M. Reagin, A. Merla, D. Meyers; Amersham Biosciences, 800 Centennial Ave, Piscataway, NJ 08855

Preparing plasmid templates for DNA sequencing is the most time consuming step in the sequencing process. Current template preparation methods rely on a labor intensive, multi-step procedure that takes up to 24 hours to produce DNA of varying quality and quantity. The TempliPhi DNA Sequencing Template Amplification Kit has been developed that eliminates the requirement for extended bacterial growth prior to sequencing and saves you hands-on time by eliminating the centrifugation and transfer steps currently required by older preparatory methods. In addition, costly purification filters or columns are not necessary as amplified product can be added directly to a sequencing reaction. Starting material can be any circular template from a colony, culture, glycerol stock, or plaque. Based on rolling circle amplification (RCA) and employing bacteriophage Phi29 DNA polymerase, the process can produce 2-3 ug of template directly from a single bacterial colony in 4 hours. We will demonstrate how the implementation of these procedures in a laboratory or core sequencing facility can decrease costs, save on tips, plates, and other plasticware, while at the same time increasing throughput.

C. Jecmen, J. T. Medalle, M. Randesi, B. S. Imai; The Rockefeller University, 1230 York Ave., New York, NY 10021

Core facilities constantly search for products that enhance and improve their services to the research community. The bottom-line for acquiring these products relies on the ability of the facility to work within their budget. Some facilities are fortunate to receive funding from a university; but those without such funding or budget conscious facilities must find ways to stretch their dollar. As a cost cutting measure many labs choose between store-bought and homemade reagents. Sequencing facilities, from low to high throughput, prepare reagents such as 10X TBE or 5X BigDye buffer for diluting the enzyme. Fluorescent dye terminator kits for automated DNA sequencing, such as BigDye Version 3.0 offered by Applied Biosystems, are vital reagents used by sequencing facilities. Diluting BigDye with 5X BigDye buffer one-to-one can defray the costs that arise from the use of BigDye chemistries; a 5000 reaction kit can be optimized to a 10,000 reaction kit. This cost-effective strategy will be discussed through various dilution examples of ABI Big Dye V3.0 on different types of samples handled by core facilities.

H. G. Hills, M. Blomberg-Wirschell, G. B. Witman, B. Rajeshkumar; University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655

Samples containing high "G-C content" often present problems when using ABI BigDye chemistry. The ABI dGTP generally sequences these areas with little or no difficulty but often results in less than optimum data due to the number of compressions. Using the BigDye and dGTP chemistries together often produce better results even though ABI does not support mixing the two chemistries. Using 3 or 4 parts of BigDye with one part of dGTP mix will often produce very good results. The combination chemistry was used to sequence a 240 base micro satellite repeat region from Chlamydomonas reinhardtii. The Amersham Dynamic ET Terminator Kit sometimes sequences high "G-C" templates better than the BigDyes but not better than the BigDye and dGTP combination. Areas of high "AT" content are often sequenced better with the Amersham chemistry. The Amersham chemistry also generally reads through a polyT area with fewer errors after the polyT. The Amersham chemistry can be used with the "E" filter set, although some loss of resolution may occur. This has the advantage that both chemistries can be run on the same gel.

R. Pershad, A. Scarpa; University of Texas. M.D.Anderson Cancer Center, 1515 Holcombe Blvd, Houston, Texas 77030

One of the goals of the investigators at the Cancer Center is to be able to rapidly screen patient tumor samples and families with a history of cancer for mutations.

In an effort to eliminate the need for individual laboratories to perform labor intensive SSCP (single stranded conformation polymorphism) screening and MDE (Mutation Detection Enhancement) gel analysis, the use of the WAVE instrument has been implemented in the DNA Core facility to provide a centralized service for mutation screening.

In this Core the WAVE has been used successfully to identify deletions, insertions and heterozygote mutations associated with familial cancers. The facility has screened over 22 different exons for 12 principal investigators. All the variants identified by WAVE analysis have been confirmed by sequencing. Here we will present one scenario of how to operate the WAVE as a core instrument.

L. Wen; San Diego State University, 5500 Campanile Drive, San Diego, CA 92182

I recently demonstrated that combining the annealing step with the extension step at one temperature (60^oC) reduces premature terminations in DNA sequences that regularly contain premature terminations when the three temperature steps are used (Molecular Biotechnology 17, pp. 135-142 (2001)). I have studied a novel class of 100 base pairs in length DNA sequences upstream from the point that cause premature terminations. Thermodynamics of 49 DNA sequences with premature terminations at three temperature steps were determined from DNA mfold profiles. Sequencing results of the 28 samples were improved by the two step cycle sequencing and the 21 sequences were not improved or severed by the two step cycle sequencing reactions. Nearest neighbor thermodynamic parameters for all 49 sequences were compared at temperature 50^oC and 60^oC. The parameters predict thermodynamic free base (external loop) energies DD G^o) are significantly different between these two groups. DNA templates that have improved the premature terminations by the two-step cycle sequencing method, the values for DDG^o60 for the external loop are higher than DDG^o50. In contrast, DNA sequences that have not improved the premature terminations by the two-step cycle protocol, the values for DDG^o60 for the external loop are no longer higher than DDG^o50. The results of the investigation are reported here.

G. J. Wiebe, E. Tanaka, D. N. Drechsel; Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany

We have been evaluating the production of high-quality sequencing template using the TempliPhi^TM kit from Amersham Pharmacia Biotech. This kit utilizes rolling circle amplification to replicate plasmid DNA directly from bacterial colonies or cultures with minimal handling. The protocol consists of a few simple steps performed within a single tube: a small amount of bacterial culture is denatured, incubated with enzyme overnight, and finally heat-inactivated. A small volume may then be added directly to a sequencing reaction without further purification.

Successful results have been obtained from high and low copy number Amp^R plasmids cultured in different types of growth media; Kan^R plasmids still require further investigation. A 96-well format has been maintained throughout, in anticipation of future high-throughput applications. This method may offer an alternative to costly robotics based template preparation.

S. P. Yadav¹, T. Haqqi², X. Zhao², A. Panciu²; ¹The Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195, ²The Lerner Res. Institute, Cleveland, OH

The presence of several copies of the same class of repetitive element in DNA templates increases the probability of ambiguous base calling caused by band compression artifacts in dye terminators sequencing method. The presence of di-, tri- and tetranucleotide repeats and short tandem repeat sequences all over in the genome pose a daunting routine sequencing roadblocks in the Core laboratories where samples originate from a variety of DNA templates. These compressions are due to the formation of secondary structures, such as hairpins and intramolecular base pairing between guanine and cytosine bases, on the nascent strand resulting in to anomalies in the migration of certain DNA fragments.

To overcome these undesired sequencing artifacts and formation of secondary structure several modifications of the sequencing reactions parameters have been applied in the past, including replacement of dGTP nucleotide by base analogs dITP, or 7-deaza-DGTP in the reaction mix, and adding denaturants in the reaction mix. We have earlier reported that the use of 1M betaine in the reaction mix is advantageous to sequence through AACCCC-type repeats found in telomeres. In this study we report that adding 1M betaine in the reaction mix also results in reduction of band compressions in other repeat elements and thus provides a higher accuracy of sequencing.

D. Adam, F. Dobie, H. McDonald; University of British Columbia, Rm.237 - 6174 University Blvd., Vancouver, B.C. V6T 1Z3 Canada

Commercial suppliers frequently develop new and improved DNA Sequencing premix, attempting to provide a more robust chemistry suitable for core facilities which encounter a wide range of samples. Templates which display characteristics such as high GC or AT content, homopolymer regions, G-stops and profound secondary structure have proven very difficult to sequence with manufacturers' recommended protocols. We present the data obtained when evaluating the performance of Applied Biosystems' Big Dye Chemistries (BDv1.0, BDv2.0, BDv2.0 dGTP and BDv3.0) and Amersham's DYEnamic ET Terminators, with and without facilitators such as DMSO and Betaine, on a wide variety of templates. The findings of our study are presented here.

F. H. Wolven, B. Etzel, A. M. Segall, L. Wen; San Diego State University, 5500 Campanile Drive CSL326, San Diego, CA 92182-1030

A large increase of samples from plasmid isolation and sequencing projects, cultured in 96 well plates, has created significant challenges in achieving high throughput DNA sequencing for a small core facility with staff of two. The experiences we learned were: (1) replacing the ABI377 with an ABI 3100 CE sequencer increased throughput while eliminating staggered workshifts, (2) it is difficult to isolate enough plasmid template grown in 2 mL deep 96 well blocks using standard protocols; to obtain sufficient yield requires chloramphenicol amplification and 2X LB, (3) when transitioned from 377 to the 3100, it was discovered that a second wash of 70% ethanol, with subsequent removal of residual ethanol, was needed to produce good data on the 3100, and (4) clones containing bacteriophage inserts were difficult to grow and would not sequence with our standard protocols due to low copy number. The thermocycle program was modified, and data collection injection time increased from 30 sec to 90 sec. to achieve optimal signal strength on the 3100. Details of our protocols, descriptions of the problems we encountered along the way, and their solutions will be presented.

S. Chen, G. Amparo, E. Kuo, K. Hacker, B. Johnson, S. Spurgeon; Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404

The new ABI PRISM® dGTP BigDye™ Terminator v3.0 Ready Reaction Cycle Sequencing kit with AmpliTaq® DNA Polymerase, FS was introduced earlier this year with the BigDye Terminator v3.0 Cycle Sequencing kits. The dGTP BigDye Terminator v3.0 sequencing kit, like the dGTP BigDye Terminator Cycle Sequencing kit, was introduced as a special chemistry solution to overcome certain problems with difficult templates. These are templates which when sequenced with the BigDye Terminator v3.0 sequencing kit or BigDye Terminator sequencing kit fail to give good read length due to certain sequence motifs that result in stops in data.. Since the introduction of the dGTP BigDye Terminator v3.0 Cycle Sequencing kit we have further studied this chemistry and made improvements to protocols for use with this chemistry. In this presentation we will discuss our study and findings.

I. Y. Hlede, S. Wegener, R. T. Pon; University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1 Canada

Our primary goals as a university core service laboratory are to provide accurate (99.9%) results, instruction in DNA sequencing analysis and sample preparation, and troubleshooting of results. We have optimized throughput, data quality, and communications through a number of changes. These affect the way samples are processed and how results are communicated to clients. These changes include: software and sequencer upgrades, use of Sephadex spin columns, switching from TBE to TTE (TRIS, TAPS, EDTA) buffer, sample (sequencing and genescan) loading on porous combs, template quantitation on a UV plate reader, synthesis of home-made lane guide, offering results in 3 data formats on a FTP/Fetch server, Apple Script short cuts, and email responses with web-references to sequencing customers.

