospital Drive N.W., Calgary, AB T2N 4N1, Canada
A new procedure for solid-phase synthesis of multiple oligonucleotides linked end-to-end (tandem synthesis) on the same solid-phase support has recently been developed in our laboratory. Upon cleavage and deprotection, the oligonucleotides are released from each other yielding a mixture of two or more sequences in the same solution. This procedure has practical benefits for applications requiring large sets of oligonucleotides, especially PCR amplification, since the number of individual oligonucleotide syntheses to be set-up and the number of individual oligonucleotide samples handled is reduced by half. Fluorescently labelled primer pairs for automated genotyping are also possible by incorporating a fluorescent dye phosphoramidite onto the 5'-end of the terminal oligonucleotide. PCR primer pairs with FAM, HEX, and TET fluorescent labels have been prepared by tandem synthesis and used to amplify known microsatellite markers from (C57BL/6 X NOD) mouse genomic DNA. Automated microsatellite analysis using GeneScan and an Applied Biosystems Prisim 377 DNA sequencer was then performed.
P14-M
Economical usage of value-added phosphoramidites.
J.B. Hobbs; Univ. of British Columbia, #237-6174 University Boulevard, Vancouver, BC V6T 1Z3, Canada
A method is described for economical use of value-added (dye, biotin, etc.) phosphoramidites. The method was developed on an Applied Biosystems 380B synthesizer. The amidite is placed in a glass autosampler vial which is located in a plastic jacket inside a 10 ml glass vial. The amidite is dissolved in situ using a user-variable microdilution adapted from a bottle-change procedure. Typically 5 mg. of amidite are dissolved in 100 microlitres of acetonitrile. The amidite is used in conjunction with Applied Biosystems LV columns and synthesis cycles adapted for use with LV columns which use a similar "multiple-coupling" approach to that seen in "LV cycles" on later (392, 394, etc.) Applied Biosystems synthesizer models. These cycles are also described. Good tagging efficiency can be obtained for 40 nmole or 200 nmole scales using 5 mg. of amidite in this way, and the wastage of large quantities of expensive amidites is avoided. While the methods are applicable to later model synthesizers, the relatively modest flow rates, gas pressures and line volumes of the 380B render it a good platform for processes of this type.
P15-T
Quality control in a core oligonucleotide synthesis facility.
R.R. Muhlhauser, C.G. Miller, A. Yeung; Fox Chase Cancer Ctr., 7701 Burholme Avenue, Philadelphia, PA 19111
Our facility makes about 400 oligonucleotides each month. For the past 16 years, we have done quality control on each oligonucleotide prior to delivery. Our users do not want the quality of oligonucleotides to ever be a variable for troubleshooting in their experiments. Even a failure rate of 1% in oligonucleotide synthesis would have caused four experiments to fail each month. Such a low rate of synthesis failure is difficult to isolate in a statistical approach of quality control by random sampling. Analyzing every oligonucleotide also allows us to catch instrument and reagent problems more quickly. By analyzing each oligonucleotide immediately after deprotection, we reject 1 to 3% of the oligonucleotides we make that do not meet our quality standard. These oligonucleotides are resynthesized without delay. The oligonucleotide analysis method we use is anion-exchange at pH 12.5, using the Mono Q system on an FPLC (Yeung, A. T. and Miller, C. G. Anal. Biochem. 187:6675, 1990), assisted by a home-made autosampler and autoinjector. At that pH, both n-1's and deprotection problems are visible. We highly recommend this practice because it does not add appreciable expense or effort to the cost of the oligonucleotide, but provides the facility personnel with assurance in the quality of their products.
P16-S
Incorporation of pseudouridine and 4-thio-uridine into RNA oligonucleotides using 5'silyl-2'-ACE-orthoester chemistry.
S.A. Scaringe1, D. Kitchen2, J. Qui2; 1Dharmacon Res. Inc., 3200 Valmont Road #5, Boulder, CO 80301, 2Dharmacon Res., Inc., Boulder
The ability to incorporate a wide range of modified ribonucleotides into RNA oligos is an essential requirement for RNA chemical synthesis methodologies. Many modified bases have already been incorporated into RNA oligos using 5'-Silyl-2'-ACE-orthoester chemistry. We report here the incorporation of two more important modified bases, pseudouridine and 4-thio-uridine.
Pseudouridine was converted from the nucleoside to the protected nucleoside phosphoramidite in five steps in 45% overall yield. During synthesis the amidite coupled in 99% stepwise yields. The high yields for both the amidite synthesis and the oligonucleotide coupling have allowed pseudouridine to be incorporated into RNA on a routine basis. For example, a series of RNA oligonucleotides containing 1-3 pseudouridines were synthesized to study the 1920 loop region of E. coli 23S RNA.
4-Thio-uridine was incorporated into RNA oligos using the convertible nucleoside approach. The protected 4-triazole-uridine phosphoramidite was synthesized from uridine in six steps in 32% yield. The amidite coupled in 99% yields. Following oligo synthesis the support bound oligonucleotide was treated with thioacetic acid, buffered to pH 8.0, for 6 hours at 55°C. The support was washed and then treated with 10% DBU methanol at room temperature for 24 hours. The oligonucleotide was desalted and 2'hydroxyl protecting groups were removed using a pH 3.8 aqueous buffer for 30 minutes at 55°C. The resulting oligonucleotides contained 98% 4-thio-uridine at the desired position as assayed by anion exchange HPLC analysis. This is significantly higher than the results using cyanoethyl protection of 4-thio-uridine. All oligonucleotides containing 4-thio-uridine clearly exhibited the characteristic 330 nm absorption peak. 4-Thio-uridine is now being routinely incorporated in RNA oligonucleotides for use in several collaborative studies.
P17-M
New methods for the synthesis of multiple oligonucleotide sequences either singly or linked end-to-end in tandem.
R.T. Pon1, S. Yu1, Y.S. Sanghvi2; 1Univ. of Calgary, 3350 Hospital Dr. NW, Calgary, AB T2N 4N1 Canada, 2Isis Pharmaceut.
Solid-phase oligonucleotide synthesis requires attachment of the 3'-terminal nucleoside to the support, usually through a 3'-ester linkage. Phosphoramidite methods for performing this attachment through a phosphate linkage on "Universal" supports have been developed. However, these methods introduce a terminal 3'-phosphate residue which is difficult to remove. We have developed new linker phosphoramidite reagents which eliminate this problem by incorporating a cleavable 3'-ester linkage within the linker arm. Thus, only products with 3'-OH ends are produced and no extra conditions for 3'-dephosphorylation are required. These new reagents provide an alternative to our previous method for automated nucleoside attachment using HBTU and DMAP (Pon and Yu, Synlett 1999, 1778). The linker phosphoramidites are especially suited to high-throughput synthesis in 96-well plates since inexpensive, underivatized CPG can be used as a "Universal" support. Nucleoside attachment to the 5'-OH group of a prior sequence is also possible. This allows multiple oligonucleotides linked end-to end in tandem to be prepared in a single synthesis. The linked oligonucleotides are released from each other upon cleavage from the support and used together as a mixture. Sequences prepared in tandem can be PCR primers, forward and reverse sequencing primers, or duplex DNA fragments. The total number of bases is only limited by the support's pore size. We have prepared strings of oligonucleotides up to 96 bases long (4 X 24 base primers) and combinations of up to ten shorter sequences.
P18-T
Performance of high-speed oligonucleotide synthesizer in 96-well format.
D. Luk, A. Tuyet-Doan, S. Hall, J. Koh, R. Guettler; GeneMachines, 935 Washington St., San Carlos, CA 94070
Oligonucleotides are essential elements of genomic research. The PolyPlexTM oligonucleotide synthesizer makes 96 different oligos in standard 96-well format, which is amenable to downstream high-throughput processing and handling. Operator time is minimal and synthesis time is less than 3 hours for a 96-well plate of 20-mers. By eliminating flushing of reagent lines through a parallel dispensing technology, optimal synthesis time and reagent consumption are achieved. PolyPlex's low reagent consumption generates oligos quickly, at low costs and small scales as low as 10 nmol. Synthesis costs, including all consumables, are less than $0.10 per base. The PolyPlex synthesis chamber provides an inert-gas environment where synthesis progress can be monitored by using full-plate trityl collection after any base addition. PolyPlex utilizes fully licensed chemistry and generates oligos with greater than 98% coupling efficiency. Versatile, easy-to-use software provides powerful synthesis control along with extreme simplicity.
P19-S
Production of antigen specific MHC class I tetramers as a core facility service.
P.S. Adams, T.B. Miller, M.J. Dobrzanski, R.J. Hogan, D.L. Woodland; Trudeau Inst., 100 Algonquin Avenue, Saranac Lake, NY 12983
Major Histocompatibility Complex (MHC) Class I tetramers are composed of a tetrameric complex of the MHC Class I molecule and a specific antigenic peptide. The Class I heavy chain has been modified to contain a BirA sequence that allows biotinylation and subsequent tetramerization of the complex with a fluorescently tagged strepavidin. These tetramers can be used to identify antigen specific CD8+ T cells involved in immune responses using flow cytometry. The Molecular Biology Core Facility prepares these tetramers as a core facility service to aid the researchers at the Trudeau Institute. One project involves the characterization of the underlying mechanisms and immunoregulatory roles of tumor antigen-reactive Tc1 and Tc2 effector cell sub-populations, which can provide insight into the modification and/or development of novel immunotherapeutic approaches to disseminated malignancies. Another project involving viral immunity has demonstrated that a substantial population of antigen specific CD8+ T cells can be found not only in the secondary lymphoid organs, but also in the peripheral tissues after resolution of a primary respiratory virus infection. These data have significant implications for vaccines that induce systemic rather than local immunity. The production procedure and examples of the usefulness of MHC Class I tetramer technology will be presented.
P20-M
Advantages of directionally immobilized 6xHis-tagged proteins for capture assays.
K. Steinert, F. Schäfer, S. Wahle, A. Hoffer, P. Söhnlein, J. Ribbe; QIAGEN GmbH, Max-Volmer-Strasse 4, Hilden, Nordrhein-Westfalen 40724, Germany
Assay signals and reproducibility can be greatly enhanced by immobilizing the assay components in a directed manner. For example, to set up an interaction assay it is essential that the immobilization of the capture molecule occurs in active conformation to allow binding of the interaction partner. Similarly, oriented binding in immunoassays results in optimal accessibility of antigenic regions and therefore increased signal strengths. Directed immobilization of assay components can easily be achieved by using 6xHis-tagged recombinant proteins and immobilizing them via their 6xHis tags. The use of this interaction is well established for efficient purification of 6xHis-tagged proteins, but is also advantageous for immobilization of recombinant proteins in highly sensitive and reproducible protein-based assays. In addition, relying on the 6xHis-Ni-NTA interaction for immobilization of assay components simplifies assay development, because immobilization conditions no longer have to be optimized for each individual protein, but rely on the same biochemical characteristics of the 6xHis-Ni-NTA interaction for each protein. As an alternative to Ni-NTA, monoclonal antibodies recognizing the 6xHis tag can be used for highly specific and oriented immobilization of 6xHis-tagged proteins. The effect of specifically oriented binding on assay results is demonstrated by comparison of ELISA experiments employing proteins passively adsorbed to polystyrene plates and proteins immobilized via their 6xHis tags, as well as by a study on the interaction of two chaperones and development of a protease assay.
