M. Gunthorpe, S. Cai, F. Chehab;
UCSF Medical Center, San Francisco, CA, United States.

Few clinical laboratories test for b -thalassemia, an anemia which results from decreased or absent production of the b -globin polypeptide chain of hemoglobin. The molecular diagnosis of b -thalassemia is complex due to the vast number and types of characterized mutations. Protocols that rely on PCR amplification of the b -globin gene followed by hybridization to allele-specific probes, such as in the reverse-dot blot hybridization technique, require production of numerous primers and are limited to screening for the most common 30-40 mutations. In order to screen in clinical samples for all the mutations known to cause b -thalassemia, we devised a DNA sequencing strategy based on sequencing the entire b -globin gene (about 2 kb) with dye-terminator chemistry. This procedure involves the amplification of the gene first by generating two PCR products, and then determining their DNA sequence with a total of 8 primers. Characterized mutations and polymorphisms were rapidly compiled by an assembly software and tabulated for inclusion in a clinical report. The assay was validated by blindly sequencing patients with previously characterized mutations. In addition, this technique will uncover b -globin gene DNA polymorphisms, which are valuable for ruling out gene deletions when homozygosity for a mutation is found in a patient. Mutation analysis by DNA sequencing greatly simplifies the diagnosis of b -thalassemia disorders caused by subtle changes in the b -globin gene structure. However, this approach is refractory to the detection of the few rare deletions which encompass a large region of the b -globin gene cluster. This DNA sequencing strategy is currently being implemented in a clinical setting and is used as an adjunct to the determination of b -thalassemia mutations that are not identified by standard allele-specific probe assays.

D. Bond, A. Swei, K. Lee, R. Nutter, C. Swartz, A. Yang, K. Louie, Y. Lei, J. Lee, K. Perry, J. Teare, E. Sun, J. Vuong, C. Heiner, J. Cassel;
Applied Biosystems, Foster City, CA, United States.

Fluorescent dideoxy sequencing has advanced greatly since the early days of automated DNA sequencing about 15 years ago. Cycle sequencing using thermally stable DNA polymerase enzymes provided a robust method of sequencing plasmids over a much wider and lower range of concentrations than was previously possible. Fluorescent dideoxy terminators reduced the number of steps, equipment and supplies required to perform sequencing reactions, and BigDye terminators provided several fold additional sensitivity. These advances greatly facilitated the rapid sequencing of the human genome. Researchers today are challenged to finish the difficult-to-sequence areas of the human genome and other organisms, and to obtain highly accurate sequences of individuals in studying polymorphisms. We have recently released two new sequencing kits that will help to address these challenges. Here we present BigDye^® Terminators v3.1 and 1.1 Cycle Sequencing kits. We will illustrate improvements with both our work as well as the results of our conformation test sites.

N. Panayotova, E. C. Almira, W. G. Farmerie;
University of Florida Biotechnology Program, Gainesville, FL, United States.

High-throughput capillary array DNA sequencing instruments have been in service since their introduction in the late 1990s, yet few improvements have been made to standard operating conditions and protocols. Many small facilities, especially those with a single sequencer, continue using the same operating conditions they used initially. In slab-gel sequencing, most common consumables, such as gel matrices, running buffers, and glass plates, may come from various sources. However, capillary sequencer consumables are mainly proprietary, making it difficult for core facilities to find potential alternatives. Still, in an effort to improve services while keeping the costs to a minimum, many budget-conscious core facilities hope to discover compatible but cost-effective consumables. We recently tried and tested three alternative running buffers (A, B and D) in our MegaBACE 1000 using standard plasmids and production DNA templates generated via the Templiphi rolling circle amplification process. Compared to the current running buffer (C), buffer D caused a moderate but significant increase in the number of total phred Q20 bases per plate. Data analysis revealed an increase in sequence readlength rather than passing rate. Results with buffers A and B were either similar or inferior to those of C. A much more consistently improved sequence data is now being generated after switching to buffer D.

D. A. Bintzler, S. I. Sherwani, Y. Song, M. Jordan, J. Ackerman;
DNA Core Facility, University of Cincinnati, Cincinnati, OH, United States.

In order to use a fine quality of DNA for various molecular biology techniques like sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (random prime labeling), etc., quantitation of DNA is extremely important to have the defined template concentration. Failure to produce results from the above techniques can sometimes be attributed to an incorrect estimate of the DNA template used.
A resource facility that provides automated DNA sequencing as a service can apply several techniques to measure the quality and the quantity of DNA templates submitted for sequencing. Scanning a template using a spectrophotometer is one technique used in various resource facilities to determine contaminants such as phenol and salt and can be applied quickly. However, this technique is limited to locating, but not quantifying, the amount of contaminant in the template. We have also found that it is quite difficult to quantify smaller PCR fragments due to the sensitivity of the spectrophotometer. Several commercial suppliers also provide quick strips that can quantify PCR products with a certain degree of accuracy. However, the process can be labor-intensive and expensive. Therefore, we decided to further develop two known techniques that can be used in a resource facility that can be less labor-intensive and can improve sensitivity. Further, we have found pre-testing a sample to be a small source of extra income, while it saves clients the cost of sequencing contaminated templates by reducing sequence failures.
Further research is warranted so that resource facilities could introduce such highly sensitive yet simple services at a minimum cost to the customer.

A. Greco, I. Y. Hlede, R. T. Pon;
University of Calgary, Calgary, AB, Canada.

University core service laboratories constantly attempt to provide the best possible results for the wide variety of DNA templates they receive. DNA templates that give poor or limited sequencing results are always a major problem for our clients and us. Our facility performed a series of reactions with the recently released new formulation of BigDye Terminator sequencing kit, version 3.1 from Applied Biosystems which is expected to produce a marked improvement in sequencing difficult templates. We have examined a variety of GC rich, AT rich, and other templates, which sequenced poorly or not at all with the BigDye v.3.0 kit. Results will be presented comparing how the v.3.0 and v.3.1 sequencing kits perform on the ABI 377 DNA sequencer.

B. T. Coullahan¹, K. D. Eluwa², O. Zimerman²;
¹Genomic Analysis and Technology Core, University of Arizona, Tucson, AZ, United States, ²Edge BioSystems, Inc., Gaithersburg, MD, United States.

The Genomic Analysis and Technology Core at the University of Arizona has evaluated a new magnetic dye terminator removal technology on a Biomek FX workstation in conjunction with an ABI 3730XL DNA Analyzer. In this method, sequencing reaction products are adsorbed to magnetic particles, washed with alcohol, and eluted in water. The system is adaptable for single tube as well as 96 and 384 well plate configurations. In a 96 well plate configuration, processing times were typically less than 20 minutes. Sequencing reaction volumes of 5 to 20 μL were evaluated, with DNA template ranging from 25 to 500 ng, and BigDye™ premix volume ranging from 0.125 μL to 8 μL. Sequence quality is excellent throughout much of the range of template, volume and premix described, with long read lengths. This technology offers a convenient and inexpensive alternative to costly and/or unautomatable purification methods and provides substantially better reproducibility than ethanol precipitation.

T. Mamone;
Amersham Biosciences, Piscataway, NJ, United States.

The TempliPhi™ DNA sequencing template amplification kit relies on Phi29 DNA polymerase to drive rolling circle amplification (RCA) of circular input DNA. Several micrograms of template DNA are generated from picogram amounts of input. The method requires only two steps to generate sequence quality templates, potentially bypassing both the requirement for liquid culture growth and the conventional steps for preparing plasmid and phage DNA for use as templates in sequencing applications.
This method is of enormous use to sequencing facilities in several aspects. Time is saved by omitting traditional multistep plasmid preps. Significant cost savings are achieved by reduced plasticware. Space devoted to bacterial growth and prep can be put to better use. Most significantly, success rates can be near 100% for plasmids, regardless of the source, due to the normalization of template amount. Supporting data will be shown, in addition to difficult template sequencing improvements.
An extension of this method in development, called GenomiPhi™, is appropriate for amplifying large constructs, bacterial genomes, and human chromosomal DNA.

M. J. Reagin, D. M. Meyers;
Amersham Biosciences, Piscataway, NJ, United States.

The majority of failures in core lab sequencing facilities are due to either poor quality of customer plasmid DNA prep or the miscalculation of DNA concentration. For core labs that offer resequencing of failed reactions, not only is the cost passed onto the customer, but also valuable time is spent by lab personnel to reproduce data that might once again fail. TempliPhi DNA amplification is a process that employs rolling circle amplification (RCA) and Phi29 DNA polymerase to produce microgram amounts of circular template from picograms of DNA in as little as 6 hours. We will show that TempliPhi can produce enough template from an empty tube that once held plasmid DNA, to generate a DNA sequence of at least 500 bp. We will also show sequence produced on a capillary electrophoresis instrument that failed due to an insufficient amount of starting plasmid template. By adding small aliquots of the failed sequencing reactions to TempliPhi, improved sequence quality and longer readlengths upon resequencing are obtained. Carryover primer from the failed sequencing reaction does not interfere with RCA and the rapid amplification by the Phi29 DNA polymerase provides enough template for a successful sequence by the next day with minimal manipulation.

E. J. Thompson, A. Anderson, R. Pershad;
The University of Texas MD Anderson Cancer Center, Houston, TX, United States.

At The University of Texas M. D. Anderson Cancer center, mouse models are frequently used to help identify genes related to cancer. The ability to genotype mice in high throughput format has been mostly limited to manual PCR. This process is time consuming and laborious. Separate pcr assays must be assembled and multiple thermocyclers are required to accommodate the unique annealing temperatures of different primer sets. In addition a Southern analysis must be performed to identify copy number.
The DAC at M. D. Anderson has developed a method to rapidly genotype mice using Taqman hydrolysis probes in 5' exonuclease based real-time polymerase chain reaction assays. This method allows mice to be rapidly screened in 96 well format. The key advantages of this technology are that multiple assays can be designed to work under the same conditions and that assays can be multiplexed.
Here we show a method to differentiate wild type, heterozygote and null mice using Taqman Assays.