M. Vazquez; City College of New York, 140th and Convent. Ave.; T-250, New York, NY 10032

The flexibility of the microfabricated format creates unique opportunities for study of the electrophoretic process. This understanding is essential to the design of all electrophoresis apparati including electrophoretic microdevices. The present work utilizes digital images to capture the motion of DNA samples during loading and injection. A systematic study of strong sample stacking (sample concentration effects) was performed in order to analyze realistic DNA analysis conditions within microdevices. These effects are shown to be highly useful and can be separately enhanced using high-voltage injection. The sample profiles within the injector have been analyzed by deconvolving the geometrical sample profile into different velocity groups. This analysis illustrates the evolution of molecular separation into distinct migrating populations within the injector itself. The present study performed DNA injections within microfabricated devices imposing run voltages between 85 V/cm - 850 V/cm. Data from 3 different offset lengths of a double-T cross-injector, 10 different applied voltages, and 2 different sample preparation protocols are presented.

C. Potter¹, M. K. Johansson², D. Dick², R. Cook²; ¹Biosearch Technologies, 81 Digital Dr., Novato, CA 94949-5750, ²Biosearch Technologies, Inc., Novato, CA

Passive reference (PR) dyes are frequently used in real-time PCR to normalize fluorescence of reporter dyes & correct for fluctuations in fluorescence that are non-PCR based. An ideal PR dye maintains steady & constant fluorescence, does not affect the PCR, & has a fluorescent signal that can be distinguished from that of any reporter & quencher dyes used in the reaction. PR dyes that emit around 600nm, such as the commonly used ROX, can be distinguished from reporter dyes in real-time PCR, because they are further red than the reporters most often used. Because these dyes are not efficiently excited by a 488nm excitation source, this can be problematic due to the fact that several of the most popular real-time PCR instruments are limited to this single excitation source. To generate sufficient signal, high concentrations of PR dye must be added, which may lead to signal instability, such as the 'ROX drop.' This study evaluates a variety of PR candidates, including a novel dye with a tremendous Stokes shift, which can be efficiently excited at 488nm & has an emission maximum of 620nm. All data is gathered from the 7700 to assess all the PR candidates, based on stability of signal & reproducibility.

L. M. Overbergh, A. Giulietti, D. Valckx, R. Bouillon, C. Mathieu; KULeuven/UZ-Gasthuisberg, Herestraat 49, O&N8, Leuven, 3000 Belgium

The analysis of cytokine profiles helps to clarify functional properties of immune cells. Real-time RT-PCR is becoming a widely used method to quantify cytokines from cells, body fluids or tissues. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step.

Using the TaqMan chemistry and the 7700 SDS, we validated a very large panel of murine and human cytokines. Although the method allows a fast, sensitive and accurate quantification, different control assays are necessary for the method to be reliable. By construction of cDNA plasmid clones, standard curves are generated which allow direct quantification of every unknown sample. Furthermore, the choice of a reliable houskeeping gene is very important. Finally, co-amplification of contaminating genomic DNA is avoided by designing primers located in different exons, or on intron/exon junctions.

In summary, the real-time RT-PCR technique is very accurate, sensitive, allows a high throughput and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.

I. V. Foulds¹, A. Kapoor¹, A. Castle², C. Gubala¹, U. J. Krull¹, P. A. Horgen¹; ¹University of Toronto, 3359 Mississauga Rd. N., Mississauga, Ontario L5L 1C6 Canada, ²Brock University, St. Catherines, Ontario Canada

The presence of waterborne pathogens in drinking (lakes/reservoirs as well as bottled water) and recreational water is traditionally quantified by time consuming culturing techniques or by chemical characterization. We have developed a more rapid, real-time PCR-based detection method for Escherichia coli, and toxic microcystin-producing Microcystis aeruginosa. Dual-labeled TaqMan probes have been designed for regions of the lacZ and microcystin synthetase genes, respectively. We have transferred our technology to the analysis of field collected water samples and commercially sold bottled water, and we are able to detect down to 3 gene copies per sample. Detection of pathogens can be done within about 2 hr after DNA extraction and sample setup, rather than up to several days needed for traditional detection methods.

W. C. Kusser, B. Lowe, M. Gleeson; Invitrogen, 1600 Faraday Ave., Carlsbad, CA 92008

A real time PCR method based on a novel design of fluorescent primers is presented. The reporting system is based on a single fluorescent dye molecule attached close to the 3’end. A primer design software was developed that identifies a primer with a suitable labeling site in a target of interest, based on a set of rules for optimum signal development. Subsequently it attaches a short tail (3-5 nucleotides) to the 5’end of the primer that is complementary to the 3’end. The resulting hairpin oligonucleotide and the sequence context around the fluorophore result in an initially low background fluorescence. Typically, JOE and FAM have been used as fluorescent dyes coupled to the primer. Upon incorporation of the primer into an amplicon during real time PCR, the fluorescence of the dye molecule increases several fold and provides an efficient reporting system compatible with most real time PCR instruments. We present results on the successful use of this system (termed Lux for "Light Upon extension") on a series of genes including house keeping genes, cytokines, signal transduction, cancer and apoptosis genes.

M. Brisson, L. Huiet, R. Park, L. Tan, K. Hamby; Bio-Rad Laboratories, Hercules, CA

Proper experimental design for real-time PCR is essential for studies involving relative or absolute comparisons between two or more genes amplified in the same tube. Several parameters must be evaluated and optimized independently to achieve accurate quantitation. Low amplification efficiency can reduce the dynamic range of quantitation for a particular target and compromise the data. For gene expression studies that require precise and accurate quantitation of multiple genes in the same samples, the iCycler iQ displays powerful multiplexing capabilities. Here we demonstrate 4-gene multiplexing capabilities. Calculated threshold values for a single gene target were identical when amplifying alone or with 3 other genes under optimized conditions. This was true for plasmid or genomic DNA targets. In multiplex, individual targets can be reliably quantitated over at least 6 orders of magnitude for a plasmid or 4 orders for a genomic DNA target. In addition, wide target concentration differences between 2 amplified gene products could be shown under optimized conditions. We quantitated as few as 10²copies of one gene in the presence of up to 10⁹copies of the second gene target.

R. T. Pon, S. Yu; University of Calgary, 3350 Hospital Drive NW, Calgary, AB T2N 4N1 Canada

New reagents with cleavable 3'-ester linkages use phosphoramidite coupling chemistry to attach the 3'-terminal nucleoside to a solid-phase support. Inexpensive amino supports may then be used as "Universal" supports in high-throughput DNA synthesizers. The linker phosphoramidites also allow multiple oligonucleotides, linked end-to-end in tandem, to be made. Tandem synthesis allows two or more oligos to be produced in a single run. Thus, a pair of PCR primers or both strands of a duplex DNA can be made in one synthesis. This doubles the number of oligonucleotides which can be produced per run and halves the number of individual samples processed. The tandem products can be used as a single mixture without separation, so end users also have fewer samples to handle. Both individual and tandem syntheses can be performed simultaneously and no changes to conventional synthesis or deprotection conditions are required.

A. T. Yeung¹, B. P. Holloway², P. S. Adams³, K. Mills⁴, S. Scaringe⁵, G. A. Buck⁶, K. Lilley⁷, M. Gunthorpe⁸; ¹Fox Chase Cancer Center, ²Centers for Disease Control, ³Trudeau Institute, ⁴Millennium Pharmaceuticals Inc., ⁵Dharmacon Research, Inc., ⁶Virginia Commonwealth University, ⁷University of Cambridge, ⁸University of San Francisco

The high cost of DNA probes for Fluorescence Resonance Energy Transfer (FRET) experiments has long impeded the full utilization of real-time PCR in genomic research. The high cost is partly due to limitations in the traditional synthesis chemistry that demanded extensive purification of these probes. Limited access to proprietary quencher reagents has also discouraged the development of better synthesis protocols. Fortunately, an opportunity has arisen with the recent availability of non-fluorescent quencher reagents to core DNA synthesis facilities. We are testing the synthesis of real-time PCR probes labeled with a fluorescence reporter on the 5' termini and with either a non-fluorescent quencher or tetramethylrhodamine on the 3' end. Both crude products of DNA synthesis and the purified probes will be tested in real-time PCR to judge the effect of probe quality on assay outcome. If improved synthesis protocols are found, we propose to verify their utility by expanding the testing to all interested laboratories.

M. Lemaitre¹, J. Truffert¹, N. Thelwell², L. Brown²; ¹EUROGENTEC SA, Herstal, Belgium Belgium, ²OSWEL Ltd, Southampton, United Kingdom

We have developed a number of novel analogues for use in real-time PCR systems. Development of these analogues was undertaken to improve fluorescent probing systems and to resolve problems that have arisen with the standard probing systems, such as Scorpion primers and TaqMan probes. The oligonucleotide modifications discussed will include dark quenchers that can be used in either of the 2 systems to quench a wide range of dyes across the spectrum. Data comparing quenchers will be shown for TaqMan probes and Scorpion primers. In addition, a system that incorporates FRET into a Scorpion primer for detection in channel 2 of the LightCycler will be described. This system works in both the standard stem-loop Scorpion format and the new Duplex Scorpion, which will be described in detail.

1) Whitcombe,D., Theaker,J., Guy,S.P., Brown,T. and Little,S., Nature Biotechnol., 1999, 17, 804.

2) Thelwell,N., Millington,S., Solinas,A., Booth,J. and Brown,T., Nucleic Acids Research, 2000, 28, 3752.

F. Schäfer, K. Steinert, C. Feckler, J. Drees, J. Ribbe; QIAGEN GmbH, Max-Volmer-Str. 4, Hilden, 40724 Germany

The TAGZyme System allows efficient and precise exoproteolytic removal of N-terminal affinity tags from proteins. In combination with Ni-NTA technology, the TAGZyme System provides high-purity proteins free of vector-encoded amino acids for use in applications such as preparation of pharmceuticals that demand recombinant reagents, an absence of non-specific cleavage, and a complete removal of all impurities from the target protein preparation.

New vectors, based on the pQE series for highly efficient expression of 6xHis-tagged proteins, have been constructed. The vectors’ multiple cloning site sequences have been designed to allow complete exoproteolytic removal of the encoded N-terminal affinity tag regardless of the restriction site used for cloning. Numerous examples of obtained expression rates and the successful downstream processing of the Ni-NTA–purified proteins are shown.

The extremely efficient cleavage reaction and removal of the His-tagged TAGZyme enzymes by subtractive Ni-NTA chromatography is demonstrated for 6xHis-Il-1β and 6xHis-TNFα. In both cases, more than 99.8% of TAGZyme proteolytic activity was separated from recovered, detagged proteins. N-terminal analyses by Edman degradation revealed the predicted sequences of the native proteins and indicated a purity >99%. Furthermore, data on the successful and fast removal of tags of between 8 and 25 amino acids in length are shown. Finally, the processing of high molecular weight protein complexes and their recovery by subtractive Ni-NTA chromatography is presented.

U. Schweiger-Hufnagel, M. Lubeck, C. Baessmann, A. Ingendoh; Bruker Daltonik GmbH, Fahrenheitstrasse 4, Bremen, 28359 Germany

Expression of recombinant proteins in biochemical laboratories can be complicated with certain degrees of primary sequence errors. Such deviations could result in modified enzymatic activities or more profoundly in modified 3-D structure.

LC-MS/MS provides ultimate sensitivity and specificity. Capabilities of full automation and exact analysis of the primary sequence can be utilized to provide a fast and reliable approach to confirm successful expression. We have developed a system that yields high sequence coverage and a sophisticated software package that would allow matching of signals from an LC-MS/MS run to a known sequence. Here, an esquire3000plus ion trap was used in combination with nano-LC for confirmation of the expected amino acid sequence of recombinant proteins.