P21-T
Versalinx chemical affinity tools for purification in protein analysis.
J.P. Wiley, M.D. Ferguson, A. Gall, K.A. Hughes, G. Li, K.P. Lund, M.L. Stolowitz; Prolinx, Inc., Bothell, WA, 22322 20th Ave. SE, Bothell, WA 98072-8341
Fractionating complex biological mixtures is one of the challenges in proteome analysis. MALDI-TOF (matrix assisted laser desorption ionization time of flight) mass spectrometry analysis, for instance, has a high mass accuracy but requires a relatively pure sample for positive structural identification. Prolinx® Inc. has developed Versalinx™ Chemical Affinity Tools, and has demonstrated success using this technology to isolate and purify proteins of interest from complex mixtures. Versalinx Chemical Affinity Tools are based upon the interaction of phenyldiboronic acid (PDBA) and salicylhydroxamic acid (SHA) to form a complex that is reversible under certain conditions. Immobilization of PDBA-modified proteins can occur on a variety of SHA-modified surfaces. The system is stable over a wide pH range and is compatible with a number of denaturants, detergents and organic solvents. With Versalinx Chemical Affinity Tools, purification of biological molecules is rapid and reproducible.
P22-S
Fully automated 96-well protein purification and magnetic bead-based assay using 6xHis-Ni-NTA technology.
K. Steinert, H. Lubenow, S. Wahle, R. Stoll, J. Ribbe; QIAGEN GmbH, Max-Volmer-Strasse 4, Hilden, Nordrhein-Westfalen 40724 Germany
The purification of proteins from expression libraries requires a method that performs reliably regardless of the characteristics of the proteins to be purified. Using Ni-NTA for purification of 6xHis-tagged recombinant proteins provides a one-step purification method that is robust and meets the challenge presented by the need to purify thousands of proteins with differing structure and characteristics. To allow this method to be performed in high-throughput applications, automated protocols were developed which run on BioRobot systems.
Ready-to-run protocols covering a wide range of protein assay and purification applications are available. They rely on 6xHis-Ni-NTA technology based on the high affinity and specificity of nickel ions immobilized on matrices bearing nitrilotriacetic acid (NTA) for recombinant biomolecules with a tag of six consecutive histidine residues (6xHis tag).
Ni-NTA Magnetic Agarose Beads allow flexible interaction or diagnostic assays with structurally active immobilized 6xHis-tagged proteins as well as micro-scale purification of up to 15 µg of 6xHis-tagged protein. The purification and assay protocols based on Ni-NTA Magnetic Agarose Beads may even be carried out directly in series.
If much larger amounts of purified proteins are needed, the Ni-NTA Superflow 96 BioRobot Protocol provides a convenient high-throughput method for purification of approximately 100 µg of 6xHis-tagged protein per well in a 96-well format. Examples of purification and assay applications using both methods are shown and data on reproducibility and cross-contamination free processing are given.
P23-M
Detection of residual isopropyl-1-thio-beta-D-galactopyranoside in proteins by HPLC with pulsed amperometric detection.
X. Ji, L. Couch, A. Pennetti, K. Venkat, J. Mozdzanowski; SmithKline Beecham Pharmaceut., 709 Swedeland Road, King of Prussia, Pa 19406
Isopropyl-1-thio-beta-D-galactopyranoside (IPTG) is an inducer of the lac operon and may be used in fermentation processes for the production of protein biopharmaceuticals in E. coli. Use of this compound in the fermentation process creates the need for the determination of residual IPTG in the final product. Chemical and optical properties of IPTG (e.g. absorbance in the UV region) do not permit sensitive detection using traditional techniques. Hemiacetal group of galactose is substituted by a 2-thiopropyl group and thus eliminates an option for simple derivatization reactions leading to fluorescent derivatives. IPTG is a sulfur-containing galactoside, and therefore, properties of sulfur and sugar may be utilized for electrochemical detection. With this mode of detection, residual IPTG can be detected in the presence of high concentrations of protein. This required development of a modified HPLC separation method permitting the use of high concentrations of acetonitrile to remove protein from the column during a wash step. HPLC conditions developed for the separation are free from interference with protein and a so-called "oxygen dip" typically observed with the gold electrode. The waveform developed by William LaCourse for the Dionex electrochemical detector (ED-40) with the gold electrode can be used for the determination of IPTG at a concentration as low as 0.05 µM (12 ng/mL).
Reference:
William LaCourse, Determination of Isopropyl-beta-D-thiogalactoside (IPTG) by HPLC-Pulsed Electrochemical Detection. Pittcon-2000 presentation.
P24-T
Construction of a tagging system for subcellular localization of proteins encoded by newly discovered open reading frames.
W-J. Syu; Natl. Yang Ming Univ., 155 Sec 2, Li-Long St., Pai-Tao, Taipei, Taiwan 112, Taiwan
We have previously characterized a monoclonal antibody (SC1D7) that is directing to maltose-binding protein (MBP) of Escherichia coli and other closely related enteric bacteria. SC1D7 does not cross-react with proteins in eucaryotes and appears to be a highly specific tool in immunochemical analyses. To better map the epitope, we took advantage of an available plasmid pMAL-c2 that encodes E. coli MBP-coding sequence and constructed plasmids to express MBP fragments. A construct containing the N-terminal portion of MBP does not react with SC1D7 whereas a second construct expressing glutathione-S-transferase fused with the C-terminal half of MBP does react with SC1D7. To precisely define the epitope, random peptides displayed on M13 were used to react with SC1D7. Sequences of reactive peptides were aligned, and a consensus sequence of XDXRIPX was deduced. This sequence matches to MBP with an amino acid stretch of KDPRIAA. To consolidate the mapping result, a sequence encoding this epitope was inserted into an expression vector and the resulted recombinant protein did react with SC1D7. Thereafter, this epitope was incorporated into an eucaryotic expression plasmid that contains a previously defined hepatitis delta virus epitope for protein tagging. The so constructed two-epitope-tagging vector is useful in various molecular analyses. We demonstrated its usage in localization of a bacterial virulence factor in host cells. This vector should be applicable for high throughput characterization of new open reading frames found in genome sequencing.
P25-S
Truncated midkine (tMK): its molecular cloning and expression in Escherichia coli, and the detection of native tMK in Wilms' tumor by an anti-tMK monoclonal antibody.
S. Paul1, W. Dansithong1, T. Mitsumoto1, Y. Asano1, M. Kato2, M. Kato2, T. shinozawa1; 1Gunma Univ., Japan, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 Japan, 2Tottori Univ., Japan
Midkine (MK) is a heparin binding growth factor identified as a product of a retinoic acid-responsive gene; it is frequently expressed at high levels in many human carcinomas. Although the expression of the mRNA encoding truncated MK (tMK) in unique human cancer cells has been reported, the tMK polypeptide itself has not yet been identified. In order to clarify the biological role of tMK, recombinant tMK was expressed in Escherichia coli and purified. Purified recombinant tMK showed the same extent of proliferative activity for the Wilms' tumor (G401) cells as full length human MK. A mouse hybridoma cell line producing an IgG2b monoclonal antibody (mab) against this purified recombinant tMK was also established. This anti-tMK mab (MiStMK-V3) did not crossreact with synthetic full length (or c-half) human MK. A native tMK, showing the same apparent molecular mass as the recombinant tMK in SDS-PAGE, was identified in G401 cells using this mab. These results suggest that the structure of the recombinant tMK is same as that of native tMK. The expression of tMK in Wilms' tumor patient specimens was also detected in an immunohistochemical study using the anti-tMK mab. The usefulness of this mab (MiStMK-V3) for the detection of tMK in Wilms' tumor was demonstrated.
P26-M
Information management systems for molecular biology.
T.M. Smith, D. Campbell, R. Connelly, A. Leonard, S.G. Porter, J. Slagel; Geospiza, Inc., Seattle, 2442 NW Market St. #344, Seattle, WA 98107
Technological advances in molecular biology have made it possible for laboratories to generate unprecedented amounts of data. DNA sequencing, for example, has seen an increase in throughput of over 400-fold in recent years. The accelerated production of data has produced a corresponding need for better data management.
The increased need for data management is partly due to institutional changes. Many institutions have been able to economize by establishing core facilities which offer laboratory services to researchers on a per sample or project basis. Core facilities are cost-effective because they provide several researchers with access to expensive equipment and skilled personnel. Because these core facilities handle a variety of data types and a large number of samples from multiple sources, their information management needs provide a worthwhile model for laboratory information management systems (LIMS).
LIMS are collections of software, communication devices, and computers that acquire, store, analyze, and present data and information about laboratory samples and their processing. LIMS centralize data storage, automate data analysis, and provide quality assurance reports for process monitoring. The central component of a LIMS is a database with software interfaces for entering, viewing, and processing information.
The level of complexity needed in a LIMS depends on factors such as the mission of the laboratory, the number of samples to be processed, analysis requirements, and workflow complexity. LIMS development presents many challenges and may take years to complete. Most labs have no need for a custom LIMS and lack the wherewithal to create one, and thus will benefit from commercial LIMS
The general features of LIMS will be presented along with case studies of Geospiza's Finch™-Suite LIMS solution illustrating how a commercially available software system can be used to meet the information management needs of DNA sequencing facilities.
P27-T
Performance evaluation of a novel application for comparative DNA sequencing analysis and mutation detection.
C.A. Kosman, L. Johnston-Dow, H. Breu, G. Chappell, S. Kumar, B. Iasnopolski, B. Kshirsagar, P.A. Suri, R. Paul, G. Mason, D. Siu, E. Sword, M. Schoppe, V. Bawge; Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404
Various software tools are available for the analysis and interpretation of de-novo DNA sequencing data. While such applications work well for sequencing for discovery many of the underlying assumptions in these tools are not valid when applied to comparative sequencing data.
We have developed a new practical application for comparative DNA sequencing analysis. The focus of this new application is the discovery or identification of sequence variants in a dataset of sequence derived from a single locus in various individuals. This tool allows for the rapid and accurate analysis and alignment of multiple sequence comparisons containing mixed base positions against a reference sequence. While NT-based, this software is compatible with data generated from all of Applied Biosystems sequencing platforms (ABI PRISM® 373, 377, 310, 3100 and 3700).
This application uses procedures and algorithms better suited to the unique nature of comparative sequencing data. Specifically, the prior knowledge of a reference sequence available in many comparative sequencing projects is used to streamline and automate portions of the analysis. The software performs sequence analysis and aligns the analyzed sequence to a reference sequence. It also aligns imported sequences that contain variants. It will then analyze the compared sequences, provide protein translation and report the analysis in a convenient format.
Here, we present the results from the performance evaluation of this new software tool on several types of comparative sequencing datasets. For these analyses, this new application produced results concordant with those previously obtained using standard DNA analysis tools while providing substantial performance advantages over these tools. In all cases the analyses were completed in significantly less time and required less manual manipulation of the data. The innovative approaches employed in this tool result in less user intervention needed to achieve a better level of mutation detection.
P28-S
A Web interface for BioLIMS System--data and user management for sequencing core facility.