M. A. Robertson, R. Scholl, M. Klein, R. M. Cawthon;
University of Utah, Salt Lake City, UT, United States.

Single nucleotide polymorphisms (SNPs) are abundant in the human genome and appear at a frequency of about 1 in every 500 to 1000 bases. SNPs can be used as genetic markers for genome scans in pedigree-based linkage analysis of families, in population studies where the haplotype distributions and linkage disequilibria can be used to map genes by association methods, and in case control studies to identify functional SNPs associated with a particular phenotype. Many more SNPs than microsatellites are required for these kinds of large studies so the methodology used has to be as automated and as cost effective as possible. This poster will compare two different methods of SNP genotyping using the 7900HT instrument as a detection platform. One method uses allele specific-PCR and melting curve analysis with Sybr Green I dye to distinguish alleles. Another method makes use of the Taqman assay with fluorescent primers and probes from Applied Biosystems. Both methods will be compared with a "mini-sequencing method" using the SnapShot™ kit from Applied Biosystems. This poster will address the ease of use, the robustness of the technology, the quality of the results and the ability for automation for each of the methods.

A. Khvorova;
Dharmacon Research, Lafayette, CO, United States.

RNA-mediated interference (RNAi) is an increasingly important method for analyzing gene function. Chemically synthesized double-stranded, short interfering RNA (siRNA), can effectively bypass the mammalian interferon response and sequence-specifically silence gene expression. Some of the factors that greatly affect the success of RNAi experiments are the quality of synthetic RNA, rapid access to candidate siRNA duplexes and the parameters implemented in the design of the duplexes.
2’-ACE RNA technology allows for rapid, dependable and scalable synthesis of any sequence regardless of nucleotide content. In addition, the technology provides a stable, well-behaved intermediate that is readily deprotected in aqueous buffer. The speed and versatility of 2’-ACE chemistry coupled with laboratory automation facilitates high-throughput siRNA applications for genome wide projects.
By analysis of a large number of functional and nonfunctional siRNAs and coupling the information with a systematic high throughput screening approach we developed a method allowing identification of highly active siRNA’s. Empirical testing of the method demonstrated that a >50% reduction in gene expression occurred with 99.97% of the selected sequences. Furthermore, in 97% of our experiments, rationally designed gene silencing reagents delivered >80% reduction in gene expression.
The ability to knock-out gene function via siRNA-mediated methods represents an exciting and valuable tool for gene functional analysis that will accelerate critical investigations across a broad range of biomedical and biological research.

M. Swope Willis, T. Fox;
Vertex Pharmaceuticals, Cambridge, MA, United States.

Chemogenomics, a driving force of drug-discovery research at Vertex Pharmaceuticals, is a parallel process allowing the evaluation of a wide variety of targets within a single gene family. The protein requirements to drive this process are substantial in their breadth and number. We have optimized our protein production process by modifying or automating several steps from expression to purification. However, when the expression of soluble protein is not possible, refolding of the inclusion bodies may be required to obtain functionally active protein. Refolding proteins is often a difficult and highly unpredictable process. Published conditions resulting in the successful refolding of one protein often tend to be protein-specific and are not always applicable to other proteins. Using semi-automated dilution equipment, we have developed a system for evaluating the effects of varying pH, osmolytes, reducing and thiol shuffling reagents, and chaperones, on protein refolding. Applying this method to multiple proteins representing five gene families, we have successfully refolded members of each family. This rapid dilution method has enabled the systematic screening of an expanded number of refolding conditions. We have identified trends in the data correlating the key additives with various proteins being refolded.

M. Kuschel¹, P. Barthmaier², E. Chernokalskaya³, E. Benson³;
¹Agilent Technologies, Waldbronn, Germany, ²Agilent Technologies, Palo Alto, CA, United States, ³Millipore Corporation, Danvers, MA, United States.

The Agilent 2100 bioanalyzer was the first commercially available microfluidic device for the molecular biology lab. Developed by Agilent Technologies in collaboration with Caliper Technologies the bioanalyzer can be used for the automated analysis of proteins, nucleic acids and cells. As with all microfluidic devices smaller sample sizes and faster run times are achieved on the bioanalyzer, compared to SDS-PAGE. With its speed and low sample consumption the Agilent 2100 bioanalyzer is the ideal instrument to use in conjunction with the Millipore Zip Tip_Ni. To aid in the purification of His tagged proteins Millipore has developed Zip Tip_Ni. The affinity interaction between Ni⁺⁺ ions and histidine is well—known. Nickel ion chelated chromatography resins have proven to be effective in capturing recombinant proteins that have 4 — 8X histidines fused to the N or C terminus of a protein. The His-tag /Ni⁺⁺ is becoming increasingly important in proteomics for expressing/isolating low abundant proteins and generating sufficient quantities of proteins for interaction studies and structure analysis. We report on the use of a 10 μl pipette tip containing ca. a 0.5 μl of Ni⁺⁺ chelated resin immobilized at the distal end (ZipTip_Ni). This device was effective in isolating 50 — 100 ng of native or denatured His-tagged protein from ca. 10 to 50 μl of cleared cell lysate in about 5 minutes. The pipette tip format offers a convenient sized tool for the rapid isolation of proteins that can subsequently be characterized on the Agilent 2100 bioanalyzer all under 1 hour.

A. T. Yeung¹, C. A. Oleykowski¹, A. Godwin¹, S. Henikoff², E. Sokurenko³, E. Nicolas¹;
¹Fox Chase Cancer Center, Philadelphia, PA, United States, ²Fred Hutchison Cancer Research Center, Seattle, WA, United States, ³University of Washington, Seattle, WA, United States.

CEL I, a nuclease from celery, has high specificity for DNA mismatch base-substitutions, insertions and deletions. It has been used to develop a robust mutation detection assay that is easy to automate. We have demonstrated its utility in the screening of unknown mutations and polymorphisms of BRCA1, BRCA2, ARSC, MED1, and TDG genes, of hundreds of individuals. Multiple unknown mutations in one PCR fragment can be simultaneously identified. This fragment analysis assay has been adopted for high throughput screening of EMS induced mutations in several plant genomes (Tilling procedure, over 600 Arabidopsis mutants have been obtained) to facilitate plant genetics studies. Similar approach is ongoing for zebra fish and mice in user labs. In contrast to knockouts that are less informative, Tilling produces a panel of mutants for investigating the multiple domains and phenotypes of a protein. CEL I mismatch endonuclease is also used for the detection of mutations in large genomic regions of microorganisms (Giraff procedure). Current effort is aimed at applying this assay for the genomic scanning of mutations in mice, the detection of mutations in pathogens such as Bacillus anthracis, and early cancer detection. The enzyme can be obtained through Transgenomic Inc. by sending an email to AT_Yeung@fccc.edu.
http://web-apps.fccc.edu/fccc/yeung/index.html

A. Swei, C. Kosman, P. Baybayan, S. Bay, S. Chang, S. Dodgen, L. Esfahani, S. Fang, T. Hatch, J. Labrenz, J. Pone, R. Sefanov, P. Suri, D. Xu, K. Yung;
Applied Biosystems, Foster City, CA, United States.

The Applied Biosystems 3730 DNA Analyzer and the accompanying control and analysis software packages are specifically designed to increase throughput and efficiency while reducing the data entry and processing complexity of diverse DNA projects. To streamline sample input and extraction, a new Data Collection Software architecture is implemented which automatically tracks and stores plate records, run folders, and analysis parameters within a searchable database. Via an auto-analysis function the sample files are processed using one of several application-specific Applied Biosystems software packages: Sequencing Analysis Software v5.0 contains an improved base calling algorithm that provides per-base Quality Value assignment and sample trimming via these Quality Value scores, along with automatic length of read estimation and several output file formats offering compatibility with various downstream post-analysis programs; SeqScape™ v2.0 Software provides integrated base calling, trimming, sequence assembly and alignment for fast and accurate sequence comparisons and variant identification; GeneMapper™ v3.0 Software offers automated peak detection and allele calling with process quality values to simplify the confirmation and review process for microsatellite analysis. With the introduction of the Applied Biosystems 3730 DNA Analyzer, the data input and analysis pathways have been simplified in order to increase the sample processing efficiency of diverse laboratories.

R. J. Chalkley¹, L. Huang¹, K. C. Hansen¹, N. P. Allen², M. Rexach², A. L. Burlingame¹;
¹UCSF, San Francisco, CA, United States, ²Stanford University, Stanford, CA, United States.

This work presents a detailed analysis of a complex mixture of proteins using a selection of multi-dimensional separation strategies. Our group has previously studied the protein-protein interactions of yeast nuclear pore complex proteins using 1D SDS-PAGE followed by mass spectrometric analysis of discrete bands observed. This work has now been extended to study changes in interactions during the cell cycle.
Isolated protein complexes have been characterized using the same 1D SDS-PAGE followed by mass spectrometry approach employed before, but also have been analyzed using multi-dimensional liquid chromatography followed by mass spectrometry. Following strong cation exchange as a first dimension of separation samples were analyzed both by LC-MALDI-MS (TOF-TOF) and by LC-ESI-MS (QSTAR). The new cleavable ICAT technology (Applied Biosystems) was also employed to quantify changes in protein ratios.
The results show a substantial overlap between the proteins identified with the different approaches, but each approach provides additional data. LC-ESI-MS provided more comprehensive information on the sample content, but the off-line analysis of sample by LC-MALDI-MS allowed a more focused analysis of those peptides which are of particular interest. The 1D gel provides a clearer representation of dramatic changes in protein levels between samples. However, ICAT technology is better for studying subtle changes, and does not suffer from the potential complication of a protein that is changing in concentration co-migrating with another protein.
In conclusion, for comprehensive analysis of a complex mixture a combination of approaches is most informative.
This work was supported by NIH grants NCCR 01614 and RR14606.