Isolated and purified proteins were digested and separated on a nanobore column. Eluted peptides were analyzed by automated nano-ESI-MS/MS. Pepmap Creator, was used to generate a combined MS and MS/MS peak list, which was then automatically compared with the expected protein sequence in Biotools. We show here that this intuitive software is a powerful tool for detecting synthesis errors during protein expression.

A. P. Wulandari; THe University of Japan, Yayoi 1-1-1, Bunkyo-ku, Tokyo, Tokyo 153-8657 Japan

The lysS gene encoding for homocitrate synthase , the key enzyme involved in the lysine biosynthesis of T. thermophilus was over expressed in E.coli. SDS-PAGE shows a single protein specific of Mr 43kD. The multimeric structure of the native enzyme were confirmed by cross-linking with glutaraldehyde followed by electrophoresis. The enzyme follows Michelis-menten kinetic to 2-oxoglutarate (Km= 0.031 mM) and exhibit allosteric condition with acetyl Co-A (nH= 2.6 and S[0.5]=0.032mM.

Thermodymic of denaturation enzyme exhibited that the reaction catalyzed by HCS from T. thermophilus is temperature dependence reaction. The enzyme strongly inhibited by lysine with Ki =0.0094mM, and showed the additive effect in the present of arginine

Withdrawn

X. Zhang, X. Xu; Zhengzhou University Medical College, 40 Daxue Road, ZhengZhou, Henan 450052 China, People's Republic of

Xiaoli Zhang Xia Xu Lexun Xue *

E-mail: lxxue@public2.zz.ha.cn

Tel/Fax: 86-371-699-9548

P. S. Adams¹, T. B. Miller¹, D. Vignali², K. Vignali², L. S. Cauley¹, L. Haynes¹, P. Arnold², S. L. Swain¹, D. Woodland¹; ¹Trudeau Institute, ²St. Jude Children's Research Hospital

MHC class II molecules play a critical role in the presentation of peptide antigens on the cell surface for recognition by CD4+ T cells. However, it has proven difficult to reliably express soluble forms of MHC class II molecules to study their role in infectious disease. To resolve this problem, we have generated fusion proteins in which the extracellular MHC class II a and b chains are linked via a fos/jun leucine zipper, and attached to the hinge and Fc domain of murine IgG2a. These were expressed as either ‘empty’ dimers or loaded with specific peptides recombinantly attached to the MHC class II b chain. We have generated 22 different MHC class II:Ig multimers using four alleles (H-2A^k, H-2A^b, H-2A^g7, H-2E^k). All are expressed as functional dimers by Drosophila S2 cells and can be purified by conventional protein A chromatography. Multimerization of peptide loaded MHC class II:Ig multimers with fluorescently labeled protein A offers a useful tool for tracking CD4+ T cells by flow cytometry. A preparation scheme and examples of experimental use of multimers representing several alleles and peptide antigens will be presented.

J. VanEe, A. Mannochia, T. Thannhauser, T. Stelick, R. Halpin; Cornell University

Large scale, multi-user DNA sequencing facilities face special challenges. Acceptable levels of customer service and reasonable prices must be maintained: Only by increasing operational efficiency can a facility meet these challenges. Cornell University's BioResource Center DNA Sequencing Facility has faced sustained exponential growth, leading to the development of several generations of data management systems. The latest, the "BRC Online" web portal, provides the ability to manage every step of the sequencing process. MySQL provides the database backbone of the system. PHP (PHP Hypertext Processor) and an Apache Web Server provide the interface for the portal. Standard networking technologies coupled with server based scripting integrate various instrumentation with the central database. Special functions include automatic robotics work list generation, creation of .plt files for ABI 3700 sequencers and generation of several quality control metrics for efficient review and interpretation of outgoing data. This system provides managers with an overview of the operation's status and an audit trail for troubleshooting.

R. Ehrnström, A. Palm, S. Wallenborg, M. Gustafsson, P. Andersson, A. Hedström, E. Togan-Tekan, G. Ekstrand; Gyros AB, Uppsala Science Park, Uppsala, SE-75183 Sweden

The sensitivity of MALDI mass spectrometry has increased immensely since its introduction and the technique is widely used for rapid protein identification. The need for improvements in sample preparation methods of protein digests prior to identification by mass spectrometry is well recognized. Until now an automated process for reliable, routine preparation of peptide digests at concentrations approaching the detection limit for mass spectrometry, has been missing. We have developed a system in which key steps for sample preparation prior to MALDI analysis are integrated and run in a CD microlaboratory. Protein digests are concentrated, desalted, mixed with matrix and crystallized on MALDI target areas on the CD. Up to 96 samples are processed in parallel. The CD is transferred to a MALDI instrument and no separate target plate is required. By optimizing the entire process we have significantly enhanced sample recovery and concentration so that analysis is performed close to the detection limit of commercial MALDI mass spectrometers. Results with sensitivities in the attomole to low femtomole can be achieved, facilitating the identification of low abundance proteins.

I. P. Smirnov, P. L. Ross, L. R. Hall, L. A. Haff; Applied Biosystems, 500 Old Connecticut Path., Framingham, MA 01701

In the past decade MALDI-TOF MS has emerged as one of the main analytical tools for proteins, peptides and short DNA fragments. The main obstacle of this method is its susceptibility to salts and surfactants, usually present in the sample as buffer components. Their elimination requires laborious desalting procedures and substantially decelerates overall analysis time. Here we report a novel solution to this problem by employing MALDI sample plates coated with various ion-exchange polymers, such as polyacrylic acid and polyethyleneimine. By using their ability to bind biomolecules sample isolation and purification can be done directly on the MALDI plates, thus significantly simplifying the sample preparation step. Various polymer films have been characterized for peptides, proteins and oligonucleotides of various size. The importance and role of different factors have been studied, such as composition of ion exchange film and compatibility of the MALDI matrices together with mass accuracy optimization in order to obtain the best performance. This method makes possible a high throughput sample preparation for MALDI analysis in proteome research.

M. E. McComb, A. Lim, T. B. Prokaeva, L. H. Connors, M. Skinner, C. E. Costello; Boston University School of Medicine, 715 Albany Street, Boston, MA 02118

Amino acid substitution in TTR, a transport protein in plasma, may result in the formation of amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with deposition of TTR amyloid fibrils in tissues and organs. More than 80 variants are known. Effective treatment requires liver transplantation, thus correct diagnosis is critical. We have characterized TTR variants using mass spectrometry (MS). Intact TTR and peptide mapping suggests the variant. For conclusive identification it is often necessary to sequence the individual peptides. To this end, we have explored the use of tandem MS (MSMS) on an ABI QSTAR quadrupole-orthogonal-time-of-flight (QoTOF) MS equipped with MALDI and ESI sources. Using MALDI-MS, ESI-MS and on-line capillary LC-ESI-MS with information dependent acquisition (IDA) we have successfully characterized several TTR variants by MSMS. Whereas manual MALDI-MSMS and ESI-MSMS may be tedious, LC-ESI-IDA-MS/MSMS affords the ability to purify, separate, obtain peptide maps and sequence information on an entire proteolytic digest in one automated experiment. We demonstrate this while characterizing amyloidogenic TTR variants.

R. Tyldesley¹, D. O. Gostick¹, J. I. Langridge², J. B. Hoyes³, K. Schneider⁴, A. O. Wattenberg⁴; ¹Micromass UK Ltd, Floats Road, Wythenshawe, Manchester M23 9LZ United Kingdom, ²Micromass UK Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ United Kingdom, ³MICROMASS UK LTD, Almere, ⁴GSK, Harlow, Essex United Kingdom

Protein identification by mass spectrometer is now a routine in many biochemistry laboratories. There are several alternative methods for producing an identification of an unknown protein, but these nearly always require the mass measurement of peptides. The analysis of the peptides is typically performed by either ESI or MALDI. These two ionisation techniques have until now always been performed on different types of mass spectrometers. In this presentation we compare the MS and MS/MS spectra from the analysis of protein digests using ESI and MALDI on the same instrument. This is now possible using the dual source available on the Q-Tof mass spectrometer.

The two ionisation techniques are known to produce different MS and MS/MS spectra from the analysis of the same sample. In this poster we investigate these differences and illustrate their complimentary nature for protein identification using database searching and bioinformatics.

B. Wu¹, P. Bondarenko¹, A. Land¹, V. Kovtoun¹, G. Stafford¹, S. Swedberg², K. Miller¹, P. Shieh¹, B. Hancock¹; ¹ThermoFinnigan Corp., 355 River Oaks Parkway, San Jose, CA, ²ThermoFinnigan, San Jose, CA

Protein identification using peptide mass maps and database searching has become an important technique for proteomic studies. MALDI-TOF MS has been used extensively to identify samples extracted from gel spots by this approach. Also proteins purified from multi-dimensional separation techniques have been characterized by off-line coupling with MALDI-TOF MS. However, MALDI-TOF MS has the disadvantage that MS/MS is better performed on tandem mass spectrometers. The power to fragment selected ions further can be key to assigning the position of modifications as well as determining the structure of the modification.

As an alternative, this report described an atmosphere-pressure MALDI source coupled to an ion-trap MS (LCQ-Deca-XP), which was used to assign peptide sequences by both MS and MS/MS spectra. The structure of modifications (e.g. disulfide linkage or phosphorylation) was determined by further fragmentation in this device (e.g. MS to 3 in the ion trap).

This new MALDI-Ion Trap is commercially available and compatible with the current Xcalibur control software and Bioworks data-handling software, which allows samples to be run and data to be analyzed automatically.

M. D. Person, K. Tikoo, T. J. Monks, S. Lau; University of Texas at Austin, 2501 University Avenue, Austin, TX 78712

Identification of post-translational modifications of proteins remains a major challenge in mass spectrometry. Our approach uses a comparative analysis of the proteolytic digest and MALDI-MS spectra of control and treated proteins to target differences due to modifications. Peptide mass mapping is used to identify peptides present. Accurate mass measurements indicate potential modification types for the peptides. HPLC-ESI-MS/MS is then used to sequence peptides and determine the type of modification present. This approach presents a broad strategy for identifying modifications without initial assumption as to type or residue localization. The MALDI screening step is used to reduce the analysis time. The MS/MS allows determination of sequence and modification. Successful characterization of a multiply modified histone protein is given as an example of the use of this strategy. Hyperacetylated histone 4 is separated on a gel into 5 bands representing different levels of acetylation. Bands are digested with chymotrypsin in-gel and then analyzed by MALDI-MS and HPLC-ESI-MS/MS to identify the sites of methylation and acetylation.