L. Liu, L. Roinishvili; Univ. of Illinois, Urbana, 330 ERML, 1201 W. Gregory Dr., Urbana, IL 61801
Sequencing core facility serves many customers. Samples in a sequencing core facility are very heterogeneous although the volume of samples from each customer is relatively small. Data and user management can be a challenge task. The BioLIMS database system from PE Bioinformatics provides a smooth data flow from sequencer to database. However, it really lacks of friendly user interface for both core facility and customers. To provide an easy management system for the core facility at the W. M. Keck Center for Comparative and Functional Genomics, we implemented a web interface for BioLIMS system. This interface serves two purposes in the core facility. One is for core facility staff to manage customers and their data. The other is for customers to view and retrieve their own data from the web. In customer management, we allow super user and sub user relationship. Super user will have access to all the data of his or her sub users. In data management, a customer can only view and retrieve his or her own data unless others give him or her privilege to view their data. The most convenient feature is web based. Customers can access their data from anywhere at anytime.
P29-M
Protein/DNA technology DSL version 2000 is a set of shareware protocols for sample and data management.
J. Medalle, M. Randesi, B. Imai; Rockefeller Univ., 1230 York Ave., Box 105, New York, NY 10021
Physical sample and data management are major concerns for a DNA sequencing core facility. Especially one with a small staff and limited space are affected the most. Samples can overtake the limited freezer space. Paper trail can overfill office and lab spaces. Both can cause a quick turnover of DNA sequencing staff. DNA Sequencing Lab's (DSL) version 2000 objectives are to alleviate repetitive computer tasks for DNA Sequencing staff and eliminate physical paper trail. DSL 2000 is a set of protocols for sample and data management. Its major components are comprised of online submittal forms at http://protein13-pc.rockefeller.edu/, scripts from Apple Script, and standardized forms and macros from Microsoft Excel 98, and Outlook Express 5. The DSL funnels the online sample submissions into sample statements for each investigator. This is a file containing sample IDs, names, account numbers, primer info, template info, etc. DSL also transforms the web submissions into logs for prep and for slab or capillary sequencing reactions. These logs direct the staff on how to process incoming samples. The set of protocols streamlines the sample flow from the investigator to the machine as well as the data flow from machine back to the investigator. DSL's protocols are "hands on" to help the staff create e-mails for investigator notifications and "a click of a button" to compress and to transfer investigator's sample statements or data files into their web account. DSL 2000 is a freeware application and can be integrated into other small core facilities with a limited budget.
P30-T
A laboratory information management system for a small DNA sequencing core facility.
M.J. Miller; NCI, NIH, Bldg 37, Rm 3C28, MSC 4255, Bethesda, MD 20817-4255
The DNA Sequencing MiniCore facility of the NCI's Division of Basic Sciences currently services over 300 investigators. The facility is designed to support small scale, short-term sequencing. In the past year we ran over 25,000 samples with an average turnover time of less than one day. While this represents a 60% increase in samples over the year before, it is still a relatively modest operation.
Keeping all these users and their data organized, as well as minimizing the amount of paperwork involved is a major concern. Although several Laboratory Information Management (LIM) System software packages exist, they are often too expensive or too inflexible for a small operation such as ours.
I describe here a LIM system built using components of the Microsoft Office software package. Users enter sample information data into an Internet form and this data is automatically entered into the database after inspection by the MiniCore staff. Programs built into the database determine which sample sets can be run together on the same gel, and what parameters (such as which virtual filter and which dye/primer set settings) should be used with each sample. Samples are assigned to a particular gel and the "sample sheet" for that gel is automatically generated by the database. By keeping track of when samples are submitted, how many samples there are, and how many basepairs of read the user requests, the database aids the facility's operators in determining sample priority. An email message is automatically sent when samples have been processed and data deposited in the user's data-destination directory. The database also keeps track of charges and automatically sends this data to the NIH's accounting system. The facility thus runs in a virtually "paperless" environment. There are no hardcopy forms to fill out. All information is maintained by the computer system.
P31-S
The analysis of complex tryptic peptide mixtures by multidimensional LC-MS/MS on a hybrid quadrupole orthogonal acceleration time-of flight (Q-TOF) mass spectrometer.
A. Millar1, C. Hughes1, T. Andresson2, T. Hemesath2, J.I. Langridge1; 1Micromass UK Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ, United Kingdom, 2deCODE genetics Inc., Reykjavik
Advances in both HPLC and mass spectrometry instrumentation have allowed the analysis of protein complexes which have not been separated on a two dimensional gel. These experiments involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data directed switching between the MS and MS/MS modes. Protein identification is then achieved via databank searching of the ESI-MS/MS, providing qualitative information on the proteins that are present. Hundreds of MS/MS spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single LC-MS/MS experiment1.
If, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity is advantageous. We have, therefore, adopted a 2D LC-MS/MS approach using a capillary LC system (CapLC) operating at nanoliter per min flow rates coupled to a Q-Tof 2 mass spectrometer. By placing a strong cation exchange (SCX) cartridge followed by a C18 trap cartridge it is possible to pre-fractionate the peptides before separation on an analytical C18 column. After loading the sample, discreet fractions are sequentially eluted from the cation exchange cartridge using a salt step gradient; the eluted peptides are then retained on the trapping C18 cartridge whilst they are desalted. Finally the peptides are eluted from the C18 pre-column, at 200 nL/min, onto a 75 µM ID X 10 cm Waters Symmetry analytical column for separation and elution into the mass spectrometer.
This analytical approach will be discussed with examples where this methodology has been used for the analysis of standard protein mixtures and for the analysis of cell lysates and sub-cellular fractions.
1. Yates et al., Nature Biotechnology (1999);17, (7), 676-682.
P32-M
Identification of in vivo phosphorylation sites in Drosophila armadillo by tandem mass spectrometry.
C.S. Raska, R.M. Pope, D. Rubenstein; Univ. of North Carolina at Chapel Hill, 4 Casabelle Ct, Durham, NC 27713
Phosphorylation is one of the most important reversible modifications of eukaryotic proteins. Often, proteins which are associated with uncontrolled cell growth, and ultimately cancer show an anomaly in their phosphorylation/dephosphorylation pathways. In humans, a protein, beta-catenin, plays a central role in the development, organization, and regulation of epithelial tissues. Aberrant regulation of beta-catenin is associated with malignancies. For example, alterations in beta-catenin gene structure have been identified in colorectal and breast carcinoma, and in a large number of melanoma cell lines. Specifically, beta-catenin signaling function is constitutively active in many melanomas due to mutations that remove phosphoresidues at the amino terminus of the protein. Armadillo, a protein found in Drosophila melanogaster, is 71% identical to beta-catenin at the amino acid level. Alpha-catenin from Drosophila and from human keratinocytes binds to armadillo and beta-catenin, respectively, and this binding has been found to be influenced by phosphorylation. Thus, we have been using armadillo as a model system to study beta-catenin regulation. To further define the regulatory function of phosphorylation, we are using mass spectrometry to map post-translationally modified amino acids in armadillo. Armadillo was isolated by immuno-affinity and ion exchange chromatography. 1-D SDS-PAGE, and in-gel tryptic digestion were performed to produce a mass spectral fingerprint on a triple quadrupole instrument using nanoelectrospray. MS scans confirmed peptides matching both armadillo and alpha-catenin from one gel spot. Neutral loss scans identified potentially phosphorylated peptides. MS/MS generated enough sequence coverage to specifically identify phosphorylated residues. We note that phosphoresidues are located within the region where binding sites for cadherin, dTCF, and dAPC are located. We plan mutation studies to further elucidate the role of phosphorylation in these systems.
P33-T
A targeted approach for more sensitive and accurate protein identification.
D.K. Leung, M.J. Horn; BioMolecular Technol., Inc., 525F Del Rey Ave., Sunnyvale, CA 94086
Selective isolation of peptides containing low abundance amino acids such as tryptophan, offers a direct way to simplify protein digest mixtures prior to mass spectrometric analysis. This approach provides benefits for both in-gel digests and digests of protein mixtures.
To this end a solid phase reagent capable of selectively binding to tryptophan has been developed. This solid support material binds covalently to tryptophan or tryptophan containing peptides, allowing for their selective separation from mixtures of peptides and proteins. Bound peptides can be released from the solid-support by a reducing reagent to give analytical samples for further analysis (LC, MS, etc.). The specificity of the binding chemistry coupled with mass spectrometric analysis by MALDI affords improved accuracy in data base search and in protein identification.
References
1. Shechter, Y., Rubinstein, M. and Patchornik, A. (1976), Biochemical and Biophysical Research Comm. {4} (70), 1257.
2. Early, S. L., Magil, S. G., Novak, C. and Horn, M. J. (1989), Techniques in Protein Chemistry, Academic Press, Inc., 439.
3. McEldoon, W., and Horn, M.J. (2000), 14th Protein Society Symposium, {August}.
P34-S
Approaches to validating SEQUEST database search results.
R.E. Moore, M.K. Young, T.D. Lee; Beckman Res. Inst., City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010
Tandem mass spectrometry and database searching is a very powerful approach to protein identification. Unfortunately, the wealth of data that makes the technique so powerful also requires extensive analyst time for data reduction and validation of search results. When analyzing complex samples, it can easily take more time to validate and reduce the data than it did to generate it. This time requirement is a serious problem because the technique is otherwise well suited to high throughput analysis.
Two aspects of result validation are analyzed: validation of search results for individual tandem mass spectra and validation of composite results for sets of individual spectral matches. Statistical analysis and experiments with falsified databases demonstrate that there is a significant chance of generating multiple incorrect matches to the same protein when examining large data sets. This makes it imperative to validate the individual spectral matches, as simple reliance on multiplicity of matches to a single protein does not guarantee a correct result.
Several approaches to validating individual spectral matches were tried and compared to the existing standard of manually comparing actual and predicted spectra. The most successful approach was to search each spectrum twice, once using tryptic specificity and once using no enzyme specificity, and correlate the results. When both searches generate the same result, that result is almost always validated by manual examination. When the non-specific search generated a higher cross-correlation score the result of the search using tryptic cleavage was almost never manually validated. Surprisingly, there were a large number of searches in which the search using tryptic cleavage resulted in a higher cross-correlation score than the search using non-specific cleavage. This third group yielded a moderate number of spectra that were manually validated.
P35-M
Guanylation of lysines as a means to enhance confidence of protein identification in proteomics.
S. Krishnan1, M. Lin2, K. Waddell1; 1Applied Biosystems, Foster City, CA, 500 Old Connecticut Path, Framingham, MA 01701, 2Applied Biosystems, Framingham, MA
The completion of several genomes, including that of the human, has shifted the emphasis to determining the proteome counterpart of biological systems. The current approach for such a proteomic analysis is the mass fingerprinting of tryptic digests of proteins (generated by ingel digest of 1 or 2-D gel spots or of chromatographic fractions) using MALDI-Tof mass spectrometry followed by database searching of the masses so obtained. It has recently been noticed that the arginine containing peptides ionize better under MALDI conditions compared to lysine containing peptides. We present here an approach to use this as an advantage in enhancing confidence of the database search matches by modifying the lysines to homoarginines. The increase in confidence is achieved due to increase in the number of peptides seen in the mass spectrum resulting from the guanylation of lysine e-amino group (addition of 42 Da) and also the fact that such a modification indicates the lysine containing peptides in the tryptic digest mix. Tryptic digests of BSA, Enolase, alpha lactalbumin, and cytochrome C were generated by routine procedures and mixed in various combinations. The digest mixtures were than analyzed by MALDI-Tof mass spectrometry followed by database searching of the peptide masses for identification of proteins. A portion of the digest mixture was then reacted with O-methyl isourea sulfate to convert the lysines to homoarginines. The modified digest mixtures were analyzed by MALDI-Tof mass spectrometry followed by database searching. The analysis before and after the modification helped identify the lysine containing peptides in the digest and hence a more comprehensive identification of the protein mixture.