K. G. Huwiler, D. F. Mosher, M. M. Vestling;
University of Wisconsin, Madison, WI, United States.

While establishing the location of the disulfide bonds in thrombospondin type 1 repeats (1), using partial proteolysis, high performance liquid chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and reduction of disulfide bonds, we found that the choice of matrix can significantly enhance the appearance of cystine- and cysteine-containing peptides. The ready in-source-decay (ISD) of disulfides using 2,5-dihydroxybenzoic acid (dhb) and a-cyano-4-hydroxycinnamic acid (CHCA) as matrices is well known (2-6). The appearance of ions corresponding to the thiols making up the disulfide in the spectrum of a disulfide is often annoying and complicates spectral interpretation. In our hands, the cleanest spectra of disulfides were obtained using mixtures of CHCA or dhb and 2-(4-hydroxyphenylazo)benzoic acid (HABA). Experiments using different mixtures and methods of mixing points up the need to consider the role of radicals and electrons in the MALDI process. In the presence of HABA, a dramatic reduction in the intensity [M].+ with respect to [MH]+ is seen for CHCA. With our conditions, disulfide containing peptides and recombinant proteins can now be easily characterized using MALDI-TOF-MS.
(1) K. G. Huwiler, M. M. Vestling, D. S. Annis, D. F. Mosher, Biochemistry, in press.
(2) S. D. Patterson, V. Katta, Anal. Chem. 66: 3727-3732 (1994).
(3) D. L. Crimmins, M. Saylor, J. Rush, R. S. Thoma, Anal. Biochem. 226: 355-361 (1995).
(4) M. D. Jones, S. D. Patterson, H. S. Lu, Anal. Chem. 70: 136-143 (1998).
(5) V. Katta, D. T. Chow, M. F. Rohde, Anal. Chem. 70: 4410-4416 (1998).
(6) V. Schnaible, S. Wefing, A. Resemann, D. Suckau, A. Bucker, S. Wolf-Kummeth, D. Hoffmann, Anal. Chem. 74: 4980-4988 (2002).

O. Chertov, T. Boronina, V. M. Hoang, T. P. Conrads, T. D. Veenstra, R. J. Fisher;
SAIC Frederick, NCI Frederick, Frederick, MD, United States.

Most reliable methods of protein identification by mass spectrometry require the generation of peptides using highly specific proteases. The confidence of protein identification improves with the increase of the number of analyzed protease-specific peptides. To generate the maximum number of peptides, the disulfide bonds should be disrupted to ensure a complete enzymatic digestion. The most popular method of disulfide bond cleavage is the reduction of proteins using thiols with subsequent alkylation of the free sulfhydryl groups. To maintain protein solubility high concentrations of chaotropic agents are used, however, they must be removed prior to analysis. These steps can lead to losses of analyte. Another method for disulfide bond cleavage is based on oxidation of proteins by performic acid. Cysteine and cystine are oxidized to cysteic acid, methionine to sulfone, tryptophan is transformed into multiple forms including N-formylkynureine. The phenolic group of tyrosine and hydroxyl groups of serine and threonine can also be oxidized and other residues could be formylated.
We investigated if this method could be modified to reduce undesirable modifications, yet still be sufficiently effective for disulfide bond cleavage yielding well-defined oxidized derivatives of Cys, Met and Trp. We found that the treatment of different proteins with 0.3 % hydrogen peroxide in concentrated formic acid for 15 min at 4° C cleaves intramolecular disulfide bonds, while converting cysteine and cystine into cysteic acid, Met into Met sulfone, and Trp is oxidized to Trp-2O.
We have utilized this method as a quick and efficient preparatory step before digestion of proteins for mass spectrometric identification. Contract NO1-CO-12400.

T. Lee¹, K. A. Resing¹, N. G. Ahn^1,2;
¹University of Colorado at Boulder, Boulder, CO, United States, ²Howard Hughes Medical Institute, Boulder, CO, United States.

Signaling pathways involving protein kinases are critical for many aspects of cell function. One regulatory mechanism for these pathways involves enzyme docking interactions in protein complexes. Recent studies identified two potential docking domains in ELK1 that target unknown docking sites on extracellular signal regulated kinase (ERK2). Mutations of the ELK1 docking domain increases the Km of ERK2, suggesting that docking interactions regulate catalysis. In order to identify the docking site on ERK2 and gain insight into how catalysis is activated, we carried out hydrogen exchange mass spectrometry (HX-MS), where the protein is incubated in D₂O for various times to measure the deuterium exchange at the back-bone amides. HX reflects conformational flexibility in proteins, as well as changes in solvent protection upon ligand binding. Experiments were carried out with activated ERK2 (ppERK) and two peptides with sequences derived from the two candidate docking domains of ELK1 (D-domain and FXFP-motif). Measurements were made with a QSTAR® (API), using LC/MS to resolve peptides; the high resolution and signal sensitivity of this system improved data quality. We also implemented a Visual Basic program which determines the weighted mass average for each peptide at each time point; this information was then fit to a three exponential model using DataFit (Oakdale Engineering). HX-MS data show these peptides each bind in distinct regions of ppERK, and both peptides altered the exchange rates near the active site and on the opposite side of ppERK. We propose that conformational mobility changes in the hinge and near the activation lip induced by docking interactions are important for ERK kinetic mechanism.

D. Chelius, C. Shieh;
ThermoFinnigan corp., San Jose, CA, United States.

The detection limits for peptide detection by mass spectrometery has improved significantly. This is important for proteomics research. Higher sensitivity allows the detection of low abundance proteins. Nano-electropray coupled with mass spectrometry is the method of choice for maximum sensitivity and separation of peptides. Peptides are separated on a reversed-phase column (75 μm ID), which is placed directly in front of the mass spectrometer, limiting peak dispersion after the column. In this paper, we describe a method that allows fast loading and highly efficient separation of peptide fragments. Peptide fragments are loaded on a peptide trap at high flow rate. After loading, the trap is switched to a low flow, reversed phase gradient with a C18 packed tip and ion trap mass spectrometer. Peptides from a bovine serum albumin digest can be detected at 500 amol. This method allows fast loading and high sensitivity detection of peptide fragments.

F. A. Abdi, S. Bhardwaj, S. Hattan, M. Lin;
Applied Biosystems Inc., Framingham, MA, United States.

Global analysis of cellular proteomics recently gained ample interest of many researchers. However, conventional methodologies for protein separation and identification are still not comprehensive enough as low abundant proteins and membrane proteins are less represented and more difficult to detect. In addition, the speed, accuracy and sensitivity at which such analyses are carried out play important role in such studies. To address this, multidimensional liquid chromatography interfaced with MS has been introduced as new technology to generate more comprehensive data. Applied Biosystems’ 4700 Proteomic Analyzer with TOF/TOF® optics was developed to provide high speed and sensitivity associated with MALDI with the ability to analyze selected ions by high-energy fragmentation. We demonstrate a mechanism enabling LC MALDI MSMS by spotting eluent from LC separation onto MALDI plates followed by analysis on ABI’s 4700 Proteomic Analyzer.A protein mixture from a living organism, obtained from the Swiss Proteomics Society, was used as a collaborative study to independently evaluate members’ abilities in the field of protein identification from complex mixtures by MS. Sample was separated using one-hour gradient on LC Packings Ultimate micro-capillary HPLC system interfaced with LC packings Probot and deposited on a single 576 spot MALDI plate. MS and MSMS analyses of each spot were carried out using ABI’s 4700 Proteomic Analyzer. Protein identifications were accomplished using ABIs’ GPS Explorer software in concert with Mascot database search engine. Sample origin was found to be Snake Venom as more than 25 proteins were identified belonging to this origin. Additional protein families were identified by database search and De Novo sequencing. This study illustrates the advantage of ABI’s 4700 Proteomic Analyzer and its versatility in LC/MALDI MS/MS studies.

C. Van Pelt;
Advion BioSciences, Inc., Ithaca, NY, United States.

Detection of non-covalent interactions by electrospray ionization mass spectrometry (ESI/MS) provides important information on molecular mass and binding stoichiometry of protein-ligand complexes. Conventional methods employ pulled-capillary nanoelectrospray; a tedious and time-consuming technique that requires a highly skilled user. The NanoMateä 100 and ESI Chipä, an automated nanoelectrospray system, have been developed to improve the efficiency and quality of such ESI/MS methods. Advantages of the system include low sample consumption, conservation of sample not consumed in the analysis, one-time spray optimization, enhanced spray stability, and no carryover.
The system aspirates samples from a 96-well microtiter plate using disposable, conductive pipette tips, and then delivers samples to the inlet of the ESI Chip, a microarray of 100 ESI nozzles microfabricated in silicon. A pressure seal is formed around a through-chip channel at one nozzle and nanoelectrospray is initiated by applying a spray voltage and psi pressure to the sample in the pipette tip.
A non-covalent interaction experiment for the binding of cytidine 2'-monophosphate to ribonuclease A was performed to demonstrate the capability of using the NanoMate system to detect non-covalent interactions. Ribonuclease A was titrated with cytidine 2'-monophosphate in 10 mM ammonium acetate, pH6.8. Scatchard plot was used to determine the dissociation constants. The result is in good agreement with the previous report. The same approach is used to study the dissociation constants for an endocelluase system. An inactive mutant of endoglucanse catalytic domain from the T. fusca bacterium was titrated with ligands cellotetrose and cellopentose. This is the first report using an ESI/MS approach to study the interaction of cellulase and its oligosaccharides. The results demonstrate that the system can be used to rapidly screen potential drug candidates in discovery programs.

U. Schweiger-Hufnagel¹, M. Lubeck¹, D. Suckau¹, P. Kovalski², C. Baessmann¹;
¹Bruker Daltonik GmbH, Bremen, Germany, ²Bruker Daltonics Inc., Billerica, MA, United States.

In proteomic research de-novo sequencing of peptides becomes a central issue when database search fails. This might happen due to post-translational modifications, sequence errors in the database or protein sequences missing in the database. For those cases a new de-novo sequencing was integrated in proteome analysis software.
Proteins from 2-D gels from various organisms were enzymatically digested, and MS- and MS(n)-spectra were acquired on an electrospray ion trap mass spectrometer and on a MALDI TOF/TOF mass spectrometer. The software used the automatically created peaklists to generate peptide sequence proposals, considering all provided hints including possible modification. The resulting peptides sequences were scored against the experimental spectrum or used for homology searches.
The presented results include the the recognition and localization of modifications, e.g. the double oxidation of tryptophane. Homology searches based on the obtained de-novo sequences were applied to detect errors in the sequence database and to identify proteins whose sequence differed from the one contained in the database.