S. H. Bhattacharya, T. J. Raiford , K. K. Murray; Lousiana State University, Choppin Hall, Baton Rouge, LA 70803

Desorption/ionization on porous silicon (DIOS) is a form of laser desorption mass spectrometry in which a silicon surface acts as the energy-absorbing medium. Conventional MALDI methods employ an organic matrix for analyte desorption and ionization which can lead to matrix interference in the region below m/z 1000. DIOS allows the analysis of low molecular weight analytes since there is no low mass matrix interference. UV DIOS requires the preparation of a porous silicon surface by electrochemical etching in a hydrofluoric acid solution. We have recently employed a tunable mid-IR infrared laser to perform DIOS on unmodified single crystal doped silicon surfaces. Using the IR laser at 2.9 micrometers, it was possible to ionize peptides up to 2000 Da on an untreated single crystal silicon (100) surface with no additional solid or liquid matrix. The analyte was deposited on the surface in an ethanol solution, or in some cases, simply sprinkled onto the surface in powder form. In ongoing work, the effects of laser wavelength and surface treatment are being investigated.

S. J. Berger¹, D. Wall¹, R. Karol¹, J. W. Finch¹, H. Y. Xiong², S. A. Cohen¹; ¹Waters Corporation, Milford, MA, ²Micromass Inc., Beverly, MA

MALDI and ESI are the predominant ionization methods for introducing biomolecules into the gas phase prior to mass analysis. Until recently, ESI has monopolized the online analysis of complicated mixtures of proteins due to the ease in coupling liquid phase separations to ESI sources. However, the propensity of MALDI to generate ions with fewer charge states is appealing for the analysis of protein mixtures, as multiple overlapping charge states often arising from ESI complicate spectral interpretation. Here, we demonstrate a device for continuous deposition of eluent from analytical/capillary scale RPHPLC protein separations onto MALDI plates precoated with matrix. MALDI analyzed separations of intact proteins, protein mixtures, and complexes are presented, and comparisons to LC/ESI-TOFMS analyses are shown. Our results show that LC-MALDI plate capture of separations can be automated, and that captured separations can be archived for re-examination. Furthermore, we demonstrate that automated LC-MALDI plate collection of proteins is accomplished without sacrificing highly resolved LC separations.

N. Leymarie, E. Berg, P. O. OConnor, J. Grogan, F. G. Oppenheim, C. E. Costello; Boston University School of Medicine, 715 Albany Street, R806, Boston, MA 02118-2526

The salivary proline-rich proteins (PRP) are involved in the maintenance of tooth integrity. Recognition of the difficulties in obtaining PRP structural information has led to the development of methodologies based on mass spectrometry. These methodologies utilize enzymatic digestion followed by the characterization of PRP by classical MS/MS techniques (CID, post-source decay). The abundance of y-type fragment ions associated with proline (proline-effect) gives rise to difficulties in obtaining complete sequence data.

Recently, a new method of fragmentation, electron capture dissociation (ECD), has emerged. It cleaves peptide backbones at the Ca-N bonds rather than at the amide linkages.

The goal of this poster is to demonstrate the power of ECD for the determination of the structure of an acidic PRP and to obtain more complete sequence information of the proteolytic peptides derived from it. Due to the cyclic structure of proline, ECD spectra do not contain c and z· product ions on the N-terminal side of proline. Additionally the localization of post-translational modifications using ECD and the complementarity of ion fragmentation methods such as CID and ECD will be discussed.

W. Sandoval¹, K. O’Connell¹, J. T. Stults², W. J. Henzel¹; ¹Genentech, South San Francisco, CA, ²Genentech, Inc., South San Francisco, CA

We have explored the use of both Lys-C and Lys-N endoproteases to obtain more complete sequence coverage for the de novo sequencing of peptides from MS/MS spectra. Lysine-C from Achromobacter lyticus is a well-characterized endoprotease that is stable in a variety of denaturants including urea and SDS. Endoproteases that cleave at the N-terminal side of lysine have also been purified from several fungal sources. We have compared MS/MS data obtained from digestions of model proteins with Lys-N from Grifoloa frondosa and with Lys-C from > Lysobacter enzymogenes. Each enzyme produces peptides with the same sequences except the lysine residues are at opposite end of the peptide. This different placement of a basic residue results in significantly different MS/MS fragmentation patterns. The complementarities of these spectra result in higher sequence coverage and/or higher confidence in sequence assignment.

H. A. Sawyer¹, B. T. Ruotolo², J. T. Marini², G. F. Verbeck, IV¹, K. J. Gillig², D. H. Russell²; ¹Texas A&M University, College Station, Texas 77840, ²Laboratory for Biological Mass Spectrometry, Texas A&M University, mail stop 3255, College Station, Texas 77843

Metal ions play important roles in many biological processes and there is considerable interest in developing mass spectrometry methods for determining structure and binding sites of peptide/protein metal ion complexes. Ion mobility-mass spectrometry couples volume-to-charge and mass-to-charge measurements and yields information about gas phase structure. For a homologous ion series, a plot of ion drift time versus mass-to-charge over a limited mass range yields a near-linear relationship. However, alkali ion adducts of bradykinin and subunits of bradykinin (des-Arg⁹, des-Arg¹, fragments 2-7, 5-9, 1-5, 1-6, and 1-7) deviate from the expected linear relationship. This result suggests that [M+Cs] ⁺ have similar mobilities to that of [M+H] ⁺, suggesting similar cross-sections. These deviations are rationalized in terms of conformational change to the peptide upon alkali metal adduction. H/D exchange experiments as well as molecular modeling will be coupled with ion mobility data to address the conformational effects of cesium ion adduction as well as the effects of primary structure and chain length on the metal ion binding site.

B. D. Miller, C. A. Miller, F. E. Kuhlmann; Agilent Technologies, Inc., 1601 California Avenue, Palo Alto, California 94304

Protein identification by protein database search of peptide-map data produced by ion trap LC/MS/MS instrumentation is a well-established technique. In this technique it is common to employ automated triggering of MS/MS experiments based on real-time evaluation of MS spectral information content. Driven by increasingly complex proteomics research questions and expanded analytical demands, efforts continue to improve both the sample throughput and the data quality available from unattended, automated LC/MS/MS analyses of protein digest mixtures.

In this work, we demonstrate the use of two novel precursor-ion selection criteria to improve data quality and identification results in the automated ion trap-LC/MS/MS analysis of protein digest mixtures: preferred charge-state and preferred mass-list. Preferred charge-state mode provides for real-time, data-dependent exclusion of singly charged peptide precursor ions and/or preferential selection of doubly charged peptide precursor ions. Preferred mass-list mode biases on-the-fly precursor selection in favor of targeted peptide precursor ions, e.g., chemically modified peptides that may be present at low relative abundance in a complex digest mixture.

J. L. Whitney, M. E. Hail, D. J. Detlefsen; Novatia, 301A College Road East, Princeton, NJ 08540

In recent years pharmaceutical and biotechnology companies have initiated wide-reaching programs in genomics and proteomics. These ongoing initiatives are resulting in a flood of new proteins and oligonucleotides used as both targets and potential therapeutics. Characterizing these biomolecules throughout the discovery and development process is of utmost importance. Rapid and automated analytical methods are needed to address important issues of molecular weight confirmation, purity assessment, sequence determination, purification heterogeneity, elucidation of chemical and biological modifications, bio-production monitoring, etc. To answer many of these questions it is often necessary to analyse the intact biomolecule. LC/MS, utilizing electrospray ionization, has emerged as a powerful technique in these application areas. However, to date, there are no fully automated approaches for analyzing and processing data obtained on intact biomolecules using LC/MS. Here we will illustrate several applications of biomolecule characterization using our totally automated LC/MS system.

S. Leicester; Micromass UK Ltd., Wythenshawe, Manchester United Kingdom

Current databank search techniques facilitate the identification of peptides from MS/MS data. However, it is becoming clear that peptides are missed in the identification process due to non-specific cleavage, amino acid substitution, databank errors and the absence of the peptide from the databank being searched. We present a methodology that allows chaining of different search algorithms together to form an overall search strategy. By combining databank searching with PTM analysis, non-specific cleavage sites, amino acid substitution and de novo peptide sequence determination, we show that peptides can be identified that would otherwise be missed by databank searching alone.

J. Leszyk; University of Massachusetts Medical School, 222 Maple Avenue, Shrewsbury, MA 01545

Matrix-Assisted-Laser-Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry has emerged as an important tool in the identification of gel purified proteins for various proteomic studies. MALDI -TOF has been somewhat limited to the determination of intact mass information from several peptides followed by enzyme specific mass correlation analysis to make an identification. This limitation is due in part to the difficulty in obtaining good MS/MS information via Post Source Decay (PSD) fragmentation for further MS/MS database correlation analysis. I describe here the use of the Kratos Axima CFR MALDI-TOF mass spectrometer as a powerful tool for the identification of proteins from both MS and MS/MS information. The Curved Field Reflectron (CFR) is capable of acquiring complete PSD spectra with isotopic resolution in a single acquisition phase at very high sensitivity (1-2 fmols). Other types of Reflectrons, which require voltage stepping and multiple acquisitions to assemble complete spectra often, take 20 to 40 minutes to acquire a single spectrum. PSD fragmentation spectra are not easily interpreted de novo but provide numerous fragments which allow definitive identifications via the Protein Prospector MS-Tag program. A powerful attribute of this program is its allowance of internal fragments for the correlation analysis of PSD spectra. The Axima CFR does not require any tuning or extensive instrument maintenance to obtain ultra high sensitivities. MALDI TOF-MS and PSD data from silver stained protein bands obtained from both 1D and 2D gel formats will be presented.

T. A. Shaler¹, P. V. Bondarenko¹, S. Swedberg²; ¹Thermo Finnigan, San Jose, CA, ²ThermoFinnigan, San Jose, CA

The entire human serum from a commercial pooled source was denatured, reduced and digested with trypsin prior to LC/MS/MS analysis using an ion trap mass spectrometer. The digest products were eluted from a 75 μm ID C18 column with a linear gradient 0 – 60% B over 500 min at 0.1 μL/min. The mass spectrometer was operated in a data-dependent MS/MS mode, when one full-scan MS was followed by three tandem scans on precursor ions selected "on the fly" from the MS scan. Using TurboSequest search algorithm, the MS/MS data were correlated with peptide sequences from a homo-sapiens database. A half of proteins were identified by only one matching peptide. Although correlation scores for these peptides were above conventional threshold, probability of true identification using one matching peptide was still not high. To improve this probability, the entire LC/MS/MS analysis was repeated three times. Those low abundant proteins, which were identified with only one matching peptide, but detected in two or three runs and with similar elution time, were included in the final list of identified proteins. Total 200 human serum proteins have been identified using described algorithm.

Guido F. Verbeck, Brandon T. Ruotolo, Kent J. Gillig, David H. Russell

Analytical chemists are always striving for techniques that add dimensions of orthogonality with increased throughput and sample complexity. Ion mobility (IM) time-of-flight(TOF) is a system that adds new dimensions to mass spectrometry(MS). IM separates gas phase ions based on their collision cross-section. IM can be coupled with time-of-flight mass spectrometry to yield a powerful tool used in the identification and characterization of proteins and peptides. One particular area of interest is in the separation/identification of different conformers of peptide and protein ions. IM-TOF-MS can simultaneously collect a spectrum of mobility through a drift gas (drift time) and mass-to-charge (m/z). Figure 1 illustrates the orthogonality of achieved by coupling these two separation techniques. The figure contains plots of drift time vs. m/z for a sum of peptide and carbon cluster ions. The ball-like structure of fullerenes and carbon clusters means their ions have high mobility (short drift times) relative to that for the peptide ions. These trend lines are seen because ions that possess similar structural features will have a constant of proportionality between mass and collision cross-section. We can probe structural differences between proteins and peptides based on this assumption. Figure 2 shows two peptides with similar mass but different mobilities. With the aid of molecular dynamics calculations and IM predictions, proposed structures of these ions can probe these different conformations. In this presentation the fundamentals of ion mobility will be covered with emphasis on applications to structural analysis of proteins and peptides.