P36-T
Automated analysis of single nucleotide polymorphism using mass spectrometry.
M.S. Minkoff1, P. Ross1, L. Hall1, R. Jones2, D. Ledman1, L. Haff1; 1Applied Biosystems, 500 Old Connecticut Path, Framingham, MA 01701, 2Inst. for Child Hlth.
Single nucleotide polymorphisms (SNPs) are increasingly employed as genetic markers for associated studies of many inherited diseases and traits. It is apparent that approximately 3 million putative SNPs will be found in the Human Genome. In order to tackle the task of validating the significance of any of these putative SNPs the chemistry and analytical platforms must meet demanding throughput and cost requirements. The SNP assay developed to meet these criteria incorporates the use of simple, inexpensive primers and unlabeled nucleotides for single base extensions, thus keeping down the cost of analysis. Multiplexing the analysis of SNPs enhances affordability of genotyping studies as well as significantly improving throughput. The use of high throughput automated Mass Spectrometry with integrated data acquisition, genotyping, sample handling software package simplifies and speeds-up the task. In addition the top-level software application package tracks sample information throughout the workflow and stores all information in a central database. Data in support of multiplexed, automated sample handling, acquisition, analysis and reporting is presented.
P37-S
Data-directed real-time instrument feedback and control: creation of dynamic peptide include and exclude lists from on-the-fly databank searching.
A. Millar, J.B. Hoyes, R.A. Carruthers, C. Jones, A. Millar, C. Hughes, S. Leicester, J.I. Langridge; Micromass UK Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ, United Kingdom
An alternative approach to 2D gel electrophoresis for the qualitative analysis of complex protein mixtures is the use of tryptic digestion followed by electrospray LC-MS/MS. This approach has been shown to increase the dynamic range of protein identification and identify low copy number proteins. However there is often a large degree of redundant sequence information acquired, as in theory one peptide MS/MS spectrum is sufficient to identify a protein from a sequence databank. If a protein identification is obtained from a databank search of an MS/MS spectrum, excluding the rest of the theoretical tryptic peptides during an LC-MS/MS experiment may allow deeper "mining" into the protein complex being studied.
We have introduced a new search engine capable of matching a tryptic peptide from the Swissprot/TrEMBL databank to an MS/MS spectrum in one second. Based upon these results, we are able to generate dynamic include or exclude lists, based upon the theoretical tryptic peptides from the identified protein. These can be passed to the acquisition software of our Q-Tof mass spectrometer in real time. Thus, we are able to automatically steer the Q-Tof during acquisition to select and switch to the MS/MS mode only on those peaks that meet the modified selection criteria. Therefore precursor ions that belong to a protein already identified during acquisition can be avoided. This exclusion list is based upon m/z, charge state and a user defined mass tolerance. Mass measurement on the Q-Tof 2 mass spectrometer is typically better than 10ppm and therefore a tight mass tolerance can be selected, making the exclude list extremely specific.
To illustrate this methodology we present examples where Q-Tof data acquisition has been directed based upon the results from a databank search. This data will be compared and contrasted to data acquired in the normal automated LC-MS/MS mode.
P38-M
Routine identification of large (>5 kDa) peptides derived from biological sources using an ESI-quadrupol-TOF mass spectrometer.
M. Kellmann, S. Neitz, M. Juergens; BioVisioN GmbH & Co KG, Hannover, Feodor-Lynen-Str. 5, Hannover, Lower Saxony 30625, Germany
Investigating the peptide portion of a proteom results in a multitude of peptides/small proteins in the mass range from 5 to 15 kDa [1]. We developed a routine procedure for direct identification exploiting the relatively high parent and daughter ion resolution (>7500) and mass accuracy (<50 ppm) of a hybrid ESI-quadrupol-time-of-flight mass spectrometer. Data processing was made with automated charge state deconvolution based on Zhang and Marshall´s Zscore algorithm [2] and a deisotoping algorithm included in the Voyager 5.0 software from Applied Biosystems. Database searching was performed by the MASCOT search engine [3].
We will illustrate that this procedure will lead to a fast and reliable identification of several large peptides in the mass range from 5 to 15 kDa derived from blood filtrate.
[1] Kellmann, M, et al., Proceedings of the 48th Conference on Mass Spectrometry and Allied Topics, 2000, Long Beach
[2] Zhang, Z and Marshall, AG. A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra. J Am Soc Mass Spectrom 9(3): 225-33 (1998).
[3] Perkins, DN, Pappin, DJ, Creasy, DM and Cottrell, JS, Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18) 3551-67 (1999).
P39-T
Automated software for identification and relative quantification of differentially expressed proteins from isotope coded affinity tag LC-MS data.
A. Millar, P. Young, R. O'Malley, A. Millar, J.I. Langridge; Micromass UK Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ, United Kingdom
Whilst LC-MS/MS has been utilised for the identification of proteins from complexes and cell lysates (qualitative proteomics), the quantitative study of gene expression using differential display has until recently been the preserve of a 2D gel based proteomic experiment. However, recently a great deal of interest has been generated on the use of isotope coded affinity tags (ICAT) 1 for the quantitative study of gene expression at the proteome level.
The technique is based upon chemically modifying the cysteine residues of proteins isolated from cells in two different states with light and heavy isotopically labeled reagents. The two cell states are then combined, digested with trypsin and the cysteine containing peptides preferentially selected by binding to an avidin column, prior to analysis by mass spectrometry. The eluent from this column is then analysed by capillary LC ESI-MS/MS. Interrogation of the eluting peptides by tandem mass spectrometry and databank searching results in the identification of the associated protein.
We describe how ICAT data analysis has been automated within a software environment. The MS and MSMS data acquired using the QTof instrument are processed and analysed using a new algorithm which recognises related isotope clusters and quantifies their relative intensities. Based on a user defined ratio threshold the software will automatically carry out an LC-MS/MS experiment and databank search in a client-server mode and provide a report of the identified proteins and their expression ratio in the two cell states.
1. Gygi et al. Nature Biotechnology (1999) 17, 994-999.
P40-S
New method for preparation of DNA for MALDI-MS analysis.
I.P. Smirnov, L.A. Haff, P.L. Ross, L.R. Hall, D.W. Ledman; Applied Biosystems, 500 Old Connecticut Path, Framingham, MA 01701
In recent years, matrix assisted laser desorption ionization mass spectrometry (MALDI MS) became one of the main analytical tools for short DNA fragments and synthetic oligonucleotides. The application of mass spectrometry for genetic analysis (such as SNP typing) offers, compared to other methods, higher sensitivity, higher accuracy and lower costs by removing the necessity for fluorescent- labeled primers. Still, the susceptibility of MALDI-MS to the presence of salts of alkali metals requires careful and often tedious desalting procedures which complicates and slows the throughput of the MS based methods. To overcome that obstacle we have developed a novel approach to sample preparation. The idea is to extract DNA out of the solution on a solid surface coated with an anion exchange polymer (e.g. polyethyleneimine). The observed binding is strong enough to remove and concentrate DNAs from buffer solutions and hold them on the plate through extensive washing procedures if necessary. The properties of anion exchange films make possible to perform purification, desalting and concentration in a single fast step. After the DNA is immobilized on the surface and supernatant solution is removed, subsequent addition of MALDI matrix releases material from the surface, which co-crystallizes with matrix. The MS analysis can then be performed directly from that support. Such surfaces may be obtained upon glass, plastic, or metal MALDI plates. Analysis of oligonucleotides and complex multiplexed SNP typing reactions (Sequazyme-PinPoint) performed by this method shows greatly improved sensitivity and excellent resolution for wide range of DNA lengths, together with high tolerance to various buffers components, such as alkali metals and surfactants. The importance and role of different factors have been studied, such as composition of ion exchange film and compatibility of the MALDI matrix in order to obtain the best performance.
P41-M
Review of gel- and column-based separation technologies for the identification of yeast proteins using MS/MS.
V.C. Wasinger, G.L. Corthals; Garvan Inst. of Med. Res., 384 Victoria St., Sydney, NSW 2010, Australia
Proteome analysis involves the identification and quantitation of expressed proteins by a given cell type, tissue or organism and most commonly involves 2-dimensional electrophoresis (2-DE) followed by mass spectrometry (MS) for protein separation and identification. While 2-DE is unequalled in its ability to separate and resolve thousands of proteins in complex solutions, we have recently shown that many proteins of biological significance lay beyond the detection limits of 2-DE. The application of 2-D gels becomes ineffective when proteins co-migrate to the same grid coordinates, merge to become one large spot or are not displayed because their concentration is simply too low for visualisation and subsequent biochemical MS analysis.
Two important biological aspects prevent us from fully exploiting the power of the 2-DE technology: 1) Firstly, protein expression exists for up to 12 orders of magnitude (in serum). This broad range of expression critically limits the current 2-DE approach, as at best only 5 orders of magnitude difference in expression can be displayed; 2) "functional" proteins operate in association with other proteins; such that research must be directed toward the analysis of multiply interacting proteins or functional modules that regulate complex biological networks and pathways.
Alternative fractionation methods such as chromatography exist and allow proteins/peptides to be separated based on similar (charge, size) or alternative (affinity, hydrophobicity) properties to 2-DE. Used in series with MS, these technologies have the potential to fractionate large numbers of proteins and peptides to enable the comprehensive, comparative and relational analysis of proteins. Yeast is an ideal choice for this study as both genome, proteome and transcriptome data is available for evaluation.
A comparison of the separation potential based on protein numbers, protein classes and properties are described using 2-D gel electrophoresis, size exclusion and cation exchange chromatography followed by protein identification using ESI-MS/MS.
P42-T
Quantitative analysis of tumor antigens by mass spectrometry.
A. Kishiyama, D. Arnott; Genentech, 1 DNA Way, South San Francisco, CA 94080
Both genomics and proteomics are useful for comparing gene expression patterns. The discovery of receptors overexpressed on tumor cells has lead to effective cancer therapies, for example. Techniques such as DNA arrays can be used to measure mRNA levels with great sensitivity and speed. More detailed information can be obtained through 2D gels, western blotting, and immunohistochemistry, but these protein-based approaches are time-consuming, prone to biases, or require generating antibodies. A method has therefore been developed, called the mass western experiment in analogy to the western blot, to better bridge genomics and proteomics.
In this experiment, a variation on the ICAT experiment described by Gygi et al. (Nature Biotech. 1999 v.17 p.994), specific proteins (such as those found to be of interest from DNA array experiments) are detected and compared between samples. Proteins extracted from two samples are labeled with a custom ICAT reagent. The samples are mixed, digested, and the labeled peptides collected. LC-MS/MS is performed on an ion trap instrument; anticipated tryptic peptides from the protein of interest are continuously subjected to CID. Heavy and light ICAT-labeled peptides are simultaneously trapped and fragmented. Both identification and quantitation is thus obtained in one experiment.