C. Sauber;
Agilent Technologies, Waldbronn, Germany.

Oligonucleotides (ONs) are widely used as primers for polymer chain reactions (PCR) and in antisense therapy. Antisense ONs can provide useful levels of gene-specific inhibition and have, therefore, become important functional genomics tools. Significant ON-mediated inhibition has been demonstrated. ONs are analyzed with various analytical techniques including gel electrophoresis, capillary electrophoresis, MALDI/TOF and HPLC.
All these techniques could be used either for qualitative or quantitative analysis and do not allow a simultaneous confirmation and quantification of ONs. Only the on-line coupling of a separation step with mass spectrometric detection allows a rapid, sensitive and fully automated identification and quantification of ONs. LC separation is preferably used over capillary electrophoresis for this application, as LC allows using more MS compatible buffer and larger injection volumes could be used. However the sensitive analysis of oligonuclotides with LC/MS needs a carefully developed method. The hydrophilic nature of these analytes allows a gradient elution from reversed-phase columns at low organic content only, while mass spectrometric detection with electrospray requires a high pH and organic content for good ionization efficiency.
We describe a methodology based on ion-pair reversed-phase capillary LC/Ion Trap mass spectrometry for the low femtomole quantitation of ONs in biological matrices.
The method for the sensitive quantitation of oligonucleotides in biological matrices shows a limit of quantitation of 30 fmol injected. Retention on capillary LC 300 um reversed-phase columns could be achieved by using a gradient of 5 mM triethylamine acetate, pH 7 and acetonitrile, starting at 1 % organic. For best MS sensitivity 5 mM triethylamine/10 mM imidazole in 90% acetonitrile /10 % water was added post-column to further suppress adduct ion formation and aid the desolvation process with a higher content of organic modifier.

X. Guo, C. Liu, L. M. Fell, T. Covey;
Applied Biosystems / MDS Sciex, Concord, ON, Canada.

The combination methods of mass spectrometry (MS) and nanoflow chromatography have become the method of choice for the characterization of low level of proteins in biological samples, an example of such tryptic digest crude cellular mixtures. In proteomics, not only is the task of protein identification important but increasingly research has focused on the study of the biological function and specificity of proteins, such as post-translational modifications. The complete LC gradient has proven to be an essential for the better coverage of protein due to the wide distribution of degree of hydrophobicity of peptides in protein digests. Therefore, the robustness and reliability of sprayer technology has come to be an issue for convenience and throughput when performing these analyses. In this poster, we present the results of a nanoLC interface which proves sensitive and robust on a QqTOF mass spectrometer. A number of nano-LC/MS experiments of standard proteins digests are used to evaluate the interface in both positive and negative scan modes.

S. Bhardwaj, F. Abdi, S. Hattan, M. Lin;
Applied Biosystems, Inc., Framingham, MA, United States.

Proteomics has developed as the principal technology in the drug discovery area. Mass Spectrometry has become the technology of choice for analysis of biological samples from a variety of sources. One major challenge in this field is analysis of low abundance proteins from complex mixtures. As demonstrated in this study, the recently developed ABI 4700 Proteomics Analyzer with TOF/TOF® optics can overcome this challenge. It provided high sensitivity associated with MALDI and has the capacity to follow the acquisition of MALDI-TOF mass spectrum with automated tandem time of flight fragmentation and analysis of selected ions. Even though this technology seems to be an ideal alternative to traditional LC-MS/MS, LC separation is still needed for MALDI based mass spectrometry. In this study, by comparing protein identifications from sample spots containing un-separated mixtures, we demonstrated that LC separation is essential to overcome ion suppression effect on mass spectrometers. It allows in depth study of Proteomics by identifying low abundant proteins. It also largely increases the confidence of protein ID from relatively simple mixtures, such as those from 1D gels.
Protein digest mixtures that contain different number and amount of proteins were analyzed using ABI 4700 Proteomics analyzer. Comparing the MS/MS analysis from single sample spots that contain undigested protein mixtures, LC-MALDI MSMS technique not only identified more proteins, it also positively identified low abundant proteins presented at different dynamic range in each mixture. Using this approach, more peptides were also identified for each protein and therefore improved the confidence of protein identifications by providing higher sequence coverage. Protein digests from an E. Coli 1-D gel sample were also analyzed by LC-MALDI MSMS technique.

D. Suckau¹, S. Hahner¹, P. Hufnagel¹, A. Resemann¹, J. P. Kowalski², A. Holle¹;
¹Bruker Daltonik GmbH, Bremen, Germany, ²Bruker Daltonics Inc., Billerica, MA, United States.

A novel TOF/TOF design has been developed in which a potential lift followed by fragment ion post acceleration (i.e., the source of a 2^nd TOF) is the central functional element. The technique is dubbed LIFT-TOF/TOF. Besides the capability of MS on intact proteins or protein mass fingerprints, the instrument offers three MS/MS modes of operation: 1. LID (laser-induced decomposition) using the metastable ions generated by the HCCA matrix, the standard mode for protein identification; 2. CID (collision-induced decomposition) high energy fragmentations due to a gas filled into the collision cell; and 3) reflector-ISD (in-source decay) prompt fragmentation, providing monoisotopically resolved precision fragment masses of intact proteins up to 40 kDa and higher. All MS/MS spectra are obtained as single scan spectra and acquisitions for proteomics data take a few seconds. Data-dependent MS/MS acquisition and processing is fully automated.
On protein digests MS sensitivity was in the 200 attomole range and for MS/MS in the 1-10 fmole range. The high sensitivity and ready availability of MS/MS data on tryptic peaks allowed the detection of sequence errors, modifications and an overall significant improvement of the extractable information content. De novo sequencing combined with homology searching (MS BLAST) was applied to these data. Typically, LID was used for de novo sequencing and CID only, if the Ile/Leu ambiguity needs to be resolved.
ISD was used for internal sequencing of intact proteins. ISD provides monoisotopically resolved fragment ions of N-terminal residues ~10-40 based on assigned c-type fragment ions and, therefore, allowed to obtain near N-terminal sequence information irrespective of a blocked N-terminal.

I. P. Smirnov, X. Zhu, I. A. Papayanopoulos, D. J. Pappin;
Applied Biosystems, Framingham, MA, United States.

MALDI-TOF Mass Spectrometry has become one of the major analytical tools in the analysis of proteins and peptides. It offers a very high throughput rate with data acquisition speed up to few seconds per spectrum. The successful identification of the peptides depends on the signal intensity and the absence of interfering signals. MALDI is susceptible to alkali salt contamination, which reduces sensitivity and causes the matrix cluster formation (widely reported for CHCA matrix) observed as signals dominating in the range below 1100 Da in the spectrum. One way to remove these background signals especially for small peptide concentrations lower then 10 fmol/ul is to wash matrix/sample spots after crystallization on the MALDI plate with deionized water prior to analysis. This method takes advantage of the low water solubility of CHCA compare to it’s alkali salts. We report here that the application of some ammonium salt solutions, such as citrates and phosphates instead of deionized water greatly improves efficiency of such approach. Besides total matrix cluster suppression due to more complete salt removal, such approach provides substantial (~3-5 fold) improvement in the sensitivity of MALDI detection compared to unwashed sample spots. This makes the analysis of protein digest in concentration below 1 fmol/ul easy to achieve. The effect of washings of the sample spot with various ammonium salts has been studied as a function of the their type, pH and concentration. This sample preparation method provided much better peptide coverage in database search and better MS-MS data on the 4700 proteomics analyzer for subnanomolar concentration protein digests, thus is becoming a valuable approach in every day sample preparation in our laboratory.

X. Zhu, I. Smirnov, I. A. Papayannopoulos;
Applied Biosystems, Framingham, MA, United States.

Introduction
Alpha-cyano-4-hydroxycinnamic acid (CHCA) has been widely used as matrix for the analysis of proteins and peptides by Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry. CHCA also ionizes and matrix cluster ions and their salt adducts often interfere with the detection and measurement of peptide ions. We found that these CHCA adduct ions are suppressed in MALDI mass spectra when certain ammonium salts are added to the CHCA matrix.
Method
Proteins were digested with trypsin. Samples were diluted to a series of concentrations from 1 fmol/μL to 1 pmol/μL. Each of a number of ammonium salt solutions was mixed, at various concentrations, with CHCA to make MALDI matrix solutions. Protein samples were mixed with the matrix solutions and spotted on MALDI targets ("dry-droplet" method). Samples were analyzed with the Applied Biosystems Voyager DE™-STR mass spectrometer or the 4700 Proteomics Analyzer. Mass spectral data were interpreted using the GPS Explorer™ software.
Results
CHCA sodium and potassium adduct ions in MALDI-TOF mass spectra were significantly suppressed if protein digests were mixed with CHCA containing an ammonium salt such as ammonium phosphate or ammonium citrate. The signals of peptides present in low fmol to high amol levels were enhanced with this method. Both MALDI MS and MS/MS data were readily matched to proteins using GPS Explorer™ and the MASCOT™ search engine.
Conclusions
The addition of certain ammonium salts to CHCA results in suppression of matrix salt adducts in MALDI-TOF mass spectra and the enhancement of peptide signals as well, which benefits especially low concentration samples.

S. Ackloo¹, E. Denoyer², J. Dicesare², A. Loboda¹;
¹PerkinElmerSCIEX, Concord, ON, Canada, ²PerkinElmerSCIEX, Shelton, CT, United States.

The widening scope of proteomics and the increasing demands on high-throughput analysis has driven the need to design an instrument with improved performance and ruggedness. A new MALDI-TOF instrument — equipped with both collisional cooling and orthogonal injection — has been developed. This design effectively decouples ion production from time-of-flight measurement making mass analysis more robust. Collisional cooling conditions, laser energy and pulse rate may be varied to optimize ionization conditions, suppress fragmentation, and increase ion yield for different matrix/analyte combinations. At the same time, high mass accuracy is achieved and maintained without the need for internal calibration; a critical success factor for protein analysis.
Decoupling ionization conditions from mass analysis permits more flexibility in the design of a sample target. Traditionally, samples are prepared on reusable metal targets where sample carryover is a concern. Single-use sample plates, in microtiter format, were developed to assure reproducible analysis without contamination. The new single-use plates perform similarly to traditional stainless steel targets.
The performance of this new instrument will be demonstrated using proteins and protein digests prepared using traditional methods. Performance criteria including mass accuracy, mass resolution, and sensitivity will be examined. Mass accuracy in the low ppm range and sensitivity in the low fmol range provide the opportunity to successfully analyze low quantities of sample. Mass spectra of a mixture of proteins, ranging in mass from 1.5 kDa to 30 kDa and acquired at one set of instrument parameters, will be presented. These data show that a wide mass range can be acquired without sacrificing mass resolution; a unique capability of the instrument and which is directly applicable to biomarker identification.