J. M. Hettick, D. H. Russell; Laboratory for Biological Mass Spectrometry, Texas A&M University, TAMU 3255, College Station, TX 77843-3255

Tandem mass spectrometry is a powerful tool for the identification and characterization of proteins, and may be used to sequence peptides de novo or to increase the specificity of a peptide mass mapping/database searching experiment. Tandem MS requires that the analyte of interest be activated prior to fragmentation. Conventional approaches rely upon the collision of the analyte with a neutral target molecule(s). Research in our laboratory has focused on the development of a MALDI tandem TOF MS that utilizes radiation from a 193-nm ArF excimer laser for ion activation. Both photodissociation and conventional collision-induced dissociation (CID) produce sequence-informative fragment ions from protonated peptide molecules ([M+H] ⁺), however the types of fragment ions produced can be quite different. In particular, photodissociation produces side-chain cleavage ions important for differentiating isomass residues. In this study, the fragmentation produced by photodissociation is compared to that produced by CID of electrosprayed ions in a commercial quadrupole TOF instrument. The utility of the two methods for proteomic analysis will be discussed.

C. A. Miller, S. M. Fischer; Agilent Technologies, Inc., 1601 California Avenue, Palo Alto, CA 94304

Proteins are commonly separated by RPLC using wide-pore silica-based columns. Typically separations are done using acetonitrile/water gradients with TFA modifier. The limiting factor in reverse-phase protein separations is mass transport, i.e., diffusion of the proteins in and out of the pores. This was improved by the development of non-porous 2-micron silica-based columns. However, the limitation for this packing material was capacity. The development of superficially porous silica-microsphere packing with a porous outer shell yielded silica-based columns with the capacity advantage of traditional protein columns and the rapid mass transport of 2-micron non-porous columns. Using this column technology with electrospray mass spectrometry, rapid separation of protein mixtures with molecular weight determination can be done. This work investigates separation conditions that optimize mass spectrometric detection with minimal chromatographic impact.

K. Bernardo, N. Pakulat, O. Krut, M. Macht, H. Frank, H. Seifert, S. Fleer; University of Cologne, Joseph Stelzmann 9, Cologne, Cologne D-50931 Germany

Katussevani Bernardo^§, Norbert Pakulat^§, Marcus Macht^$, Silke Fleer^§, Oleg Krut^§ and Martin Kroenke^§.

M. E. Hail, J. L. Whitney, D. J. Detlefsen; Novatia, 301A College Road East, Princeton, NJ 08540

The recent increase in genomic and proteomic discovery has increased the need for high-throughput automated tools for biomolecule characterization. The flood of new protein targets will soon demand more efficient tools for evaluation of expressed proteins for drug discovery and structural biology studies. Intact protein molecular weight measurement using electrospray ionization mass spectrometry (ESI/MS) remains one of the most important methods to assess identity, purity, and the possibility of post-translational modifications. However, to date, there are no fully automated systems to perform these types of analyses. Here we describe a system, called ProMassXcali^TM, which utilizes on-line LC/MS, automated biomolecule deconvolution and web-based reporting for totally automated biomolecule molecular weight profiling. This system can be used to process data from a wide variety of biomolecules including large proteins, oligonucleotides, peptides, etc. An overview of ProMassXcali^TM will be presented along with selected applications that highlight its utility in a high-throughput environment.

M. Lubeck¹, C. Baessmann¹, U. Schweiger-Hufnagel¹, M. Shen², A. Ingendoh¹; ¹Bruker Daltonik GmbH, Fahrenheitstrasse 4, Bremen, 28359 Germany, ²Bruker Daltonics, Inc., Billerica, MA

Automated data dependent LC-MS/MS strategies have been utilized for analyses and subsequent identification of digested proteins. One of the challenges in working with physiologically derived proteins is that sample amount could be a limiting factor. We have demonstrated previously that nano-LC systems coupled on-line with esquire3000plus ion trap can provide an effective solution to protein identification in low femtomole and even attomole ranges.

The unparalleled high scan power of esquire3000plus facilitates fast switching between MS and MS(n) modes. Even so, the time needed for automated MS(n) is limited by typical peak widths of up to 30s.

We show here that "peak parking" was integrated by implementing a switching valve that could be controlled seamlessly through HyStar. The eluted peaks were recognized and preferentially selected by their charge states. The valve was used to block the flow from the pump when a peak was detected, and the peak was sent to the mass spectrometer by an additional pump with an 8-fold lower flow rate. Several proteins were digested and analyzed, and sequence information was obtained by de novo sequencing in Biotools using MS/MS and MS(3) spectra.

A. H. Henschen-Edman; University of California, Irvine, Biological Sciences 2, Irvine, CA 92697-3900

Cyanogen bromide cleavage was used to analyze posttranslational modifications in human fibrinogen, a 340 kDa dimeric protein with 33 Met per monomer. CNBr cleavage was performed in 75% formic acid at a protein concentration of 10 mg/ml. CNBr concentrations compared were 100, 10, 1 and 0.1 mg/ml. Reaction times were 2 or 24 h. Extents of cleavage and possible side reactions were evaluated by gel filtration chromatography, HPLC, N-terminal sequence analysis and MALDI-TOF mass spectrometry. The most complete cleavage was only obtained with 100 mg/ml for 2 h and 10 mg/ml for 24 h, i.e. concentrations considerably higher than described in the literature. The shorter cleavage time caused less oxidative damage to Trp residues. About half of the Met residues occurred to a small part in an uncleaved, modified form that was resistant to the acid in gel filtration, HPLC and mass spectrometry as well as to SDS-PAGE. However, these modified Met residues always cleaved during N-terminal sequencing, the sequence following the specific Met appearing both in the first and the second cycle indicating cleavage on both sides of these residues.

S. Wong, W. J. Henzel; Genentech, South San Francisco, CA

We have designed a hydrolysis sample preparation station (HSPS) which is used with PICOTAG hydrolysis vessels (Waters Corp.) to control the rapid succession of vacuum and nitrogen purging in the hydrolysis vessel. The HSPS is attached between the vacuum pump and the sample hydrolysis vessel and consist of several relays and an electronic counter. An electronic vacuum gauge containing outpoint relays allow nitrogen gas to be delivered to the hydrolysis vessel before bumping can occur. The counter is set to the number of purging/vacuum cycles desired. Sufficient cycles are required to prevent bumping when the last cycle is reached and high vacuum is applied. The hydrolysis vessel is then removed from the device and placed in an oven.

An improved sample clean-up procedure to process large number of samples containing high salts and buffer components for amino acid analysis has been developed. This procedure utilized the new Millipore Multiscreen 96-well sample plate with high capacity PVDF-psq membrane. Multiple samples may be applied to the plate and can be processed at the same time for further washing or concentrating.

These protocols have greatly reduced the time spent on sample preparation and improved the quality of analysis.

P. Jandik, J. Cheng, M. Weitzhandler, D. Farnan, C. Loran, N. Avdalovic; Dionex Corp., 445 Lakeside Drive, Sunnyvale, CA 94085

Anion exchange chromatography with IPAD detection (AEC/IPAD) is a recently introduced new tool for amino acid analysis. It avoids derivatization as a detection-enabling step. Amino sugars and amino acids can be separated and quantified in a single chromatographic run.

We have evaluated the use of this new tool for amino acid analysis after gel electrophoresis and electroblotting and also in fractions collected from HPLC.

In the study of electroblotted PVDF spots, we followed the format of the 1999 ABRF Amino Acid Survey Report. Several different levels of a test compound were analyzed and resulting data were used to search the PROPSEARCH and ExPASy sites for protein identification.

To assess the applicability of AEC/IPAD for amino analysis of HPLC fractions, we carried out two-dimensional separations (cation exchange/ reversed phase) of intact proteins and enzyme digested proteins. Using the same approach as with PVDF spots, we submitted the compositional data obtained from HPLC fractions to the Internet sites to test the reliability of identification.

K. D. Linse, T. E. Taub-Montemayor, J. W. Kent, M. Rankin; University of Texas at Austin, 2500 Speedway Blvd, Austin, Texas 78712

Reversed phase HPLC has traditionally been used to purify neuropeptide hormones from the corpora cardiaca glands (CC) of insects. Using RP-HPLC, we have previously identified an Adipokinetic Hormone (AKH) from the CC of the migratory grasshopper Melanoplus sanguinipes. This hydrophobic decapeptide has the same primary structure as locust (Lom) AKH I. Initially, only one HPLC peak with the same retention time as Lom AKH I was detected; however, we found and characterized a second putative AKH within this fraction using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) and HPLC tandem Electrospray Ionization Mass Spectrometry (ESI-MS). Armed with this information, we were able to improve the resolution of the native AKHs by HPLC. Our results show the power of coupling HPLC and MS methods to resolve and identify very similar peptides in minute amounts.

S. Kwak, N. Laliberte, M. Hincapie, R. Chicz; Zycos, Inc, 44 Hartwell Ave, Lexington, MA 02421

We present a rapid and efficient automated immunoaffinity chromatography method for the purification of Major Histocompatibility Complex (MHC) molecules. The speed of purification allows for greater sample throughput and better preserves the integrity of the non-covalently bound peptide repertoire of these receptors. Up to 4 different antibody columns can be serially connected, allowing simultaneous purification of multiple human leukocyte antigen (HLA) allotypes from a single cell source. High purity protein is isolated from these HLA specific antibody columns and the HLA molecules are free of contaminant proteins.

The recovery of purified HLA can range from one microgram to several milligrams depending on the protein expression level from as little as one gram of harvested cultured cells. This is illustrated by the purification of both HLA class I and class II protein from multiple preparations of human lymphoblastoid preparations. This method has been successfully applied for the isolation and subsequent sequence analysis of both HLA class I and class II T cell epitopes.

J. Masuda¹, E. J. Unsworth¹, D. M. Maynard¹, M. Nishimura², T. Ueda³, J. A. Kowalak¹, S. P. Markey¹; ¹NIMH/NIH, 10 Center Drive Room 3N321, Bethesda, Maryland 20892-1262, ²Shimadzu Scientific Instrumets, Inc., Columbia, Maryland, ³Shimadzu Corporation, Kyoto, Kyoto Japan

Over the past few years, several studies have been published on two-dimensional HPLC separation methods for proteome analyses. In general, a combination of strong cation exchange (SCX) chromatography and reversed phase (RP) chromatography is used for a two-dimensional HPLC with the sample fraction eluting from a cation exchange column being directly loaded onto a pre-equilibrated RP column. Analytical methods combining two orthogonal separation techniques have been directly coupled to electrospray ionization tandem mass spectrometers for the analysis of proteolytic hydrolysates of complex protein mixtures. Constituent proteins are subsequently identified using algorithms that utilize MS/MS data to search genomic databases.