This approach has been validated by the comparison of cell lines expressing known tumor antigens in different amounts. Overexpression of the receptor Her-2 in breast cancer cell lines was shown, and by factors in agreement with other measurements. Other potential tumor antigens have likewise been detected.
P43-S
Characterization of differential protein expression between wild type and Rce1 knockout mouse embryonic fibroblasts using 2-D SDS PAGE and MALDI TOF mass spectrometry.
S.C. Hall1, D. Smith-Beckerman2, M. Lobo2, S.G. Young3; 1Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, 2San Francisco State Univ., 3UCSF
Ras proteins play an important role in transmitting growth signals from membrane receptors to the nucleus, triggering the transcription of genes involved in cellular proliferation. Ras proteins are membrane bound, guanine nucleotide-binding proteins with GTP-ase activity. Many human cancers contain mutationally activated Ras proteins that transmit growth signals in an uncontrolled manner, triggering neoplastic transformation and uncontrolled cellular proliferation. The proper intracellular location of the Ras proteins depends on a series of posttranslational modifications: isoprenylation of a C-terminal cysteine, endoproteolytic release of the carboxyl-terminal three amino acids, and methyl esterification of the carboxyl-terminal isoprenylcysteine. Each of these processing steps represents a potential target for cancer therapy. The endoproteolytic cleavage step is carried out by the product of the Rce1 gene. Recently, the Rce1 gene was inactivated in mice, completely blocking the endoproteolytic processing of the Ras proteins. We used 2D-PAGE to assess differences in protein expression in primary embryonic fibroblasts from wild-type and Rce1 knockout mice. First, we wanted to detect substrates for Rce1. Altered electrophoretic mobility of individual protein spots on the gel indicated the possibility of aberrant post-translational processing. Second, we wanted to detect proteins whose expression level was affected by the Rce1 knockout mutation. MALDI TOF mass spectrometry was used to generate peptide mass fingerprints to identify several proteins having identical molecular weight, pI, and relative abundance in both wild-type and Rce1 knockout mice. Additional confirmation of the identities of these proteins was obtained by performing post-source decay analysis on selected tryptic peptides. It was important to identify these "marker" proteins as they will be used as migration reference points when comparing future 2D-gel separations. Furthermore, they permitted optimization of protocols for the MS analysis of Ras proteins from wild-type and Rce1 knockout fibroblasts.
P44-M
Intelligent data acquisition and automated sample analysis via orthogonal MALDI- QqTOF, a new tool for protein identification.
C.M. Lock; MDS-Sciex, 71 Four Valley Drive, Concord, Ontario L4K 4V8, Canada
The application of a novel UV-MALDI ionisation source coupled to an Applied Biosystems/MDS-Sciex QSTAR Pulsar QqTof mass spectrometer for protein sequencing and identification is described. The coupling of these two devices enables collision induced dissociation spectra of singly charged MADLI ions to be generated, with all the associated QqTof benefits of high mass accuracy and resolution. The inherent pulsed nature of the o-MALDI source is converted into a pseudo continuous beam of ions by collisional cooling in the Q0 region. The o-MALDI source is completely decoupled from and has no influence on the orthogonal Tof analyser.
High mass accuracy and resolution is thus maintained simultaneously over the full mass range when switching between MS and MS/MS modes as opposed to conventional MALDI post source decay experiments.
The application of the technique to the analysis of low femtomole unseparated protein tryptic digests is demonstrated using an automated data acquisition approach. The software developed enables the intelligent acquisition of data from sample plates with minimal user intervention.
The high speed data acquisition capabilities of the o-MALDI source in combination with the high performance of the QqTof offers unique possibilities for rapid identification of proteins. Rapid analysis and identification of proteins via a peptide-mass fingerprinting approach and MS/MS sequence information will be shown.
P45-T
Identification of changes in protein expression in Deinoccocus radiodurans using isotope-coded affinity tags.
E.A. Panisko1, T.P. Conrads1, T. Veenstra1, L. Pasa-tolic1, G.A. Anderson1, R. Aebersold2, R.D. Smith1; 1Pacific Northwest Natl. Lab., P.O. Box 999, K8-98, Richland, WA 99352, 2Univ. of Washington
The isotope-coded affinity tag (ICAT™) strategy was used to identify the effects of irradiation on relative protein abundances in the highly radioresistant organism, Deinococcus radiodurans (D. radiodurans). The ICAT™ strategy uses two distinct isotopic versions of a cysteine-specific reagent; a "light" version and a "heavy" version in which eight hydrogen atoms are substituted for deuterium atoms. Proteome samples extracted from the organisms just prior to, as well as 30 min. and 3 hours after treatment with ionizing radiation were labeled with either the heavy or light isotopic version of the ICAT™ reagent. After mixing the samples in various combinations the cysteine containing polypeptides (Cys-polypeptides) were extracted using immobilized avidin chromatography. The Cys-polypeptides were initially identified by capillary reverse phase liquid chromatography (LC) using a conventional mass spectrometer (MS) operating in the tandem mass spectrometry (MS/MS) mode. The masses of the identified peptides were subsequently identified in a capillary LC separation coupled on-line with Fourier transform ion cyclotron resonance (FTICR) MS. Changes in relative protein expression were measured using the results obtained by LC/FTICR. The results show that most proteins undergo a decrease in expression when D. radiodurans is subjected to ionizing radiation.
P46-S
Quantification of neurosteroids using NCI GC/MS.
R.L. Fitzgerald; VA Med. Ctr., UCSD, VAMC-113, 3350 La Jolla Village Dr., San Diego, CA 92161
Electron capture negative chemical ionization (NCI) is one of the most sensitive ionization techniques available and is especially well suited for quantitative analysis of target compounds in biological extracts. For the analysis of small molecules, NCI is often several orders of magnitude more sensitive than traditional techniques such as electron ionization or positive chemical ionization. There are several important prerequisites for performing quantitative analysis using NCI, including an electronegative functional group, stable isotopic internal standards, and good method validation. We synthesized deuterium labeled analogs of neurosteroids and developed a NCI GC/MS method for quantification of neurosteroids in biological samples using isotope dilution.
Neurosteroids have distinct neurotransmitter mediated effects and consequently it is important to be able to identify and quantify individual compounds. Previously, the determination of neurosteroids in biological matrices involved complicated purification protocols or did not use appropriate internal standards. We added deuterium-labeled internal standards to brain (100 mg of cortex homogenate) or plasma (300 µL). Samples were homogenized in methanol, centrifuged and diluted to contain 5% methanol and then applied to C-18 columns. After washing the column with methanol/water (50/50), steroids were eluted with methanol. Following evaporation, steroids were converted to pentafluorobenzyl oxime/trimethylsilyl ether derivatives. The extracts were analyzed using SIM. The present method allows simultaneous quantification of pg amounts (100 pg in 300 µL of plasma and 250 pg in 100 mg of brain tissue) of neurosteroids and will be helpful in elucidating the role of neurosteroids in health and disease.
P47-M
Use of stable isotope amino acid labels to simplify MS/MS peptide analysis.
S.J. Berger, S-W. Lee, Y. Shen, G.A. Anderson, R.D. Smith; Pacific Northwest Natl. Lab., 902 Battelle Blvd. Box 999 MS: K8-98, Richland, WA 99352
Tandem mass spectrometry (MS/MS) is a primary analysis tool for the identification of peptides from proteolytic sample digests. When coupled with online separations, one or more peptides from a single 2D-gel spot digest can uniquely identify a given protein, or more complex digests can yield a global view of the proteome. The identification of individual fragment ions from a parent peptide is indirect, and the resulting peptide identification is strictly correlative. The ability to add sequence and composition constraints to assign peptide identity should significantly narrow the possible assignments of ions, and simplify interpretation of MS/MS spectra. Here we describe an application of stable isotope labeling in conjunction with MS/MS analysis of proteolytic digests that permit significant improvements assigning ions in the resultant MS/MS spectrum. Auxotrophic cells grown in parallel in the presence of a labeled or unlabeled amino acid are combined prior to cell disruption. Peptides derived from proteolytic digests generate pairs of peaks separated by a known mass difference during an initial LC/MS scan. These identified peptide pairs are isolated, and analyzed by LC/MS/MS in subsequent scans. When using a protease that cuts adjacent to the labeled amino acid, the paired/unpaired daughter ion pattern permits simplified identification of the parent peptide.
This work was supported by OBER (U.S. DOE) and PNNL Laboratory Directed R&D. Battelle Memorial Institute operates PNNL for the U.S. DOE under Contract DE-AC06-76RLO 1830.
P48-T
A comprehensive proteomic analysis of human cilia using nanoscale capillary LC/MS/MS.
M. Moyer1, K. Blackburn1, W. Burkhart1, A. Moseley1, L. Ostrowski2, R. Boucher2; 1Glaxo Wellcome, 5 Moore Drive, Research Triangle Park, NC 27709, 2Univ. of North Carolina
Ciliated cells play an integral role in the defense mechanisms of the respiratory system. By the coordinated beating of their cilia they provide the force necessary to clear potentially harmful material from the airways. In order to better understand the protein composition of cilia, human cilia were subjected to a comprehensive proteomic analysis. Cilia were isolated from cultures of airway epithelial cells and component proteins separated by 1D or 2D gel electrophoresis. Bands or spots were excised, subjected to in-gel proteolytic digestion, and component proteins identified by nanoscale capillary LC/MS/MS. Alternatively, proteolytic digests of intact cilia were analyzed directly by nanoscale capillary LC/MS/MS with or without the use of cysteine-specific affinity tags. Data will be presented on proteins identified as components of cilia as well as details of the analytical methodologies.
P49-S
4-(4-Hydroxystyryl)pyridine, a rationally designed negative ion matrix for MALDI-TOF mass spectrometry.
J.C. Lopez, P.A. Liddell, J.W. Lippert, D.C. Brune; Arizona State Univ., PO Box 871604, Tempe, AZ 85287-1604
To date, most matrices for MALDI-TOF mass spectrometry have been discovered by trial and error experiments, and through broad investigation of compounds chemically related to known matrix molecules. The most widely used matrices have been used to analyze proteins and other analytes in the positive ion mode. We chose to work with negative ion matrices because this area has received less attention, and because we occasionally analyze samples for which a good negative ion matrix is required. In this investigation, we synthesized and studied 4-(4-hydroxystyryl)pyridine. This compound resembles those derived from 4-hydroxycinnamic acid in having an electron-withdrawing moiety (in this case a pyridyl group) separated from a 4-hydroxyphenyl moiety by a vinyl group. In the electronically excited state, charge density delocalized across the vinyl group onto the pyridyl group will make the latter more strongly basic, favoring abstraction of protons from both the analyte and other matrix molecules. Preliminary experiments on Fmoc and other modified amino acids have shown that this matrix works as well as 3-aminoquinoline, one of the best negative ion matrices reported so far. Further comparisons involving structurally related matrix molecules are in progress and will be reported. A characteristic for negative ion matrices is a shift to longer wavelengths under acidic conditions.
P50-M
LC-MS of oligonucleotides using polystyrene supports and API-TOF mass spectrometry.