R. Zeng¹, C. Shieh², H. Tran², B. Wu²;
¹Shanghai Institute for Biological Science, Shanghai, China, ²ThermoFinnigan Corp., San Jose, CA, United States.

Mass spectrometry has become an important tool for the protein analysis and peptide sequence determination. Typically, a protein mixture is digested by trypsin, analyzed by mass spectrometry, and then searched against a relevant protein database where identification can be made by comparing experimental MS/MS peptide spectra with those predicted by theory. In this paper, we report the analysis of a yeast protein digest by an AP MALDI ion source coupled to an Ion Trap Mass Spectrometer. Proteins from the yeast cell lysate were extracted by protein G beads followed by one-dimensional SDS-PAGE gel electrophoresis. Ten gel bands were cut and digest by trypsin. The resulting peptide fragments were analyzed using both MALDI-TOF and AP MALDI-Ion Trap. The MS results from MALDI-TOF were searched by Mascot software. Two proteins were identified. The MS/MS spectra from the AP MALDI-Ion Trap system were searched by SEQUEST™ software. Ten proteins were identified. The ability to generate MS/MS spectra by coupling MALDI ionization with ion trap mass spectrometry provides higher confidence and greater accuracy for protein identification compared to MALDI-TOF mass fingerprinting.

P. Perkins, D. Yi, J. Meza, C. A. Miller;
Agilent Technologies, Palo Alto, CA, United States.

With the introduction of atmospheric pressure matrix assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI trap), it is possible to easily obtain MS/MS data on a protein digest using a MALDI source. Several different protein digests were analyzed using both AP-MALDI trap and ESI-LC/MS/MS. As expected, the MS/MS spectra of doubly charged ions from ESI showed more complete fragmentation and predominantly b and y ions. AP-MALDI MS/MS of singly charged ions also generated either b or y ions, but the MS/MS spectra showed other uninformative mass losses such as water. The protein sequence coverage was incomplete for both techniques at low levels, with a large overlap in detection between AP-MALDI and ESI.

J. W. Finch, J. C. Gebler, W. Chen, S. J. Berger, S. A. Cohen;
Waters Corporation, Milford, MA, United States.

The technique of static nanospray utilizes loading of 1-3 μL of sample into needle emitters which have been fabricated from borosilicate glass capillaries pulled to a fine tip (1-2 micron i.d.) and coated with a conductive material. One key advantage of this technique is the ability to introduce samples into a mass spectrometer by electrospray ionization (ESI) at very low flow rates, typically between 20-40 nanoliters/minute, where many MS/MS spectra can be acquired from a single sample over a period of 1-2 hours. While this technique is highly sensitive, samples must be relatively free of high concentrations of buffers, salts, detergents, and other contaminants which suppress ionization of peptides and proteins, and can cause the emitter to plug. Sample pre-treatment methods currently employed to remove these contaminants include use of ZipTips™ and microcolumn purification techniques. However, these methods are cumbersome, making it difficult to handle and transfer microliter volumes without sample loss. A further disadvantage is that sample recovery from packed beds is often poor.
In this study we present results demonstrating an alternative sample preparation method for static nanospray, which utilizes a surface with unique properties that allow both pre-concentration and removal of contaminants. The surface facilitates manipulation of low sample volumes without loss of sample, and the process can be easily automated. Results are presented for intact proteins and protein digests from 1D and 2D gel spots analyzed on a quadrupole-time-of-flight hybrid (Q-Tof) instrument. Excellent sensitivity can be achieved when a data-directed analysis (DDA) protocol is utilized to automatically generate MS/MS data for each individual sample infused by static nanospray.

D. J. Rousell, S. M. Dutta, K. K. Murray;
Louisiana State University, Baton Rouge, LA, United States.

Desorption/ionisation on silicon, DIOS, has the advantage in that its matrix free approach makes sample preparation simpler. In addition, the lack of matrix reduces the number of low mass peaks considerably simplifying this section of the mass spectrum. The UV-DIOS technique requires a porous silicon surface. It has been shown that for IR-DIOS a porous surface is not necessary.
The instrument under development combines a commercial mass spectrometer and IR laser to produce a system capable of employing DIOS and MALDI techniques with IR and UV lasers. The mass spectrometer used is a Bruker "Omniflex" reflectron instrument that has been modified to be capable of triggering an external laser and sample targets were milled to accept silicon wafers. An Er:YAG (2.94 micrometer) laser is positioned so that the IR laser beam enters the vacuum chamber co-axially with the UV beam. With this configuration the system can be easily switched between IR and UV analysis.
Spectra obtained with IR-DIOS contain fewer low mass peaks when compared to spectra of identical compounds under IR-MALDI conditions. The ease of sample preparation is evident with biological analytes. Tissue can be applied directly to the silicon surface for immediate mass spectrometry analysis. Bacteria have also been nebulized from an aqueous solution directly onto silicon. Recorded mass spectra contain numerous peaks below m/z 20,000 as previously reported. In addition our mass spectra also contain peaks of significantly higher mass.
Tobacco smoke particulate has also been analyzed. Using a single stage Andersen N6 impactor sidestream smoke was directly collected onto silicon. Mass spectrometric analysis was achieved without the need for sample extraction or matrix addition.

M. Kostrzewa¹, T. Elssner¹, K. Fahr¹, D. Peters¹, T. Wenzel¹, J. Wurmbach², W. Pusch²;
¹Bruker Daltonik GmbH, Leipzig, Germany, ²Bruker Daltonik GmbH, Bremen, Germany.

After completion of the Human Genome Project, intensive exploration of genome function diversity is one of the main objectives. The variety of genomes and their function can be observed at several levels like genetic polymorphisms, mRNA expression, protein abundance and protein modification. Mass spectrometry has evolved to a key technology for proteomic approaches in the last few years. Here, we present GENOLINK, a SNP genotyping module expanding the capabilities of a MALDI-TOF MS system to create a platform for functional genomics studies. GENOLINK is the combination of a novel method for SNP genotyping, genoSNIP, a robotic system for automated sample preparation, and a bioinformatics package. The genoSNIP method is based on extension primers containing a non-nucleoside UV-cleavable building block and a biotin label at the 5’ part. After amplification of template DNA by PCR, these primers hybridize adjacent to the polymorphic site and will be converted to allele specific products by single nucleotide extension. Subsequently, primer extension products are desalted using genostrep, a new DNA purification system based on streptavidin coated microtiter plates. This sample preparation is a simple pipetting protocol performed by the puredisk, a novel 96fold pipetting robot. The purified molecules are cleaved by UV- irradiation releasing very small molecules containing the genotype information. These small products are measured in the mass spectrometer with very high precision and sensitivity. The mass spectra obtained are translated into genotypes by the genotools software. Thereby, the GENOLINK system comprises a complete solution for automated, highly accurate SNP genotyping by MALDI-TOF MS.

G. Zurek¹, A. Germanus¹, C. Baessmann¹, A. Ingendoh¹, H. U. Muccitelli²;
¹Bruker Daltonik GmbH, Bremen, Germany, ²Bruker Daltonics Inc, Billerica, MA, United States.

The major task of metabolite identification is rapid and reliable elucidation of structural changes. Liquid chromatography hyphenated to atmospheric pressure ionization mass spectrometers (LC-MS) has become an integral part of metabolite identification. Ion trap mass spectrometers have proven excellent tools, especially due to the multiple stages of MS capability. Because data acquisition is now performed in an automated fashion, the new bottleneck in the identification process is data interpretation.
We present here that LC-MS data sets of control and serum/urine samples are automatically interpreted by Metabolite Tools, which also provides significant input downstream for the processes of data acquisition and interpretation. Full integration of Metabolite Tools with the Esquire Control software enables an effortless process.
Examples are shown where the Metabolite Predict module generates candidates of biotransformation based on metabolism rules of the parent drug structure. Molecular weight information of predicted metabolites are used to generate a mass list for further AutoMSn experiments or can be transferred to Metabolite Detect for use in an LC-MS data extraction.
The Metabolite Detect module performs a sophisticated comparison of two LC-MS chromatograms using the eXpose algorithm, and rapidly delivers even low-level metabolites that are barely detectable using radioactive tracers. Retention time and the m/z value are then used for an MS/MS experiment that yields higher quality spectra than data dependant experiments. Identification of metabolites is then completed using the MSn data.
Metabolite Tools is not only essential in drug metabolite detection, but also useful in detecting subtle changes in LC-MS analysis of peptides, nucleotides and other biomolecules.

V. Pham, W. J. Henzel;
Genentech, South San Francisco, CA, United States.

We have developed a rapid method for digesting and identifying blocked proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes. The unbound protein-binding sites on the membrane are blocked using zwittergent 3-16. The proteins are then digested with trypsin, Lys-N, Lys-C, Asp-N, Glu-C, or clostripain in appropriate buffers at 37 C for 5-30 min. Peptides generated by these digestions remain on the membrane, which are then identified by N-terminal sequencing. The mixture of sequences obtained is used for database searching which leads to unambiguous protein identification. This approach can also be used for blocked proteins that have been subjected to the Edman chemistry. The membrane is removed from the sequencer, blocked and digested with Asp-N, Glu-C or clostripain.

V. P. Hanko, J. S. Rohrer;
Dionex Corporation, Sunnyvale, CA, United States.

Integrated pulsed amperometric detection with a highly selective anion-exchange column (AAA-Direct™) is a powerful technique for amino acid analysis. This technique enables the direct detection of virtually all amino acids without pre- or post-column derivatization and without compromising sensitivity. The AminoPac™ PA10 anion exchange column is designed to separate amino acids with baseline resolution. Using high eluent strength and elevated column temperature, this column provides a rapid (<12 min) separation of tryptophan (Trp) from other amino acids and sample components. We determined Trp in alkaline hydrolyzed protein and peptide samples and cell culture media/fermentation broths. High levels of carbohydrates, typical of some samples, do not interfere with this method.
In this poster we present methods for preparing protein samples for hydrolysis and subsequent Trp analysis by AAA-Direct. Techniques commonly used to increase Trp recovery during alkaline hydrolysis were also evaluated for their suitability with AAA-Direct. Direct detection allowed us to easily optimize hydrolysis conditions because we did not have to be concerned with differences in derivatization efficiencies between samples complicating our results.