In this paper, a new automated two-dimensional HPLC system is described that is based on micro-scale HPLC techniques and utilizes six capillary trapping pre-columns, CapTrap (Michrom BioResources, Inc.), mounted on a 14-port rotary valve to capture sample fractions after sequential elution from a micro-bore SCX column. SCX fractions trapped on CapTraps are effectively desalted, and sequentially transferred, by RP gradient elution, onto a pre-equilibrated capillary RP column. The chromatographic resolution of peaks that could not be separated using only RP chromatography was achieved when analyzing a tryptically digested protein mixture with ESI-ion trap as the detector. Data will be presented that illustrate the utility of a micro-scale two-dimensional HPLC system for complex protein digest mixtures in proteomic analysis.

J. S. Smith, E. R. Surriga, A. Kurosky; University of Texas Medical Branch, Rm. 2.226 BSB, Galveston, Texas 77555-0645

The Applied Biosystems 173A Microblotter has proven to be a valuable resource for isolating peptides at the pmol level after in-gel or on-PVDF membrane enzyme hyrdrolyses. On occasion and, for a variety of reasons, these samples may contain nonvolatile buffers, salts, and detergents that can interfere with the capillary HPLC performed by the microblotter. An on-line sample cleanup method for the microblotter was developed using commercially available Michrom BioResources peptide traps and nonionic detergent removal cartridges. In this procedure, we replaced the Microblotter’s HPLC sample loop with a rheodyne cartridge holder containing the peptide or nonionic detergent trap. Samples were loaded on to the trap and manually washed with 0.1% TFA prior to injection. The resulting chromatograms showed that the salt peaks and detergent artifacts were virtually eliminated and the baseline greatly improved when compared with control non-trap results. The resolution of the peptide fractions was also considerably enhanced. Finally, we have found that the traps also served as an effective guard column preventing back-pressure problems and extending the life of the column.

S. A. Cohen, R. S. Plumb, J. W. Finch, C. L. Stumpf; Waters Corporation, 34 Maple Street, Milford, MA 01757

The identification of proteins via the peptides produced from their tryptic digests is now the approach of choice in proteomics. When dealing with a complex mixture such as a protein digest it is necessary to have as much chromatographic performance and mass spectrometric sensitivity as possible. The use of nanoflow MS has significantly increased instrument sensitivity when dealing with the trace levels of material encountered in protein analysis. Nanoflow chromatography does, however, present serious problems, namely gradient fidelity at very low flow rates, band broadening due to sample loading, analyte losses due to adsorption and poor column performance. Here we will discuss the use of small trapping and focusing columns for sample introduction and discuss their benefits and drawbacks. The selection of tubing and components to reduce sample loss will also be discussed along with potential solutions. Finally we will consider the effects of the chromatographic column, and the merits of pulled tip and/or fritted columns. Procedures that produce highly efficient, reproducible 75 micron columns will be demonstrated while highlighting the beneficial effects on MS sensitivity and spectral quality.

M. Weitzhandler, H. Schwartz, C. Saini, M. Rey, N. Avdalovic; Dionex Corp., 445 Lakeside Drive, Sunnyvale, CA 94085

We have developed a new monosaccharide column that gives faster, more efficient separations of glycoprotein monosaccharides with better spacing of monosaccharides. The new column (3mm x 150mm) is based on a new chemistry that reduced the resin bead particle size from 10 microns to 7 microns. Faster, more efficient separations of glycoprotein monosaccharides with better spacing was achieved across a range of isocratic NaOH concentrations (8 – 20 mm NaOH) at lower flow rates. Sensitivity of monosaccharide detection was improved as a consequence of the higher efficiency resin and narrower column diameter, enabling routine low pmol monosaccharide determinations. Fucose, galactosamine, glucosamine, galactose, glucose, and mannose were baseline separated and eluted in less than eight minutes (cycle time 30 min) at a flow rate of 0.5 mL/min (back pressure < 800psi). Further, when used with a 3mm AminoTrap guard column, amino acids did not interfere with monosaccharide determinations, enabling the direct monosaccharide profiling of glycoproteins with low levels of glycosylation such as monoclonal antibody hydrolysates.

F. Maroto¹, M. Scigelova², J. Vazquez³; ¹Thermo Finnigan, San Jose, California, ²Thermo Finnigan UK, Boundary way, Hemel Hempstead, HP2 7GE United Kingdom, ³Centro de Biología Molecular Severo Ochoa, Madrid, Spain

Post-translational modifications (PTM) are prominent features of proteins in higher organisms. Automated LC/MS analysis of peptides is a high throughput technique for protein identification. Several strategies have been developed to detect PTMs in an automated fashion suitable for high throughput peptide characterization.

We have compared the merits of several different strategies using a correlation of either peptide masses obtained from full scan spectra or sequence tags derived de novo from MS/MS spectra. Both approaches use a systematic search of peptide mass differences that can identify a modified peptide. The key benefit of using the de novo MS/MS strategy is that it is able to detect an unexpected post-translational modification and its exact location.

J. I. LANGRIDGE¹, A. Millar², C. HUGHES³, H. VISSERS⁴, T. Dourdeville⁵, J. B. Hoyes⁶; ¹Micromass UK Ltd, FLOATS ROAD, MANCHESTER, M23 9LZ United Kingdom, ²Micromass UK Ltd., Floats Rd, Wythenshawe, Manchester M23 9LZ United Kingdom, ³Micromass UK, MANCHESTER, ⁴Micromass Europe, Almere, Netherlands, ⁵Waters Corp., Milford, MA, ⁶MICROMASS UK LTD, Almere

Mass spectrometry has firmly established itself as the primary technique for identifying proteins. Currently, the mass spectrometry technique providing the highest degree of specificity and sensitivity is electrospray LC-MS/MS. However in the case of post translationally modified peptides only a very limited sub set of the peptides present are required to be fragmented and often these low intensity peaks can be missed. A solution to this problem is a method that allows specific post translationally modified peptides to be identified during the course of an LC-MS experiment. In the case of phosphotyrosine, a low mass immonium ion at m/z 216 can be detected. This characteristic ion is used to direct the mass spectrometer to fragment potential phosphopeptide precursor ions which are present at that time point in the low energy data. In this case several precursor ions may require MS/MS interrogation at one decision making time-point and implementation of a chromatographic technique known as variable flow chromatography allows greater time to interrogate these peaks. This approach will be discussed with examples of where this methodology has been used for the targeted analysis of phosphorylated peptides.

A. S. Woods¹, K. Fuhrer², M. Gonin², T. Egan², J. A. Schultz²; ¹NIDA IRP, NIH, 5500 Nathan Shock Drive, Baltimore, MD 21208, ²Ionwerks, Houston, TX

Spectra acquired using MALDI and Ion-Mobility-Orthogonal MALDI instruments show that phosphorylated residues form salt bridge with quaternary ammonium compounds. Each phosphate group forms a non-covalent complex with one or two quaternary ammonium compounds. When both the mono- and di-ammonium compounds are present in the mixture we see MH+ of the phosphorylated compound and two mono- or di-quaternary ammonium molecules, or MH+ to which one mono- and one di-ammonium added to the same phosphate.

IM coupled with MALDI is metamorphosing from laboratory curiosity to useful analytical technique. Laser Desorbed peptide ions are drifted through 2 Torr of He by a 40 V/cm field. The ions drift with a speed related to the ratio of their volume/charge (shape), which is slightly different in many cases than their mass/charge ratio. IM allows separation based on the conformation or overall shape of the MH+. Sensitivity for peptides is not far from conventional MALDI instruments. We can separate phosphorylated peptides from non-phosporylated ones, and achieve even larger separations between peptides as a class when compared to inorganic salts and small aromatic organic molecules. This ability to do "plasma chromatography" within the time scale of the combined MALDI experiment makes this instrument attractive for analysis of complex biological samples. We will present data showing the separation of phosphorylated peptides or lipids, and their non-covalent complexes with quaternary ammonium compounds by Ion-Mobility.

R. Shen¹, P. Shieh¹, W. S. Hancock²; ¹ThermoFinnigan Corp., 355 River Oaks Pkwy, San Jose, CA 95134, ²Thermofinnigan corp., San Jose, CA

Phosphorylation is an important post-translational modification critical in various cell functions including receptor-mediated signal transduction. The often transient phosphorylation of minute amount of a protein has made the determination of site of phosphorylation rather challenging. We explored the potential of using magnet-immobilized anti-phosphotyrosine or metal chelating matrix for capturing phosphorylated proteins/peptides derived from A431 cells. The tryptic peptides were analyzed by LC/MS and the identities and sites of phosphorylation revealed by database search. Several proteins bound by the anti-pY-coated beads are proteins with known regulatory function, e.g., proteasome activator, SH3-containing adaptor, and hu MCM2, topoisomerase I, etc. The identification of two or more peptides for some of these proteins and the absence of these proteins in the control beads suggest that the binding is antibody-dependent. A phosphopeptide derived from epidermal growth factor receptors (EGFR)phosphorylated at a tyrosine reside was identified. We've also identified and mapped the sites of phosphorylation of several peptides captured by Ga+3-NTA agarose. These approaches involve minimal sample manipulation and are desirable when dealing with limited amounts of samples.

S. Wu, P. Shieh, W. S. Hancock; ThermoFinnigan Corp., 355 River Oaks Parkway, San Jose, CA 95134-1991

Purpose: To obtain detailed carbohydrate and peptide structures for a glycosylated protein using a high throughput, automated procedure.

Method: LC-MSn</ on an ion trap using data dependent ion selection and dynamic exclusion.

Results: The majority of the protein sequence and glycosylation sites were identified in one MS/MS run using a small aliquot of a tryptic digest mixture. A second experiment was performed confirming the glycoform and linked peptide structures via data dependent MS2 through MS5.

Conclusions. A. The complicated peptide mixture was easily identified through the MS/MS fragment ion match in Turbosequest search software. B. The post-translational glycosylation sites were identified and the glyco-structures were clearly elucidated by MS from 2 to 5th on the fly. C. The overlooked and low-intensity ions (e.g. glycopeptides) can still be automatically picked up for MS/MS fragmentation by dynamic exclusion software on the fly. D. The assembly of Surveyor capillary LC coupling with ion-trap MSn (LCQ) for sample analysis and equipped with Turbosequest software for data analysis is automatic, robust, and easy to use.

H. Tran¹, A. Huhmer², S. Swedberg¹; ¹ThermoFinnigan, 355 River Oaks Parkway, San Jose, CA 95134, ²Thermo Finnigan, San Jose, CA

Calmodulin (CaM) is the major high affinity Ca2+ -binding protein in most eukaryotic cells that regulates a variety of cellular Ca2+ -dependent signaling pathways. CaM has an unusual amino acid composition with a high proportion of acidic residues (> 34%) and is particularly rich in methionine (Met) residues, accounting for more than 5% of all amino acid residues. It has been demonstrated that Met residues are the primary targets of oxidative changes in CaM in vivo and in vitro. We discuss experimental approaches for data dependent MSn analysis exploiting the unique capabilities of ion-trap MS for the identification and characterization of oxidative modifications in CaM. Different data-dependent acquisition methods are evaluated to maximize sequence coverage and sequence information for those peptide fragments with two possible oxidation sites. We also discuss search strategies for the automatic identification of those post-translational modifications by TURBOSequest.