D.H. Hawke1, J.S. Nelson2, R. Vinayak2, W. Xiao3, P.J. Oefner3; 1Applied Biosystems, 850 Lincoln Ctr. Dr., Foster City, CA 94404, 2Applied Biosystems, 3Stanford Genome Res. Ctr.
Oligonucleotides may be analyzed by either Maldi or electrospray methods after suitable desalting of samples. Maldi methods are convenient and offer high-speed analysis, while electrospray methods can be directly coupled to HPLC separations. A classical application for these methods is oligonucleotide synthesis, in this case there is usually enough material available that sensitivity is not a major problem. LCMS (1) is advantagous if UV detection is required, and desalting is effectively performed on-line. A newer area is that of genotyping. In this application separation may be required prior to analysis and current ion-pairing methods have remarkable separating power for these molecules (2). We have coupled this separation technology to a high-resolution API-TOF MS system (Mariner) to give both high resolution separations and high mass accuracy measurements of oligonucleotides and labeled oligos.
1. Huber C.G., Krajete A., Anal. Chem. (1999), 71:3730-3739.
2. Underhill, P.A., L. Jin, A.A. Lin, S.Q. Mehdi, T. Jenkins, D. Vollrath, R.W. Davis, L.L. Cavalli-Sforza and P.J. Oefner. Genome Res. (1997), 7:996-1005.
P51-T
Advances in quantitation and identification of proteins using isotope-coded affinity tags and LC/MS/MS using information-dependent acquisition.
T.A. Settineri, C.L. Hunter, L. Nuwaysir, A. Patel, A. Tomaney, B. Halpern; Applied Biosystems, 850 Lincoln Ctre. Dr., Foster City, CA 94404
Recently, Aebersold et al. introduced a method in which proteins are labeled with an isotope-coded affinity tag (ICAT), digested, and the resulting labeled peptides separated and analyzed by mass spectrometry. The advantage to this technique is that the ICAT approach precludes the use of 2D gels while allowing complete automation of the entire process. In addition, the ICAT technique is generally applicable and can be applied to the global analysis of protein expression. In this study we investigate the ICAT approach using an API-QSTAR (hybrid quadrupole time-of-flight) mass spectrometer fitted with a nano-electrospray source, ICAT reagents synthesized in-house, and novel data reduction algorithms to both quantitate protein expression and identify proteins from MS/MS data acquired by using information dependent acquisition (IDA)
This approach will be demonstrated for the analysis of proteins arising from various cell lines including E. coli and Arabidopsis treated in different ways. After treatment with ICAT reagent and purification, samples are analyzed by LC/MS/MS using a QSTAR mass spectrometer using information dependent acquisition (IDA). Two new algorithms developed to identify and quantitate ICAT expression pairs are applied to the MS scans while a third new algorithm is applied to the MS/MS data to identify proteins. The first two algorithms cluster the data based on ICAT fragments, and optimally collapse adjacent spectra for maximum signal to noise. The third algorithm then identifies the protein using the MS/MS fragment ion data to perform a database search.
P52-S
High-throughput SNP genotyping by MALDI-TOF MS.
M. Kostrzewa1, T. Fröhlich1, T. Wenzel1, C. Franke1, W. Pusch2, K-O. Kräuter2, Y. Stalgies2; 1Bruker Saxonia Analytik GmbH, Permoserstrasse 15, Leipzig, Saxonia D-04318 Germany, 2Bruker Daltonik GmbH, Bremen
One of the great challenges of the upcoming postgenomic era is the determination of sequence variations, in particular single nucleotide polymorphisms (SNPs). These polymorphisms are believed to have an enormous impact in diagnosis of diseases, improvement of drugs, and forensic analysis in future. In contrast to the increasing demand for SNP genotyping, there is still a lack of highly reliable high-throughput SNP typing techniques.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful advanced technology capable of the accurate and cost-effective typing of many thousands of SNPs per day. The "GOOD assay", a novel sample preparation procedure for MALDI-TOF typing of SNPs was recently presented (Sauer et al., Nucleic Acids Research, methods online, 2000, 28, e13). This preparation uses sensitivity enhancing, chemical modifications of the allele specific products. Main advantage of the GOOD assay over other SNP genotyping preparation methods with MALDI detection is that this protocol does not require solid phase purification of the products prior MALDI analysis. SNP typing starts with a PCR encompassing a region containing a known SNP. Thereafter dNTPs are digested with shrimp alkaline phosphatase. In the following primer extension reaction using a charge tag containing extension primer, a conditioned set of ddNTPs and a DNA polymerase of allele specific products are generated. The 5' part of the extension primer is removed by an exonuclease. By alkylation the backbone phosphate groups are neutralised. This results in small singly charged product molecules which are transferred onto non-protonating matrix on the MALDI target.
Mass spectra of 384 samples were acquired in less than one hour. Allele calling was performed automatically by the novel genotools SNP manager software, online during spectra acquisition or offline. The resulting genotypes are stored in a table in the ASCII format together with a quality asse.
P53-M
Nano-LC ion-trap mass spectrometry for the investigation of low-fmol protein amounts.
U. Schweiger-Hufnagel1, A. Schneider1, A. Ingendoh1, S. Liedtke2, R. van Soest3; 1Bruker Daltonik GmbH, Bremen, Fahrenheitstr. 4, Bremen, Bremen 28359 Germany, 2Bai GmbH, 3LC Packings NL
Ion trap mass spectrometry is a powerful tool for the proteomic research since it is predestined for the connection to a liquid chromatography system, which allows the simple fragmentation of all separated peptides in one experiment. However, the limiting factor for the protein analysis so far was the chromatography, which, due to the high inner column diameter and high flow rates reduced the detection limit of the mass spectrometer. To improve this, we connected a nano-LC system to the ion trap mass spectrometer to investigate the applicability of this setup for the proteomic research.
The peptides were separated on a 75 µm ID RP column at a flow rate of 160 nl/min. To achieve the LC-MS coupling fused-silica spray needles in combination with interfaces that support the use of peek based HPLC fittings (New Objective, USA) were used. The ion trap mass spectrometer was equipped with an online-nanospray device.
Using this setup, a detection limit in the low fmol range is achieved (about 1/100 of the detection limit for a capillary HPLC column). Thus, this powerful technique can be applied to those protein amounts which are provided by the proteome researcher (e.g. obtained from 2-D gels). Therefore, the nano-LC ion trap mass spectrometer meets the sensitivity requirements of proteomic research, and it enables the use of the ion trap for diverse analytical questions. Those include protein identification, the analysis of protein mixtures, the analysis of post-translational modifications and the sequence analysis.
P54-T
Characterization of the proteome of Oryza sativa.
P.A. Haynes, A. Koller, N. Andon, J. Wei, J.R. Yates, III; Novartis Agr. Discovery Inst., San Diego, 3115 Merryfield Row, San Diego, CA 92121
As part of our ongoing efforts to enhance our understanding of plant biology in cereal crops, we have undertaken the characterization of the proteome of the major tissues and subcellular fractions of Oryza sativa (Rice). The tissues analyzed include leaf, root and seed. These were chosen because, despite certain similarities, each tissue performs very different functions in the plant life cycle and interacts with unique environmental factors. Further fractionation of leaf tissue has allowed the analysis of organelles including nuclei, chloroplasts and mitochondria, and subcellular fractions including cytosol and membrane preparations. Further fractionation of seed has allowed the analysis of endosperm and embryo fractions. Protein extracts were prepared from each sample, and proteins were visualized by two dimensional electrophoresis using immobilized pH gradient-SDS-PAGE. Proteins were then excised from the gel and digested in-gel with trypsin. Peptide mixtures were separated and analyzed using liquid chromatography-tandem mass spectrometry, and peptides were identified using computer database searching of uninterpreted fragment ion mass spectra.
We have identified several hundred proteins by searching our data against publicly available sequence data from various plant species. These include proteins common to several tissues as well as those which are unique to a particular tissue. This data represents the most comprehensive picture to date of the proteome of tissues and organelles from cereals, and in particular rice plants. The knowledge of the identity and distribution of expressed proteins will add value to our genomic sequencing efforts, and will be used to provide a baseline for future studies of which proteins are up- or down-regulated in response to environmental stress.
P55-S
ESI-ion trap mass spectrometry for the analysis of PCR products.
S. Hahner, A. Schneider, A. Ingendoh; Bruker Daltonik GmbH, Bremen, Fahrenheitstr. 4, Bremen, HB 28359 Germany
The polymerase chain reaction (PCR) is an important tool used in many applications in molecular biology for the generation and amplification of specific DNA sequences from very small sample material. PCR products are commonly analyzed by means of gel electrophoresis. Major disadvantage of this technique is that the size of a PCR product is determined by comparison of the relative electrophoretic mobility in the gel matrix to an internal size standard. In addition, a labelling of the analyte is required for detection.
Mass spectrometry (MS) has been proven to be a useful technique for the determination of both the length and sequence variations of PCR products based on the accurate determination of molecular weights. So far, PCR products have been analyzed using either matrix-assisted laser desorption- (MALDI) or electrospray ionization- (ESI) MS. The performance of both techniques is less labour- and time consuming compared to gel electrophoretic techniques. However, the application of MS is mainly limited due to the tendency of the DNA molecules for the formation of multiple salt adducts which complicate the data.
The development of a purification approach based on the adsorption of PCR products on magnetic particles has been found to be efficient for purification of PCR products prior to MS. In addition, this approach is amenable to full automated handling. The application of the magnetic bead purification technique in combination with ESI ion trap MS yields mass spectra with a resolution and accuracy sufficient to assign single nucleotide sequence deviation in PCR products up to a size of 90 bp.
P56-M
Hydrophilic anchors enable high throughput protein identification in the subfemtomole range.
P. Hufnagel, M. Schürenberg, C. Köster, K-O. Kräuter; Bruker Daltonik GmbH, Bremen, Fahrenheitstr. 4, Bremen, Bremen D-28359, Germany
Research strategies in the Proteomics field rely on both high sensitivity and high throughput spectra acquisition. In addition, there is a strong need for convenient analysis tools that allow high throughput processing, database searches and the assessment of a high number of search results.
Hydrophobically coated targets equipped with small hydrophilic anchors have shown 10-100-fold higher sensitivity for MALDI TOF MS than conventional steel targets. A droplet (0.5-3 µl) containing matrix and analyte is applied onto a hydrophilic anchor and shrinks down to the anchor's size, thereby restricting the formation of matrix crystals to an exactly defined area. This drying process increases the concentration of the analyte by a factor that depends on the droplet's and on the anchor's size. The fact that the matrix crystals are restricted to the area of the anchor is a big advantage of anchor targets when it comes to automation.
Using an eight-channel pipetting robot for sample preparation (approx. 2 sec/sample) arrays of hydrophilic anchors (as well as standard metal targets) were loaded with protein digests of varying sample amounts. The combination of automatic spectra acquisition with batchwise database searching led to an overview of the sensitivity increase (a factor of approx. 100) achieved by the anchor technology. The database search results have been automatically assessed by a fuzzy-control-based algorithm.
Here, high throughput capabilities of preparation, acquisition and analysis have been used to compare the sensitivity of standard sample preparation with the hydrophilic anchor technology on a statistical basis. Furthermore, this analysis serves as a model for the application of these fundamental techniques for the fast achievement and analysis of search results coming from proteomic research tasks.