J. A. Moss, A. R. Coyle, K. D. Janda;
The Scripps Research Institute, La Jolla, CA, United States.

Immobilized metal affinity chromatography (IMAC) has rapidly become one of the most widespread affinity purification techniques employed in recombinant protein expression. The efficacy of this technique relies on the stable coordination sphere provided by an immobilized metal (Ni²⁺, Zn²⁺) and a hexa-histidine tag appended to an expressed protein. While this method usually affords material in high yield and purity, our purifications of an antibody single chain variable fragment (scFv) expressed in E. coli were contaminated by a hydrophobic 80 kDa protein and lipopolysaccharide (LPS) endotoxin. Implementation of a diphenyl reversed-phase HPLC purification step following IMAC purification effectively removed both of these contaminants without serious adverse effects on the antibody. In addition, this orthogonal purification step furnished material that could be lyophilized and obtained as a salt-free solid amenable to long-term storage without perceivable loss in activity.

E. Chertova, J. Bess, B. Crise, R. Sowder, II, T. Schaden-Ireland, L. Henderson, J. Lifson, L. Arthur;
SAIC-Frederick, NCI-Frederick, Frederick, MD, United States.

Objective: We characterized factors influencing the gp120^SU content of retroviral virions.
Methods: To determine the Gag and Env glycoproteins content of purified virions and to calculate the ratio of Gag /Env proteins in viral samples, we used microscale HPLC methods, coupled with SDS-PAGE, immunoblot analysis, mass spectrometry, and protein sequence analysis of eluted proteins.
Results: All HIV-1 and most SIV isolates examined had Gag:Env ratios of approximately 60:1, corresponding to only 7-14 envelope-containing trimers per virion, given 1200-2500 Gag molecules per virion. SIVmne E11S showed a Gag:Env ratio of approximately 6:1, corresponding to 70-140 envelope trimers per virion. TM truncation (gp32, rather than gp41) was associated with higher envelope glycoprotein content. All HIV-1 and SIV isolates examined, except one SIV clone, had surface SU:TM molar ratios of 1:1 indicating that gp120 does not generally shed from the virus once TM is inserted into the membrane; shedding would yield a molar excess of TM over SU. Virus SU content and SU:TM ratios were not significantly changed during sucrose gradient purification.. Heating purified HIV and SIV to temperatures of 55°C or greater for one hour resulted in loss of most of the gp120 from the virus but retention of TM.
Conclusions: Envelope glycoprotein incorporation and not shedding of surface glycoprotein gp120 is the primary determinant of gp120 content of purified HIV-1 SIV.
Contract No. N01-CO-12400.

J. Chervet, Sr.;
LC Packings, Amsterdam, Netherlands.

Capillary and Nano LC columns with inner diameters ranging from 50 to 300 µm are typically packed with 3 to 5 µm particle size stationary phases. Although these column are robust and yield reasonable efficient separations there are a few limitations: the slow mass transfer in the stationary phase limits the separation efficiency and the packed bed of this granular phases results in high back pressures. In contrast to conventional stationary phases, monolithic structures are highly porous, characterized by macro and meso pores which results in better mass transfer properties and in reduced back pressure.
In this poster we report on the chromatographic performance of 200 µm i.d. polystyrene/divinylbenzene monolithic capillary columns. The improved mass transfer in the stationary phase will be illustrated by H/u curve. Efficiencies up to a 1/4 million plates per meter are obtained routinely. To achieve these high efficiencies special attention must be given to the chromatographic system. The response time and sampling rate of the data system must be adapted accordingly.
Other advantages using monolithic bed structures are the high mechanical stability of the column bed (no voiding) resulting in a superior lifetime.
Ultra-fast separation of proteins and peptides including protein digests will be shown to illustrate the performance of these monolithic capillary columns. For proteins and peptides peak width at half heights of 1 to 3 seconds are achieved routinely. Peak capacities with up to 100 peaks in less than 15 minutes in gradient mode are now possible.

H. Schwartz¹, R. Swart², I. Dragan², R. Van Ling², J. Chervet², R. Van Soest³;
¹Dionex Corporation, Sunnyvale, CA, United States, ²LC Packings, a Dionex Company, Amsterdam, Netherlands, ³LC Packings USA / Dionex, San Francisco, CA, United States.

During the last decade, capillary and nano LC columns (i.d.s ~ 50-300 um) were typically packed with 3 to 5 um particles. Although these columns are robust and yield fairly efficient separations, they do have limitations: 1) slow mass transfer in the stationary phase limits the separation efficiency and 2) the packed bed of these phases results in high back pressure. In contrast, monolithic structures are highly porous, and have macro- and mesopores resulting in better mass transfer properties and reduced back pressure. Another advantage of a monolithic bed structure is the high mechanical stability of the column bed (no void formation), resulting in superior column lifetime.
Here we report on the performance of 200 um i.d. polystyrene/divinylbenzene monolithic capillary columns. The improved mass transfer in the stationary phase is illustrated by an H/u plot. Efficiencies up to a 0.25 million plates/m can be obtained routinely. To achieve these high efficiencies, special attention must be given to the hardware of the chromatographic system: connecting tubing, flow cell, injector, and other fluidic parts must have minimal dead volume. In addition, the response time and sampling rate of the data system must be adapted. A dedicated Capillary/Nano LC system, UltiMate (LC Packings, a Dionex company) meets these strict requirements. The sample capacity for proteins and peptides, and the influence of mobile phase additives also will be discussed. Finally, ultra-fast separations of proteins and peptides will be shown. For proteins and peptides gradients, peak widths of 1-3 s can be obtained with peak capacities of ~ 100 peaks/15 min.

R. Moritz¹, N. O'Reilly²;
¹Agilent Technologies, Waldbronn, Germany, ²Cancer Research UK, London, United Kingdom.

Synthetic peptides become more and more important as drug candidates in the treatment of a variety of diseases. In order to keep pace with the growing number of newly synthesized peptides, peptide purification should not represent the bottleneck in the drug discovery process.
Mass-based fraction collection is an efficient technique for the purification of compounds with well-known masses. In contrast to less specific detectors, in each run only the compound of interest is being purified. Hence, it is not necessary to pick out target compounds out of a series of redundant fractions that have been collected. Here we demonstrate mass-based purification of a series of synthetic peptides with the Agilent 1100 Series Purification System. The modular set-up of the system, a reliable fully automated delay volume calibration and a comprehensive software package assemble a versatile platform for purification tasks in the flow range from 0 to 100 mL/min. We could successfully purify crude peptides by reverse phase HPLC from less than 1 kDa to more than 10 kDa, covering a pI range from 4 to 13.

T. Neumann, R. Moritz, M. Kuschel;
Agilent Technologies, Waldbronn, Germany.

Liquid chromatography in analytical and preparative scale plays a key role in the purification of proteins and peptides. Due to its high resolving power reverse phase high performance chromatography (RP-HPLC) is often employed as a last polishing step in a purification workflow. Besides appropriate chromatographic conditions also reliable fraction collection is indispensable. Herein we present an LC system with a fully automated delay calibration and low delay volume for reliable fraction collection of valuable samples. In a combined approach we demonstrate how microfluidics can be integrated efficiently into a protein purification workflow as an orthogonal method for purity checks. In addition the microfluidic system allows to monitor and optimise the protein purification process in a fast and easy way.

F. A. Chen¹, M. Santos¹, D. Binger²;
¹Life Sciences Division, Beckman Coulter Inc., Fullerton, CA, United States, ²Diagnostics Division, Beckman Coulter Inc., Chaska, MN, United States.

Monoclonal antibodies obtained from mouse ascites fluid and cell culture demonstrate differences in isoelectric range despite their identical genetic imprint from the hybridoma. This may influence susceptibility to chemical modification and aspects of functional behavior in immunoassays. Presumably the post-translational modification of the monoclonal antibodies by sugar moieties results in such differences. In this report, we demonstrate a practical approach for the analysis of N-linked oligosaccharide in IgG monoclonal antibodies obtained from mouse ascites fluid and cell culture and provide comparative isoelectric focussing profiles for two production methods. Purified IgG was denatured in the presence of a reducing agent and the N-linked oligosaccharides were released in the presence of peptide N-glycosidase (PNGase F; EC3.5.1.52). The released reducing sugars were derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS, 1) under mild reductive amination conditions to avoid desialylation and the detachment of fucose residues. The reaction adducts mixture was analyzed directly without any prior clean-up procedures by capillary electrophoresis (CE) with a laser-induced fluorescence (LIF) detection.
Significant differences in the oligosaccharide profiles were observed between the antibody obtained from the ascites fluid and the cell culture. These results are compared with the microheterogeneity of protein species obtained by capillary isoelectric focussing of the same monoclonal antibodies. Differential behavior of each monoclonal antibody and relationship to altered isoelectric point will be discussed. The present method of oligosaccharide profiling of IgG monoclonal antibody appears to be suitable for routine analysis.
Reference:
1) F-T. A. Chen and R. A. Evangelista. Profiling N-linked oligosaccharides of Glycoproteins. Electrophoresis, 15, 1892-1898 (1998).

A. K. Shukla, M. M. Shukla;
Glygen Corp., Columbia, MD, United States.

The desalting and cleanup of small water-soluble molecules such as carbohydrates is a challenging task. Due to their smaller size and similar molecular properties, these molecules cannot be cleaned easily by dialysis, because the small pore size (100, 500 or 1000Dalton) of the membranes allows only a very slow dialysis and may take 24 hrs or longer. Furthermore, the molecular weight cut off’s (MWCO) of these membranes of smaller pore sizes are not very sharp, therefore, it is difficult to get the sample completely desalted for further analysis (such as MALDI, MS, capillary electrophoresis, capillary HPLC). By using the activated charcoal, porous carbon or ion-exchange resin in a small filter-less column with volatile elution buffer, the sample can be cleaned up efficiently for further analysis. Using active charcoal and porous carbon, the different mono, di and oligo-saccharides can be easily cleaned up from phosphate buffer and the samples are analyzed by HPLC. The binding capacity of the active charcoal is higher than that of the porous carbon, however, the reproducibility of porous carbon is better, because porous carbon particles are commercially available with controlled particle size. However, commercially available active charcoal particles are not of controlled particle size and are a mix of various shapes and sizes. By using the same technique, other small water-soluble molecules, such as nucleotides, water-soluble dyes, peptides, glycopeptides can also be cleaned up for further analysis.