L. K. Barbacci, J. M. Moeller, M. C. Harvey, J. G. Dapron, W. K. Kappel; Sigma-Aldrich Biotechnology, 2909 Laclede Ave, St. Louis, MO 63103

The analysis of protein phosphorylation sites is a major endeavor in the field of proteomics. Current techniques commonly employ protein digests with subsequent analysis by mass spectrometry (MS). Generally there is a need to enrich the phosphopeptides prior to MS analysis, often utilizing immobilized metal affinity capture (IMAC) matrices. A novel chelate was attached to both resin and 96-well plates followed by loading with iron (Fe⁺³). Tryptic digests of β-casein were used to evaluate the specificity of phosphopeptide capture. Enrichment of the phosphopeptides (75–90% purity) was observed using HPLC-MS. Using purified monophosphopeptide (MPP) from β-casein, capacity was determined as approximately 0.5 nmole per well for the plate and 1 μmole per mL for the resin. Capacity for the purified tetraphosphopeptide (TPP) was greater than the MPP in both formats. In the peptide mixtures, preferential binding of the TPP was observed. Various eluents compatible with MS analysis were evaluated. Significant differences in recovery efficiencies were noted. Optimal utilization of elution conditions is paramount in ensuring representative recovery of complex phosphopeptide mixtures.

L. Nuwaysir, T. Settineri, C. Hunter, B. Halpern, D. Schaeffer; Applied Biosystems, Foster City, CA

Mass spectrometry has become one of the primary techniques for identifying proteins and studying protein expression levels and protein function. Many software programs have been developed to identify proteins from mass spectrometric data. The software programs Pro ID and Pro ICAT use a patent-pending algorithm called InterrogatorTM for identifying proteins from uninterpreted MS/MS data. In addition to finding specific modifications, error and modification-tolerant searching is possible by specifying a generic "zone modification" mass to add or subtract to the calculated peptide molecular weight. The zone modification can be due to specific chemical modifications, amino acid substitutions (homologous peptides), truncations, non-specific or missed cleavages, or any other modification that results in the experimental mass differing from the expected database mass. The Interrogator search algorithm returns the modification mass and the region of the peptide where the modification is localized. This powerful feature will be demonstrated with examples of proteins that have been identified from post-translationally modified peptides, non-specifically cleaved peptides, and disulfide-bound peptides.

M. Scigelova¹, G. Woffendin², M. Ward³, H. Byers³, D. Hanger⁴; ¹Thermo Finnigan UK, Boundary way, Hemel Hempstead, HP2 7GE United Kingdom, ²Thermo Finnigan, Hemel Hempstead, United Kingdom, ³Proteome Sciences plc, De Crespigny Park, London, SE5 8AF United Kingdom, ⁴Kings College London, London, United Kingdom

Phosphorylation of proteins plays very important roles in regulation of metabolic processes. Neurofilament proteins are important structural features of the neuronal cytoskeleton. Abnormal phosphorylation of these proteins is associated with some neurodegenerative diseases.

The MS/MS spectra of peptides provide a wealth of information on the assignment of peptide sequence and identification of modified residues. Phosphorylated peptides usually exhibit a prominent loss of a phosphate group in their MS/MS spectra. A neutral loss of 49 amu (for doubly charged peptides) indicates a possible phosphopeptide candidate. An identification of the peptide sequence is then obtained with SEQUEST database search engine, and the exact location of the phosphorylated residue is confirmed.

We present a simple strategy enabling an unambiguous identification of the phosphorylated residue of a peptide analysed by LC/MS/MS in a complex peptide mixture.

G. Woffendin¹, M. Scigelova², E. Tarelli³, S. Pouria⁴, P. Corran⁵; ¹Thermo Finnigan, Hemel Hempstead, United Kingdom, ²Thermo Finnigan UK, Boundary way, Hemel Hempstead, HP2 7GE United Kingdom, ³St. George's Hospital Medical School, London, United Kingdom, ⁴Kings College Hospital, London, United Kingdom, ⁵London School of Hygiene and Tropical Diseases, London, United Kingdom

Glycosylation is a prominent post-translational modification (PTM) of proteins in higher organisms. The heterogeneity of glycan population, a multitude of combinations of sugar building blocks, and a relatively large size makes the study of glycopeptides a challenging task.

The use of MS/MS is essential in analyses of PTMs such as glycosylation. We have used a straightforward routine to identify glycosylated peptides in complex mixtures by automated capillary LC/MS on an ion trap by profiling specific losses of sugar moieties. Multiple sugar losses observed under optimised fragmentation conditions afforded important structural information about the glycan portion of the molecule.

J. D. Haley¹, A. Thelemann¹, H. Pan¹, D. Fenyo², T. Settineri³, K. Iwata¹, A. Bruskin¹; ¹OSI Pharmaceuticals Inc, 106 Charles Lindbergh Blvd, Uniondale, NY 11553-3649, ²Proteometrics LLC, New York, NY, ³Applied Biosystems, Foster City, CA

Enhanced activation of the epidermal growth factor receptor (EGFr) is frequently observed in human cancers and correlates with poor prognosis. Affinity chromatography/MS (MALDI and Qq-TOF) was used to define phosphotyrosine-containing protein complexes. 638 MALDI spectra from multiple experiments and separations were evaluated (RADARs; Proteometrics LLC) allowing tentative identification of 129 proteins based on matches common to both trypsin and GluC (20-30ppm). The predominant protein identified by all methods was EGFr. Peptides phosphorylated on P1 and P2 C-terminal tyrosines were observed. Proteins from a specific internalization/ degradation pathway were confirmed by Western transfer. 34,931 MS-MS spectra identified 1813 peptides using stringent selection criteria (Pro-ID; ABI-Sciex). 34 individual EGFr peptides were sequenced from multiple MS-MS scans. 81 of 129 MALDI-assigned proteins were identified by uLC-MS-MS using established cutoff criteria. Proteins of unknown function and novel complexes were detected. ICAT labeling is being used to measure protein changes associated with the blockade of EGFr kinase activity by the specific quinazoline inhibitor OSI-774 (Tarceva).

H. A. Remmer¹, N. Ambulos², L. F. Bonewald³, J. J. Dougherty², E. Eisenstein⁴, E. Fowler², J. Johnson², A. Khatri², N. Ritter², S. T. Weintraub²; ¹University of Michigan, ², ³University of Missouri, ⁴National Institute of Standards and Technology, Rockville, MD

The Peptide Standards Project, conducted by the Peptide Standards Project Committee (PSPC) of the ABRF, entails production and establishment of 3 synthetic peptides as registered and certified peptide reference standards by collaboration with the National Institute of Standards and Technology (NIST).

The committee’s mission is to provide active support and advice to NIST in conducting this project. Therefore the PSPC focuses on the following:

(1) Large scale synthesis the standard peptides in highest purity.

(2) Packaging of the peptides in small quantities.

(3) Analysis of peptide quality by the ABRF member laboratories in a collaborative study.

(4) Stability tests for a variety of physical and chemical conditions and for long-term storage and handling.

Results of synthesis optimization and the set-up for large scale synthesis will be presented and discussed as well as peptide design, quality control and a project overwiew.

The Peptide Standards Project is the first ABRF project funded by NIST. Collaboration with NIST provides additional recognition and visibility for the ABRF. Vice versa, the ABRF membership is a valuable resource of information, data and data validation for NIST. For the Peptide Standards Project, the collaborative analysis of the peptides by the ABRF membership will yield the required certificate of analysis.

J. P. Wiley¹, K. A. Hughes², R. J. Kaiser¹, G. Li¹, D. D. Lucas¹, K. P. Lund², C. L. Pershing¹; ¹Prolinx, Inc., 22322 20th Ave SE, Bothell, WA 98021, ²Prolinx Inc., Bothell, WA

Surface plasmon resonance is a powerful, sensitive and label-free methodology for the analysis of biomolecular interactions. However, the utility of this technology is limited by the cost, throughput, and complexity of available instrumentation and surface chemistries. Prolinx®, Inc. has developed a new SPR instrument, the Octave™ Molecular Interaction Analysis System, which will address these limitations and will enable SPR biosensors to become ubiquitous in life science research and drug discovery laboratories. This technology is the result of combining the Texas Instruments Spreeta® 2000 chip with Prolinx Versalinx™ Chemical Affinity Tools. The Octave incorporates eight independent sensors operating in parallel. The gold sensor surface has been modified to minimize nonspecific binding and utilizes Versalinx Chemical Affinity Tools to facilitate the immobilization of macromolecular ligands for binding studies. Samples are introduced to the sensors using an integrated liquid handling robot, enabling walk-away automated analysis. The versatility and ease of use of the Octave provide affordability and accessibility of molecular interaction analysis for both academic and industrial laboratories.

O. Beg; University of Maryland, 701 East Pratt St., Baltimore, MD 21202

Lipoprotein Lipase (LPL) is a key enzyme in Lipid Metabolism. Recent evidence suggests that LPL could play a role in the process of atherosclerosis by retaining lipoproteins (LP) at the surface of cells, which when taken up the cells contribute in the process of atherosclerosis. In view of these functions of LPL, a study was conducted by making use of synthetic peptides corresponding to potential heparin and LP binding sites to study the role of critical residues in LPL which bind its ligands. In contrary to the site directed mutagenesis results these studies map a binding site for both LP's and heparin to a specific region in LPL. These results also suggest that peptides corresponding to specific sequences in LPL are also responsible in inducing structural change(s) to accommodate an increase in binding its ligands. These preliminary findings suggest a possible mechanism of allosteric regulation in LPL. Data from these studies will be presented to delineate a different approach to study heparin and LP binding sites in LPL.

(This work was supported by a grant from American Heart Association-TN affiliate to OUB. Author acknowledges this support).

M. Raida, M. Schirle, T. Werner, B. Kuster; CellZome GmbH, Meyerhofstrasse 1, Heidelberg, n/a D-69117 Germany

Most cellular processes are maintained by functional multi-protein complexes rather than by single proteins. In a complex, proteins of different biological functions are assembled to exercise highly specific overall function. As proteins are used as drug targets, it is advantageous to gather information about their interaction partners as to provide more insight into the molecular environment in which drug targets operate. Intact protein complexes can be purified from cells by using the tandem affinity purification. By applying specific protocols, complexes from different subcellular compartments can be purified. From yeast, complexes containing up to 100 proteins have been purified. In general, proteins are first separated by 1D SDS-PAGE and subsequently identified by mass spectrometric methods.

Depending on the complexity, different mass spectrometric approaches have to be selected. While well-separated proteins from known small genomes can easily identified by MALDI-TOF MS instruments, more complex mixtures require LC-MS/MS approaches. The application of one- and multi-dimensional LC separations coupled to either quadrupole TOF or ion trap mass spectrometers will be discussed in detail.