P57-T
Optimization of a procedure for labeling proteins with isotope-coded affinity tags.
K. Parker, S. Pillai, S. Daniels, W. Stanick, R. Lotti, B. Purkayastha, T. Nadler; Applied Biosystems, 500 Old Connecticut Path, Framingham, MA 01701
The use of the isotope coded affinity tag (ICAT™), developed by the laboratory of R. Aebersold (Nat. Biotechnol. 1999 Oct 17(10):994-9), allows one to compare the relative protein abundance between two samples by mass spectrometry. The reagent used is essentially cysteine reactive biotinylation reagent into which stable isotopes have been incorporated. One of the two samples is treated with the light reagent while the other sample is treated with the same reagent containing 8 deuterium atoms such that the samples are chemically equivalent but differ only in mass. In this way two peptides may be monitored in the mass spectrometer and quantified relative to one another by peak intensity.
A number of imporant questions concerning the use of this technique are addressed in this presentation. The reagent has been extensively characterized and the recommended protocol for its use has been optimized for ease of use and robustness. Experiments were carried out to determine the purity, stability, and reaction kinetics of the reagent. Additional experiments were performed to optimize the derivatization protocol, including measurements of the degree of alkylation obtained, determining the optimal reduction reagent, how to remove excess reagent, and measurements of stability, capacity, carryover, and recovery of biotinylated peptides from monomeric avidin columns, Finally, the reproducibility of quantitative measurements in the mass spectrometer was addressed and will be presented.
P58-S
Rapid genotyping using spectrometrically monitored selections.
J. Stoerker1, J.D. Mayo2, C.N. Tetzlaff3, D.A. Sarracino4, C. Richert3; 1Bruker Daltonics, 15 Fortune Drive, Billerica, MA 01821, 2Tufts Univ., 3Univ. of Konstanz, 4Variagenics, Inc.
Presented here is an inexpensive and rapid method for genotyping single nucleotide polymorphisms (SNPs) on PCR products using spectrometrically monitored selection from probe libraries. We have previously reported nuclease selections of modified oligonucleotides with increased RNA or DNA targets. This methodology relies on the selection of short hybridization probes complementary to the target sequence with a single-strand specific nuclease that does not attack duplexes. Conditions are described where the matched duplex is profoundly better protected from enzymatic digestion than single base mismatches. Semi-quantitative MALDI-TOF MS is used to identify the selected probes after a 10-minute digestion reaction with snake venom phosphodiesterase. We have examined sequences from exon 10 of the cystic fibrosis transmembrane regulatory (CFTR) gene, using a probe library representing a number of alleles, including: native, I506S, 1651 benign polymorphism, I506S/1651 dual polymorphism, and dF508, and dI507. Probe libraries composed of 8-mers and 9-mers annealing to PCR products have been used in our experiments. DNA from blood or cheekbrush samples was used from 10 genomic sources. The method discriminates both alleles of a heterozygote pair in a single experiment, and can discriminate alleles from multiple loci in the same sample.
P59-M
Quantitative proteomic and mRNA expression analysis of S. cerevisiae via metabolic labeling and multidimensional protein identification technology.
M.P. Washburn1, G. Oshiro2, D. Wolters1, D. Schieltz1, C. Deciu1, E. Winzeler2, J.R. Yates, III1; 1Novartis Agr. Discovery Inst., 3115 Merryfield Row Suite 100, San Diego, CA 92121, 2Genomics Inst. of Novartis Fndn., San Diego
Proteomic technologies are being developed to determine large-scale changes in protein expression levels. Several quantitative proteomic methods have recently been published where the protein expression levels are compared between two different growth conditions by metabolic isotopic labeling of proteins or post-growth isotopic labeling of proteins in a sample. To fully understand how growth conditions effect biological systems, both protein and mRNA expression levels must be analyzed. We have applied metabolic labeling strategies to S. cerevisiae in order to determine the changes in protein and mRNA expression levels. S. cerevisiae was grown in 15N enriched minimal media and compared to S. cerevisiae grown in rich media. Each sample was analyzed via mRNA expression array and multidimensional protein identification technology. By combining these methods we were able to correlate the changes in protein and mRNA expression levels of several hundred gene products.
P60-T
A software algorithm for automated quantitation of ICAT labeled peptides analyzed by mass spectrometry is described.
J. Eng, J. Ranish, R. Aebersold; Inst. for Systems Biol., 4225 Roosevelt Way NE, #200, Seattle, WA 98105
We describe a software program which automatically generates quantitation ratios of Isotope Coded Affinity Tag (ICAT) labeled peptides analyzed in an LC-MS/MS experiment performed on an LCQ ion trap mass spectrometer. The peptides are initially identified via a sequence database search. Quantitation of each ICAT-labeled peptide pair is then performed by integrating over the peptide's light and heavy elution profiles. An interactive, graphical program displays the light and heavy elution traces and allows the researcher to adjust the area of integration as needed. This program should enable and facilitate large scale, quantitative proteomics studies.
P61-S
Genopure: a novel magnetic bead DNA purification system for MALDI-TOF MS analysis.
M. Kostrzewa1, J. Bimmler2, I. Thomas2, T. Wenzel1, E. Nordhoff3, H. Rauth3, T. Fröhlich1; 1Bruker Saxonia Analytik GmbH, Permoser-strasse 15, Leipzig, Saxonia D-04318 Germany, 2Bruker Saxonia Analytik GmbH, Leipzig, 3Max Planck Inst. of Molec. Genet., Berlin
MALDI-TOF mass spectrometry has a high potential for high throughput DNA analyses because of its accuracy, speed, automation capabilities, and cost-effectiveness. Unfortunately, molecular biological reactions are performed in the presence of high amounts of salts and detergents which form adducts with DNA or interfere with matrix crystallization, respectively. Therefore, effective DNA purification is indispensable for successful and valid MALDI-TOF MS analysis. We present a novel magnetic DNA purification system which gives excellent results in subsequent mass spectrometric measurement.
Small double stranded PCR products were precipitated on paramagnetic beads using an appropriate binding solution. The binding of DNA to the particles did not require any labeling of primers or dNTPs. After several washing steps with buffers containing either ethanol or isopropanol, DNA was eluted and measured with 3-HPA matrix in a MALDI-TOF mass spectrometer. Residual PCR primers were removed while PCR products down to about 50 bp in size were recovered with high yield. Quality of the acquired spectra was superior to that of alternative purification protocols.
Alternatively, eluted PCR-Products were used for subsequent reactions like primer extension or restriction enzyme digest. Primer extension products or very small restriction enzyme digest products (about 20 bp) were again purified by the magnetic bead system to MALDI quality with high recovery using a dedicated binding buffer. The combination of both purification procedures enabled the genotyping of SNPs by performing PCR, purification of the PCR product, primer extension or digest, and clean-up of the allele specific products in one reaction tube. High quality spectra could be acquired for several model systems allowing the unambiguous determination of genotypes. The bead handling can easily be automated on standard pipetting robots thereby enabling the processing of thousands of samples per day.
P62-M
Industrial scale high throughput proteomics.
J. Brown, D. Gostick, P. Young, J. Langridge; Micromass UK Ltd, Floats Road, Wythenshawe, Manchester M23 9LZ, United Kingdom
With the completion of the Human Genome sequence MALDI-TOF-MS is increasingly becoming an established method for identification of proteins separated by 2D gel electrophoresis. Mono-isotopic peptide mass fingerprinting (PMF) has been previously shown to be amenable to full automation encompassing the process of acquisition, data processing and databank searching under full software control.
Until now the throughput of MALDI-TOF-MS for proteomics has been limited to several hundred samples in a working day and this represents approximately 5-10% of the total proteins resolved by a large format 2D gel. To reduce the number of proteins to be identified the 2D gels are imaged and analysed to determine differences in expression levels within a set of gels. Although much of the image processing is semi-automated the comparison is labour intensive as manual pattern matching has a role in the gel alignments (land marking). Increased MS sample throughput allows the possibility of identifying every protein spot in a 2D gel within a day. This could eliminate the potentially erroneous step of human gel image alignment, whereby land marking could be achieved using the MS data.
Increased sample throughput requires greater capacity and robust unattended instrument operation. In this poster we describe an integrated robotic multiple plate loader that allows overnight unattended MS operation. Other improvements include an increased laser repetition rate that allows the data capture rate to increase four fold. Sample tracking, data archiving and data reporting are essential attributes of this new technology and these aspects are outlined in the present
P63-T
Bioinformatics assessment of mass spectrometric chemical derivatisation techniques for proteome database searching.
K.S. Sidhu1, P.J. Lester1, S.J. Gaskell1, O.V. Wolkenhauer1, S.G. Oliver2, F. Brancia1, A.G. Sullivan1, P. Sangvanich1, S.J. Hubbard1; 1UMIST, UK, PO Box 88, Manchester, Lancashire M60 1QD, United Kingdom, 2Manchester Univ.
Identification of proteins from the mass spectra of peptide fragments generated by proteolytic fission and subsequent database searching is one of the most powerful techniques in proteome science. Using computer simulation, we have studied how various chemical derivatisation techniques developed in the Michael Barber Centre for Mass Spectrometry at UMIST can improve the efficiency of protein identification from mass spectrometric data. The impact of these different derivatization strategies, which promote and stabilise certain fragmentation pathways yielding additional database search information has been assessed. For example, by reliably promoting fragmentation at aspartic acid residues after homoarginine derivatisation, 85% of yeast proteins can be unambiguously identified from a single peptide with a measured mass accuracy of 500 ppm by using the associated fragment ion data. We present results here for 3 alternative techniques, both for single proteins and for simple mixtures. Additional data have also been generated to compare these "partial sequencing" methods with high accuracy mass spectrometry where peptide mass accuracy can be achieved at 50 ppm and beyond. Interestingly, for protein mixture analysis, the inclusion of limited sequence information for the peptides can compensate and exceed the search efficiency available via high accuracy searches of around 20 ppm, suggesting the particular use of this experimental approach for the simple protein mixtures that are routinely obtained from 2D-gels.
P64-S
Automated high throughput protein identification on a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer coupled with a MALDI ion source.
A. Millar, R. Tyldesley, J.I. Langridge, J.B. Hoyes, Y. Philip, R. O'Malley, R. Bateman; Micromass UK Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ, United Kingdom
The mass spectrometry technique providing the highest throughput in terms of samples per hour, is currently MALDI-TOF-MS. This technique provides a peptide mass fingerprint of the protein digests and allows rapid and accurate identification of the parent protein by comparison to a databank. However, under some circumstances, for example if the number of peptides detected is small or if the sequence coverage is poor, it is advantageous to be able to include even a short piece of sequence information to provide added specificity. In a conventional MALDI-TOF-MS instrument post source decay (PSD) can be used to try and generate sequence information, however this approach is notoriously unreliable in producing good quality MS/MS data. One reason for this is that the peptide does not undergo fragmentation in a controlled environment such as a gas cell.
An alternative approach is to couple a MALDI ion source to a hybrid quadrupole orthogonal acceleration time-of-flight (Q-Tof) mass spectrometer, which results in predictable fragmentation. In contrast to a conventional MALDI-TOF-MS instrument the resolution and mass measurement accuracy of the data is comparable between the MS and MS/MS modes. This allows superior data acquisition in the MS-MS mode compared to other MALDI-TOF systems.