A. M. DeSantis;
Virginia Tech Center for Genomics, Blacksburg, VA, United States.

Several methods are currently available for enriching phosphopeptides for analysis by liquid chromatography-tandem mass spectrometry. Most protocols involve enrichment of the phosphopeptides by immobilized metal-ion affinity chromatography (IMAC) prior to separation by reverse-phase chromatography and subsequent analysis by mass spectrometry. A methylation step for minimizing contamination of phosphopeptide solutions by acidic, non-phosphorylated peptides has also been reported. As we desired to have a routine protocol in place to rapidly enrich and analyze for phosphopeptides, we embarked on a study to compare several strategies to see which protocol, if any, offers both convenience and breadth of recovery. Two phosphopeptide purification techniques, an Fe-IMAC column procedure and the commercially available gallium-based phosphopeptide isolation discs are assessed for their abilities to obtain enriched phosphopeptide solutions from non-casein standards as well as tryptic digests. Both methylated and nonmethyated samples are used in the investigation.

T. H. Steinberg, K. J. Martin, B. J. Agnew, K. R. Gee, W. Leung, T. Goodman, B. Schulenberg, J. M. Beechem, R. P. Haugland, W. F. Patton;
Molecular Probes, Inc., Eugene, OR, United States.

Fluorescence detection methods are likely to offer the best solution to detection and quantitation of total protein profiles and post-translational modifications in proteomics. Reversible protein phosphorylation plays a critical regulatory role in biological systems, as demonstrated in the context of carcinogenesis and other disease states. Our newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins displayed on SDS-polyacrylamide gels, 2-D gels and protein microarrays. This technology is based upon small-molecule-fluorophore, phospho-amino acid recognition, and does not require antibodies. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. DNA, RNA and sulfated glycoproteins fail to be detected with Pro-Q Diamond dye. The staining is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as MALDI-TOF mass spectrometry. Pro-Q Diamond dye technology currently can detect as little as 1-2 ng of b-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein, in SDS minigels with a linear response over a 500-1000 fold concentration range. Less than 1 pg of phosphorylated protein and/or peptide can be detected in microarray format using a microarray optimized procedure for Pro-Q Diamond stain. Through combination of Pro-Q Diamond phosphoprotein stain with Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, our Multiplexed Proteomics platform permits quantitative, multi-color fluorescence detection of phosphorylated proteins, glycoproteins, and total-protein in the same 2-D gel or microarray.

K. A. Lee, J. B. Hurley;
University of Washington, Seattle, WA, United States.

On stimulation, rhodopsin, the light-sensing protein in the rod cells of the retina, is phosphorylated at several sites on its C terminus as the first step in deactivation. We have developed a mass spectrometry-based method to quantify the kinetics of phosphorylation in vivo. After exposing a mouse to a light stimulus, phosphorylation and dephosphorylation reactions are terminated by rapidly homogenizing the eye in 8M urea. The C-terminal peptide containing all known phosphorylation sites is cleaved from rhodopsin, partially purified by ultracentrifugation, and analyzed by LCMS. The relative sensitivity of the mass spectrometer to peptides with zero to five phosphates was determined using purified phosphopeptide standards, and an equation was generated to calculate these sensitivity coefficients for other phosphopeptides. Three primary phosphorylation sites, Ser334, Ser338, and Ser343 were distinguished by liquid chromatography coupled with tandem mass spectrometry (LCMS/MS). Although peptides monophosphorylated at Ser338 and Ser343 coeluted, the relative amounts of each species were determined by monitoring the ratio of specific daughter ions characteristic of each peptide. Doubly-phosphorylated and triply-phosphorylated rhodopsin peptides with different sites of phosphorylation also were distinguished by LCMS/MS. These methods were used to study the kinetics of rhodopsin phosphorylation and dephosphorylation in response to a continuous light stimulus. Sixty to ninety minutes of light adaptation were required to achieve steady-state phosphorylation. Upon light exposure, Ser343 is phosphorylated most rapidly, followed by Ser338 and Ser334. However, at the steady state, rhodopsin peptides are monophosphorylated on Ser334 seven times more than Ser343 and three times more than Ser338. After reaching the steady state of phosphorylation, mice were placed in darkness, and the kinetics of dephosphorylation were determined for each phosphorylation site.

G. M. Hathaway, J. Zhou, F. Rusnak;
Caltech, Pasadena, CA, United States.

The increasing number of known sequences makes it possible to use a new approach to determine sites of post-translational modifications (PTM) by chemically targeting them for specific proteolytic cleavage. Analysis of the digest allows one to obtain correct structures by inference. We call this approach chemically-targeted- identification or CTID.
Peptides, phosphorylated or glycosylated on serine and threonine residues, underwent ß-elimination in the presence of the nucleophile 2-aminoethanethiol to produce analogs of lysine. Both serine and threonine derivatives were specifically cleaved with lysine endopeptidase.
The products of digestion were analyzed by mass spectrometry and/or Edman sequencing. Prior knowledge of the sequence allowed site identification either from peptide mass, or from a few residues sequenced from the newly liberated amino terminus.
Oxidation of the modified peptide allowed its detection in digest mixtures by precursor ion or neutral loss linked scans. Starting with 2 picomoles of a 3 kDa glycopeptide, the CTID method successfully assigned the site of glycosylation to a single threonine residue.

M. Zou¹, C. Shi², X. Hou², R. H. Cohen²;
¹The University of Tennessee, Knoxville, TN, United States, ²Boston University, Boston, MA, United States.

We have previously reported that peroxynitrite (ONOO^-), a potent oxidant formed from nitric oxide (NO) and superoxide anions (O₂^.-) at a diffusion-controlled rate, activates 5’-AMP-activated kinase (AMPK), resulting in phosphorylation of endothelial nitric oxide synthase serine¹¹⁷⁹-P (eNOS-Ser¹¹⁷⁹-P) and acetyl CoA carboxylase (Ser⁷⁹, ACC), two known downstream targets of AMPK. Here we investigated how ONOO^- activates AMPK in cultured bovine aortic endothelial cells (BAEC). In parallel with increased AMPK (Thr¹⁷²) phosphorylation as well as that of eNOS and ACC, ONOO^- significantly increased c-Src (Tyr⁴¹⁶) phosphorylation. Inhibition of c-Src activity by PP2 or overexpression of a c-Src dominant-negative mutant blocked ONOO^--enhanced phosphorylation of AMPK, eNOS-Ser¹¹⁷⁹-P, and ACC-P. In addition, ONOO^- significantly enhanced levels of phosphorylated PDK1 and p70S6 kinase, indicating that ONOO^- activated PI-3 kinase. Furthermore, inhibition of PI-3 kinase by either wortmannin or LY294002, or overexpression of an inactive p-PDK1 mutant attenuated ONOO^--induced AMPK phosphorylation as well as that of eNOS-Ser¹¹⁷⁹-P and ACC-P. Inhibition of PI-3 kinase also prevented the co-immunoprecipitation of AMPK and eNOS induced by ONOO^-, suggesting that the oxidant, through its actions on upstream kinases, favors the physical association of AMPK and eNOS. Taken together, our results indicate that ONOO^- activates AMPK in a PI-3 kinase-dependent manner and suggest that ONOO^--induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC, HMG-CoA reductase, and glycogen synthase.

W. L. McEldoon, M. J. Horn;
BioMolcular Technologies, Inc., Sunnyvale, CA, United States.

We previously have described easy methodologies employing our Pi^3™ solid-phase reagents to selectively separate and isolate methionine-, tryptophan-, or phosphotyrosine-containing peptides from mixtures. We have applied these methodologies to digests of mixtures of proteins, peptides derived from in-gel digestion of proteins, as well as digestions of lysates. We report here the development of an improved, simplified and quicker method for phosphotyrosine, using a novel water-insoluble, solid-phase reagent that forms a covalent bond with phosphotyrosine-containing peptides. The reagent provides for the selective isolation of phosphotyrosine peptides from peptide mixtures and from mixtures containing phosphoserine- and phosphothreonine-containing peptides. These separated phosphotyrosine- containing peptides subsequently may be regenerated and analyzed by mass spectrometry or liquid chromatography. When coupled with mass spectrometric analysis and more focused database searching, these methodologies provide a significant increase in simplicity and accuracy of protein identification. Additionally, sites of tyrosine phosphorylation may be readily identified in known and unknown proteins. This new methodology has been shown to allow for the identification of phosphotyrosine peptides at the 2% occurrence level. Compared with IMAC, the phosphopeptide yields of this described method are substantially higher, and the method provides the specificity of phosphotyrosine only.

D. C. Watson, N. M. Young, J. R. Brisson, J. Kelly, H. C. Jarrell, L. Tessier, P. H. Lanthier, N. Cadotte, F. St Michael, E. Aberg, C. M. Szymanski;
National Research Council, Instiute of Biological Sciences, Ottawa, ON, Canada.

Campylobacter jejuni is a Gram-negative bacterium that is a leading cause of gastroenteritis, which in a small number of cases can be followed by a neuroparalytic disorder, Guillain Barre Syndrome. Mass spectrometry investigations of partially purified C. jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan of mass 1406Da and comprised of one hexose, five HexNAc, and a species of mass 228Da, consistent with a trideoxydiacetamido hexose. The attachment site was an Asn residue within a sequence DFNVSK, thus matching the eukaryotic N-linked glycoprotein sequon Asn-Xaa-Ser. By means of soybean lectin affinity chromatography, a mixture of glycoproteins was obtained from a glycine extract, and 2D-gel proteomics analysis led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins, all of which contained putative Asn sequons with either Ser or Thr. Glycopeptides were prepared from the glycoprotein mixture by pronase digestion and gel filtration. The structure of the glycan was determined by using nano probe magic angle spinning-NMR techniques to be
GalNAc-α1,4-[Glcβ1-3-]GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,4-GalNac-α1,3-Bac-β1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the synthesis of bacillosamine.