D. Bintzler, S. Sherwani; University of Cincinnati, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0524

Human DNA profiling, also referred to as DNA fingerprinting, is a method of fragment analysis used for assigning a genetic identity to a human being. In the US, human DNA testing has been used as evidence in police forensics, other criminal cases and parentage issues since 1988. It has also been used to settle immigration cases, disputes involving purebred dogs and in animal conservation. Since these issues can be very sensitive, absolute accuracy is the foremost requirement for DNA profiling. The University of Cincinnati DNA Core Facility provides DNA profiling as a new service. DNA profiling uses 10 certified markers to amplify 10 specified areas of the human genome, including a marker to identify X and Y chromosomes. The product sizes range from 106 base pairs to 343 bases pairs. Various protocols in method design were tested to improve upon the accuracy. The Applied Biosystems DNA profiling allelic ladder with known base pair values provided a good standard to calculate error. It was determined that the gel percentage was a key factor. Since accuracy is an extremely important factor for any laboratory providing fragment analysis as a service, results from this study are reported here.

C. R. Kemper, B. Schulenberg, W. Leung, W. F. Patton; Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon 97402

Recombinant proteins are often linked to oligohistidine tags and purified by immobilized metal-ion affinity chromatography. Typically, oligohistidine-tagged proteins must be electroblotted and probed using anti-oligohistidine antibody/ secondary antibody-reporter enzyme conjugates or biotin-nitrilotriacetic acid/streptavidin-reporter enzyme conjugates, to verify fusion tag expression and monitor fusion protein purification. Two fluorophore- nitrilotriacetate conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, permit fluorescence detection of oligohistidine-tagged proteins directly in SDS-polyacrylamide gels. Pro-Q Sapphire 365 dye exhibits bright blue fluorescence when illuminated with a UV transilluminator while Pro-Q Sapphire 488 dye exhibits bright green fluorescence when illuminated with a 470-488 nm laser-based scanner. 25-65 ng of fusion protein is detectable using either stain. Gels may then be post-stained with the red-fluorescent SYPRO Ruby protein gel stain to reveal the total protein pattern.

B. S. Miller, B. Fryksdale, G. Ganshaw, P. T. Jedrzejewski, D. Wong, G. England, A. Gaertner; Genencor International Inc., 925 Page Mill Road, Palo Alto, CA 94304

Semi-automated 2D gel systems have been developed and are entering service in a number of industrial and academic laboratories. We describe the advantages and applications of such a system applied to an industrial enzyme project in a product based biotechnology company. We discuss the generation of high quality data that allowed protein identification using a fungal exoproteome as an example. We also discuss the limitations of the technology and illustrate our findings with examples from product composition analysis, whole cell and cell fractionation proteomics experiments.

Our conclusions are based on the development of reliable analytical methods using deglycosylation of the proteins that facilitates peptide assignments via MALDI-TOF mass spectrometry. Also described are analytical investigations on the "charge trains" seen in 2D gel electrophoresis.

G. L. Hansen¹, L. Stevens¹, F. Gharahdaghi², S. Kim¹, S. M. Mische¹, V. Papov¹; ¹Boehringer Ingelheim Pharmaceuticals, 175 Briar Ridge Rd., Ridgefield, CT 06877, ²AstraZeneca, Willington, DE

Two-dimensional gel electrophoresis (2-DE) is currently the favored method for the separation of proteins not only for the highly achievable resolution but also because 2-DE allows direct visual comparison of the changes in protein expression or modification between the proteomes under study. For many years proteins separated by 2-DE have generally been stained with either the well-known Coomassie blue (CB) or the more sensitive silver staining.

The increase in popularity of proteome analysis has fueled numerous efforts to develop "new" stains and protocols in an effort to maximize the limit of protein detection while maintaining compatibility with mass spectrometric analysis. Here, we compared some of the newer commercially available stains with the stains and dyes already in widespread use. Further, we examined a double stain of colloidal CB, followed by silver stain to determine whether an enhancement in stain sensitivity could be achieved above either stain alone.

Stains/dyes and methods were compared in terms of limit of detection, compatibility with mass spectrometry and ease of use. Band intensities in 1D gels were visually compared to determine the sensitivity of each stain. The number of proteins detected in 2DE gels and mass profiling were summarized. The results of these experiments allowed us to develop a coherent strategy for the visualization of proteins for proteome comparison without sacrificing analyte sensitivity in the mass spectrometric analysis of peptides which resulted from in-gel digests of selected protein spots.

K. J. Martin, B. Schulenberg, C. R. Kemper, W. F. Patton; Molecular Probes, Inc., 4849 Pitchford Ave., Eugene, OR 97402

For specific protein identification, researchers typically wrestle with the time-consuming task of running replicate gels; one gel being stained for total protein while the other is electroblotted and probed using standard immunoblotting methods. Ambiguity arises due to difficulty in aligning the gel and the electroblot. Simultaneous dichromatic detection, on one blot, eliminates the inherent uncertainty arising from running replicate gels. A two-color fluorescence detection method is described based upon covalently coupling BODIPY TR-X succinimidyl ester to electroblotted proteins, followed by detection of target proteins using ELF 39 phosphate in combination with alkaline-phosphatase-conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic method permits detection of low nanogram amounts of protein and allows unambiguous identification of target proteins relative to the entire protein profile on a single electroblot.

N. E. Caceres¹, T. R. Brockett¹, I. Jordon-Thaden², R. L. Cerny², G. Sarath¹; ¹University of Nebraska-Lincoln. Center for Biotechnology-Proteomics Core Facility, 19 th & Vine, Lincoln, NE 68588-0665, ²Mass Spectrometry Core Facility, Lincoln, NE

Our facility handles a broad variety of samples from diverse sources. This poses several challenges for providing effective separation and imaging services. Successful 2DE greatly relies on efficient extraction and solubilization of proteins. Our goal was to develop standard protocols to achieve the best separation and reproducibility for all of the samples. However, the complexity of the material specified the necessity for customized sample procedures. Proteins from bacterial crude extracts and purified protein fractions were recovered with 10 % TCA or precipitated with cold acetone if the samples were more diluted. Spot patterns were improved when extracts were further cleaned with phenol extraction. For protein preparations from plant tissues and fungal mycelia we used 10 % TCA in acetone containing 0.07 % β-mercaptoethanol. Proteins prepared by this protocol could be focused in absence of DTT. Our equilibration buffer contained 5 mM TBP. Under these conditions, streaking in the 2nd dimension was greatly minimized. Data obtained from our experiments will be presented. Our experience revealed that most samples required substantially different conditions for extraction as well as focusing.

A. cherkasskiy¹, L. lacy¹, J. J. Broadbent², J. loveless¹, K. Appasani²; ¹perkinelmer life sciences, 100 e. dedham, boston, ma 02118, ²PerkinElmer Life Sciences, Boston, Massachusetts

Total RNA was isolated from Jurkat and HL-60 cell lines. Separate labeling reactions were run using 100, 75, 25, or 10 ug of input total RNA using the MICROMAXTM Direct Labeling Kit. Same vs. same hybridizations were performed at all mass levels using Jurkat in both channels and scatter plots were examined. The above all masses showed differential expression within two-fold limits for greater than 99% of the genes. For the lowest levels of mass (10 ug), an overall weaker signals and an increased scatter was observed, indicating that the possibility of false positive data from the weakest signals. Differential expression patterns were studied using hybridizations comparing Jurkat to HL-60 expression. Again, lowest mass levels resulted in increased scatter on the lowest signal and also somewhat reduced expression ratios on highly expressed genes. We conclude that lowering mass levels of input RNA, especially to the 10 ug range may result in a loss of sensitivity, specificity and differential expression data.

F. J. Hilerio¹, D. T. Campogan¹, K. A. Hughes², D. Spicer¹, A. L. Springer², L. R. Booth¹; ¹Prolinx, Inc., 22322 20th Avenue SE, Bothell, Washington 98021, ²Prolinx Inc., Bothell, WA

Nucleic acid microarrays allow for the profiling of thousands of genes in a single experiment and have become an increasingly valuable tool in gene expression analysis. A typical microarray is composed of a 3 x 1 glass slide containing an array of nucleic acids to which fluorescently-labeled cDNAs are hybridized. Considerable time and expense can be spent in preparing RNA from which the labeled cDNA is derived. The removal of excess dyes from the reverse transcription reactions, while maintaining recovery of message, is essential to the accuracy of experimental results. Prolinx^®, Inc. has developed a new method for the removal of excess Cy3- and Cy5-dye labeled dNTPs from reactions using direct Cy-labeled nucleotide incorporation. The superparamagnetic particle-based technology is quick to use and affords minimal loss of product, making it ideal when sample size is limited. High cDNA recoveries also reduce the time and expense of multiple RNA preps when replicates are required and allow for the possibility of improved detection of rare message. The procedure can be rapidly optimized to eliminate excess dyes while maximizing product recovery, and can be automated.

K. Appasani¹, M. Tyler¹, A. cherkasskiy², J. J. Broadbent¹, L. Lacy¹; ¹PerkinElmer Life Sciences, 549 Albany Street, Boston, Massachusetts 02118, ²perkinelmer life sciences, 100 e. dedham, boston, ma 02118

RNA was isolated from HL-60 and Jurkat cell lines, and two masses (75 ug and 20 ug) of total RNA was used. The mRNA was isolated and converted to label cDNA. After labeling and hydrolysis using the Direct Labeling kit; both Cyanine-3 and Cyanine-5 reactions were purified and hybridized to MICROMAX 4800 cDNA arrays. Our results indicated in duplicate analyses where the input mRNA was 1.4 ug and 0.4 ug, we observed ratios of ratios (ROR) for differential genes to be 99% and 98.5% respectively. When we compared the mRNA (1.4 ug) and total RNA (75 ug) labeling in identical conditions, we found that ROR agreement for differential genes was 99%, where as, it was 96%, when we used mRNA (0.4 ug) and total RNA (20 ug). The top 200 differentially expressed genes were selected and grouped into different clusters. Such analysis, pathway identification and differential expression analysis will be described in detail. In conclusion, our results indicated that input of 20 ug total RNA or 0.4 ug mRNA is sufficient enough to perform microarray experiments, and the differential analysis is equivalent in both scenarios, except that image qualities were much better in the mRNA labeling.

J. Li, A. Viale; Memorial Sloan Kettering, New York, NY

Microarray technology has changed the way scientists study biological phenomena. Whether their goal is gene discovery or pathway analysis, scientists have access to different array platforms. The recent trend is to get more information from less tissue and to monitor gene expression profiling from biopsies. However, the amount of starting material is still an obstacle in many cases. The processing of samples for the Affymetrix genechip technology involves an in vitro transcription step during which biotinylated nucleotides are incorporated into cRNA. Several biotechnology companies have taken up the challenge of labeling small amounts of total RNA samples (between 100ng and 10μg ). This study is designed to evaluate the reliability of one labeling kit in particular, the Ambion MesAmp aRNA kit. This kit permits a small amount of total RNA sample (1μg with one amplification or 100 ng with two rounds of amplification) to be labeled in comparison to the 10 micrograms used in the Invitrogen cDNA synthesis kit. After hybridization on the U74Av2.0 array, fold changes between the three conditions are gener