In this paper we demonstrate the application of the MALDI Q-TOF instrument for high throughput proteomics. A number of modifications have been made to optimise the system for high throughput proteomics, these include a MALDI source with a high-density target plate and software that has been developed for automated data acquisition in both the MS mode and the MS to MS/MS switching mode. Dedicated processing software has been developed to fully automate the post acquisition and databank searching. This software has been optimised to consider the unique nature of the data acquired from this configuration of instrument.
P65-M
Single scan PSD-MALDI-TOF analysis at high speed and sensitivity.
D. Suckau, A. La Rotta, A. Holle, P. Hufnagel, D. Suckau; Bruker Daltonik GmbH, Bremen, Fahrenheitstr. 4, Bremen 28359, Germany
The typical approach to Proteomics measurements involves the MALDI-TOF fingerprint analysis of protein digests, which allows the identification of a significant fraction of samples. PSD as it is available on a reflector TOF instrument is in our hands a very sensitive technique (1-10 fmol level) and is automated; therefore it principally is the ideal technique to continue MS/MS analysis from the same prepared sample. However, it suffers from the fact that each PSD spectrum is pasted together from a set of ca. 10 voltage segments, each consisting of 100-200 laser shots. Sample consumption is such that a maximum of 2-3 peptides can be submitted to MS/MS analysis and the process is time consuming. Here we report on the usability of single scan PSD spectra from digests for protein identification.
The acquisition of the entire fragment spectrum has been achieved for a MALDI reflector TOF mass spectrometer by introducing a "potential lift", a precursor selection device consisting of a pulsed HV cell with acceleration grid. An HV poten-tial is applied to the lift cell at the moment the precursor ion and its PSD fragment ions have entered it. By the subsequent acceleration, the energies of the fragment ions are compressed into the operating window of the reflector, which allows analysing them in a single spectrum.
With this device LIFT-PSD spectra from proteolytic digest mixture were obtained and evaluated with respect to sensitivity, mass accuracy, speed of analysis and number of PSD spectra per sample. It potential use in Proteomics is discussed.
P66-T
Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer.
T.J. Griffin1, S.P. Gygi2, B. Rist3, H. Zhou1, H. Lee1, A. Loboda4, A. Jilkine5, W. Ens6, K.G. Standing6, R. Aebersold1; 1Inst. for Systems Biol., Seattle, 4225 Roosevelt Way N., Seattle, WA 98105, 2Harvard Med. Sch., 3BioVisioN GmbH and Co. KG, Hannover, 4MDS Sciex, Concord, Canada, 5Univ. of Manitoba, 6Univ. of Manitoba
We describe an approach to the quantitative analysis of complex protein mixtures using a MALDI quadrupole time-of-flight (MALDI QqTOF) mass spectrometer and isotope coded affinity tag (ICAT) reagents. Proteins in mixtures are first labeled on cysteinyl residues using an ICAT reagent, the proteins are enzymatically digested, and the labeled peptides are purified using a multi-dimensional separation procedure, with the last step being the elution of the labeled peptides from a microcapillary reverse-phase liquid chromatography column directly onto a MALDI sample target. After addition of matrix, the sample spots are analyzed using a MALDI QqTOF mass spectrometer, by first obtaining a mass spectrum of the peptides in each sample spot in order to quantify the ratio of abundance of pairs of isotopically tagged peptides, followed by tandem mass spectrometric analysis to ascertain the sequence of selected peptides for protein identification. The effectiveness of this approach is demonstrated in the quantification and identification of peptides from a control mixture of proteins of known relative concentrations, and also in the comparative analysis of protein expression in Saccharomyces cerevisiae grown on two different carbon sources. Additionally, we have demonstrated the utility of this approach for the quantitative analysis of isotopically labeled phosphorylated proteins, and we are currently working on automating the sample preparation procedures for high-throughput applications.
P67-S
Proteomics approach to investigate the toxicity of diclofenac.
L.J. Alward, O.V. Nemirovskiy, G.S. Cavey, J.E. Carlson, W.R. Mathews, J.A. Ware; Pharmacia, 301 Henrietta Street, Kalamazoo, MI 49007
Diclofenac, a nonselective inhibitor of cyclooxygenase, is a widely used non-steroidal anti-inflammatory drug. It has been shown to produce hemolytic anemia in sensitive individuals. A proteomics approach was used to determine the mechanism of diclofenac-induced hemolytic anemia in the mouse. A flexible, high performance proteomics laboratory was assembled using commercially available instrumentation. The system used was validated with protein standards and included robotic spot excision from 2D gels, automated in-gel digestion, robotic sample preparation, automated MALDI-Tof analysis, automated nanoLC-MS/MS analysis, and automated database searching. Diclofenac was administered to mice by gavage. Liver, plasma, and RBC proteins were separated using 1D and 2D gels, and diclofenac-protein adducts were visualized by either radiolabel or immunoblot. Proteins of interest were excised, reduced, alkylated, and trypsin digested. Successful protein identification was accomplished via MALDI-Tof MS peptide mass fingerprint analysis using 20% of digest sample. Nanoscale LC-MS/MS provided comprehensive identification using 50% of digest sample. The results obtained by nano-LC-MS/MS analyses were in a good agreement with those obtained by MALDI-Tof. Many proteins were identified including fibrinogen alpha and beta, hemoglobin beta and delta chains, and RBC anion exchanger (AE1). Abnormalities in AE1 have been shown to accelerate membrane destruction. Preliminary data from these experiments is promising and may help explain diclofenac toxicity.
P68-M
Selective separation of methionine-containing peptides combined with mass spectrometry: a high accuracy technique for identification of proteins.
W.L. McEldoon, M.J. Horn; BioMolecular Technol., Inc., 525F Del Rey Ave., Sunnyvale, CA 94085
Although MALDI-TOF MS is a widely accepted technique for identification of proteins and peptides in proteomic and genomic analyses, rapid and accurate identification of a large number of proteins is still a great challenge. Previously, we reported an easy method, which uses a novel water-insoluble reagent that selectively separates methionine-containing peptides from peptide mixtures. This method, combined with MALDI-TOF analysis, demonstrates a significantly improved accuracy in the identification of proteins. In the present study, we present a total methodology for the separation and identification of methionine-containing peptides generated from 2D gel digestion mixtures. The methodology provides a "one-pot" method for sample desalting and concentration after the isolation process, giving cleaned-up samples ready for multiple MALDI or other analytical analyses.
P69-T
Efficient detergent and Coomassie blue dye removal from peptide samples prior to MALDI-TOF MS.
W. Kopaciewicz, E. Kellard; Millipore, 17 Cherry Hill Drive, Danvers, MA 01923
Advancements in Mass Spectrometry (MS) instruments over the last 5 years have reduced the amount of sample required and expanded the molecular weight analysis range. As such, MS is an increasingly valuable tool for the analysis of biomolecules in areas of proteomics and functional genomics. However, the presence of salts, detergents and dyes can decrease macromolecular ionization and/or obscure mass peaks, thus decreasing instrument performance. As such, numerous methods and devices have been developed for sample preparation prior to MS analysis.
Millipore ZipTip sample preparation devices containing C18 and C4 media reversed phase media have been demonstrated to be effective for the concentration and desalting of microliter volumes of peptide, protein and oligonucleotide samples prior to MALDI-TOF MS. Although reversed phase chromatography meets the vast majority of the sample preparation needs, there are cases where it isn't totally effective. This is especially evident when the contaminants have similar hydrophobic character as the solutes and thus co-purify.
Alternatively, a subset of normal phase chromatography termed hydrophilic interaction chromatography has been shown to be an efficient sample preparation technique for certain biomolecules in those cases where reversed phase struggles (1,2,3). The method utilizes a highly hydrophilic adsorbent that binds sample out of a high organic mobile phase (e.g. 90% acetonitrile). After washing with this solvent, bound molecules are then eluted by decreasing the organic content (e.g. 50% acetonitrile). In this poster, we demonstrate the use of a ZipTip with hydrophilic interaction media (ZipTipHPL). The device was very effective for the removal of detergents and coomassie blue dye from sub-picomole quantities of peptides. The resulting mass spectra demonstrated good signal strength and fidelity of peptide capture.
1. Alpert, A.J., J. Chromatogr. 499 (1990) p.177
2. Zhu, B.Y., et al., J. Chromatogr. 548 (1991) p.13
3. Scherer, J.P., et al., Anal. Biochem. 215 (1993) p.292
P70-S
Improved proteome coverage through the use of affinity tags with nanoscale capillary LC/MS/MS.
K. Blackburn1, W. Burkhart2, R. Davis2, M. Moyer2, A. Moseley2; 1Glaxo Wellcome, 5 Moore Drive, PO Box 13398, Research Triangle Park, NC 27709, 2Glaxo Wellcome
Because of the inherent difficulties associated with 2D gel electrophoresis (low throughput, bias against certain classes of proteins, poor peptide recovery from in-gel digests, etc.), numerous groups have explored ways to analyze complex protein mixtures directly by mass spectrometry, avoiding the gel separation altogether. In proteomic experiments where qualitative and/or quantitative data is required for complex biological samples such as protein complexes, organelles, or tissue samples, the number of proteins potentially encountered may range from tens to thousands. The number of proteolytic peptides generated from digests of such complex protein mixtures precludes "complete" proteome coverage by any 1D-LC/MS/MS analysis. Following on the work of Aebersold and Patterson, we have investigated the use of cysteine-modifying "affinity" tags with avidin affinity chromatography for the simplification of complex peptide mixtures prior to nanoscale capillary LC/MS/MS analysis. This approach reduces the number of peptides presented to the mass spectrometer to a more reasonable number, thus allowing improved proteome coverage. Data will be presented on improved sample handling procedures for labeling and digestion steps as well as qualitative and quantitative applications to complex biological samples.
P71-M
Factors affecting rapid sample processing for protein capture chips analyzed by MS.
J. Chan1, P.T. Jedrzejewski1, P. Wagner1, S. Nock2; 1Zyomyx, Inc, 3911 Trust Way, Hayward, CA 94545, 2Zyomyx, Inc.
Robust, sensitive, and comprehensive methodologies for systemic approaches to proteome analysis analogous to genomics methods (e.g., gene chips) are not available. Current methodologies (e.g., 2D-PAGE and multi-dimensional chromatographic methods) suffer from fundamental limitations. In order to overcome these limitations, we as well as others have focused on the development of alternative methods (e.g., protein capture chips) for proteomics.
Protein capture chips, consisting of arrayed capture molecules (e.g. protein, DNA, or small molecule), allow for rapid and comprehensive micropurification and analysis of proteins. Protein chips may be readily interrogated by mass spectrometric analysis. Data on the post-translational modifications (PTM), protein identification, and quantitation may be obtained with a single detection scheme without labeling of analytes.
We have been successful in microfabricating protein capture arrays, the accompanying microfluidics devices, and implementing MS analysis. We have investigated various parameters which have an effect on the utility and efficacy of the overall system for proteomics. In this presentation, we will demonstrate data on parameters affected by the type of capture molecules, digest efficiency (e.g., time), and sensitivity of analysis (e.g., LC-MS conditions). <