A. Cordoba, B. Shyong, D. Breen, S. Shire, R. Harris;
Genentech, South San Francisco, CA, United States.

Monoclonal antibodies (MAbs) typically undergo fragmentation over time in the liquid state. The purpose of this study was to investigate whether this fragmentation is due to enzymes or kinetic forces that break the weak link that connects Fab to Fc. Fragmentation occurs in the hinge region near the papain cleavage site, resulting in Fab and Fab-Fc forms. Four different Genentech MAbs were exchanged into the same buffer and stored at —20°C, 5°C, 30°C, and 40°C for 1 month. Protease inhibitors were added to one of the MAbs. Cleavage rates were measured after incubation using size exclusion chromatography (SEC). Peaks that were separated using SEC were analyzed by electrospray mass spectrometry to identify the clipping sites. The effect of host cell proteins on fragmentation was also studied. Different pools of an antibody (affinity pool, cation exchange pool, and final bulk pool) were incubated at different temperatures. After incubation their cleavage rates were measured by SEC. Cleavage rates were observed to be equivalent between antibodies. MAb clipping does not always produce complementary Fab and Fc fragments. In all the MAbs cleavage takes place within the same heavy chain hinge sequence: Ser-Cys-Asp-Lys-Thr-His-Thr. The main clip sites are between Asp-Lys and His-Thr (the papain clip site). No clipping was observed in the light chain. The use of protease inhibitors did not affect rates of cleavage or modify sites of fragmentation. Degradation was not affected by host cell protein content. In summary, fragmentation does not appear to be caused by proteases.

Z. Wu^1,2, K. Nakanishi³, J. W. Crabb^1,2;
¹Cleveland Clinic Foundation, Cleveland, OH, United States, ²Cleveland State University, Cleveland, OH, United States, ³Columbia University, New york, NY, United States.

CRALBP (cellular retinaldehyde-binding protein) serves as an 11-cis-retinol acceptor and as a modulator of 11-cis-retinol dehydrogenase in the mammalian rod visual cycle. Mutations in the CRALBP gene cause progressive retinal degenerations that lead to blindness. To better understand the molecular interactions between CRALBP and its ligand, photoaffinity labeling with retinoid analogue 3-diazo-4-keto-11-cis-retinal has been pursued and labeled sites identified by mass spectrometry. Purified human recombinant CRALBP was labeled in the dark with 3-diazo-4-keto-11-cis-retinal. Covalent incorporation of this ligand was evaluated following photolysis with UV-light (254 nm) at —196⁰C for 5s to 20 min. Protein bound retinal was reduced to retinol with NaB³H₄, radiolabeling the ligand incorporation sites. Labeled rCRALBP was denatured in 8 M urea, alkylated with iodoacetamide, digested with trypsin and peptides fractionated by RP-HPLC. Radioactive HPLC fractions were identified by scintillation counting and peptide sites of incorporation identified by MALDI-TOF MS and LC MS/MS. Short (5-40s) irradiation times yielded relatively constant incorporation levels (~1%) therefore 5s photolysis times were chosen to minimize nonspecific protein modifications. MALDI TOF MS and LC MS/MS analyses of tryptic peptides from photolabeled rCRALBP have accounted for 100% of the rCRALBP sequence. Eight photoaffinity modified residues were identified, all with variable mass additions. This variability may be due to free radical migration throughout the conjugated double bonds of the retinoid analogue. Four of the photoaffinity modified sites are in authentic retinoid binding pocket residues based upon previous studies. The other four modified residues may represent newly identified CRALBP ligand binding pocket components. The functional significance of these residues is under investigation.

M. A. Ritchie, N. Johnson, J. B. Hoyes, A. Millar, R. Carruthers, C. Jones, J. Langridge;
Micromass MS Technologies, Manchester, United Kingdom.

Asparagine (N-) linked glycosylation is one of the most common, complex, and highly variable post-translational modification. As glycoforms are the product of a series of biochemical modifications, perturbations within a cell can have profound effects on their structure. As glycosylation also plays an important role in cell signaling and recognition it’s detection and characterisation are of great importance.
Here we present a method for the detection and characterisation of glycopeptides. A proteolytic digest is analysed by reverse phase HPLC-ESI using a Q-Tof mass spectrometer. The instrument is switched at one-second intervals between low and high collision energy on the collision cell. The quadrupole operates in the non-selective rf only mode. The first data set at low energy (7eV) shows only the normal pseudo molecular ions. The second at higher energy shows their fragments. Wherever a specified product occurs in the high-energy data all its possible precursors are revealed by the corresponding 7eV data. Upon detection of the carbohydrate oxonium ions at m/z 204 (HexNAc), 366 (HexHexNAc) and 274/292 (NeuAc) the instrument is switched into MS/MS mode and ions from the low energy spectra are selected by the quadrupole for fragmentation. The high mass of glycopeptides gives rise glycopeptide ions typically of 4 or more positive charges, a feature that is exploited for their preferential selection for MS/MS. The glycosidic bonds tend to be more labile than the peptide bonds hence MS/MS spectra produce predominately glycopeptide Y-type fragment ions. Interpretation of MS/MS data is facilitated through the use of software tools.

Y. Zhang¹, R. R. Cocklin¹, K. D. O'Neil², N. X. Chen², S. M. Moe^2,3, K. R. Bidase⁴, M. Wang¹;
¹Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, United States, ²Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States, ³3Richard L. Roudebush VAMC, Indiana University School of Medicine, Indianapolis, IN, United States, ⁴Department of Pharmacology, University of Nebraska Medical Center, Omaha, NE, United States.

Beta-2-Microglobulin (β2M) amyloidosis is a destructive osteoarticular disease that affects patients undergoing long-term hemodialysis. Some of the β2M in the amyloid deposits, and circulating β2M in dialysis patients, are modified with advanced glycation end products (β2M-AGEs). Advanced glycation end products, which are made up of various glucose or carbohydrate adducts, are thought to be responsible for several diabetic and age related complications. However, to date, specific sites of AGE modification on the β2M protein remain largely unknown. We report here the use of MALDI-MS to determine the type and location of several AGEs on human β2M. A PERL script software, that was developed in-house, makes the relatively large amount of data generated by the MALDI much more manageable. The outstanding sensitivity of MALDI-MS coupled with the afore mentioned software allows such an approach to be a very useful tool in detecting AGEs and other post-translational modifications. We believe that this method could be an important option when analyzing naturally occurring AGEs in other human diseases.

C. L. Hunter¹, S. T. Weintraub², T. A. Settineri¹;
¹Applied Biosystems, Foster City, CA, United States, ²University of Texas Health Science Center, Dept. Biochemistry, San Antonio, TX, United States.

The direct combination of triple quadrupole and ion trapping capabilities in the
Q TRAP™ hybrid linear ion trap LC/MS/MS System presents new opportunities for the automated investigation of posttranslational modifications (PTMs). For the first time, the unique specificity of precursor ion (PI) and neutral loss (NL) scans for PTM identification can be automatically linked to high sensitivity ion trap MS/MS scans on an LC time scale. In addition, the ability to switch rapidly between positive and negative ion detection while performing these scans provides unique capabilities and maximum flexibility for automated PTM discovery. To identify the PTMs, Pro ID software was used to determine the protein of origin for each modified peptide and to identify the site(s) of modification on the peptides.
In this study, phosphopeptide standards containing S, T or Y phosphorylation were spiked into a characterized protein digest (500 fmole) and analyzed by nanoflow LC/MS/MS on the Q TRAP System. Several different concentration levels (20-100 fmole) were used to simulate a range of phosphorylation levels known to exist in nature. Automated PTM discovery was performed using information-dependent acquisition (IDA), where a negative ion PI scan for m/z 79 was used as a survey scan, followed by a positive ion enhanced resolution (ER) scan and a positive ion enhanced product ion (EPI) scan. At 50 fmole injected, all phosphopeptides were detected by PI scanning, and the resulting MS/MS spectra yielded positive sequence identification of 75% of these phosphopeptides. Further results will be presented on the identification and characterization of PTM sites on other modified peptides.

S. Wu, A. Huhmer, I. Jardine;
ThermoFinnigan, San Jose, CA, United States.

The analysis using peptide mass maps has become an important technique for proteomic studies. By digesting a protein from a gel spot or a separated subcellular fraction with a specific enzyme and comparing the measured fragment ions of the peptides (e.g. in tandem MS or ion trap) to those predicated by the sequence, one could elucidate the corresponding protein identification through a database search. For a targeted protein in a complex mixture, any changes in the sequence can be pinpointed to specific locations and the structures of the modifications (e.g. phosphorylation, glycosylation, methylation, and disulfide linkage) can be determined by further fragmentation (e.g. MS to 3 in an ion trap).
In this report, we will present how to use ion-trap mass spectrometer (MSⁿ) coupled withprotein identification (TurboSEQUEST^®), denovo sequencing (DenovoX™), and structure modification (Salsa) software to study the modifications of phosphorylation, glycosylation, and methylation in a complex mixture of biological fluid.

S. P. Yadav;
The Lerner Research Institute, Cleveland, OH, United States.

Unlike oligonucleotide synthesis, peptide synthesis has inherent complexities due to incredible chemical diversity in amino acid side chains. In fact each peptide made is unique in its own way. Despite the availability of several types of in situ coupling reagents and activators in solid-phase synthesis, amino acid deletion remains a major pitfall during the synthesis. To ensure that the coupling reactions reach completion, several fold excess Na-Fmoc protected amino acids are used in solid-phase synthesis. Other approaches, such as increasing the coupling cycle time for val, ile, ser, thr, gly and his including double coupling at long stretches of ser and thr have been proposed to avoid deletions. At present there are no well-defined guidelines or database available for predicting potential pitfalls in a given sequence to be synthesized. We have analyzed several synthetic peptides of various lengths by MALDI-TOF to seek information on peptide deletion products, methionine oxidation, and incomplete side chain deblocking. These preliminary data suggest that arg and asn were deleted more frequently than other amino acids in sequences that were synthesized using diisopropylcarbodiimide (DIC) as an activator.

B. Lee, S. Gupta, I. Morozova;
University of Illinois at Chicago, Chicago, IL, United States.

We have developed a novel three-dimensional sodium dodecyl sulfate polyacrylamide cube gel electrophoresis (3-D SDS-PACGE) technique to increase the resolution of protein separation. The three-dimensional technology involves the separation of proteins by isoelectric point (PI) in the first dimension, by molecular weight with a high percentage (12%) SDS-PAGE in the second dimension, and by molecular weight with a low percentage (7.5%) SDS-PACGE in the third dimension. The high percentage SDS-PAGE enhances the separation of the low molecular weight proteins. The low percentage SDS-PAGE cube gel enhances the separation of the high molecular weight proteins. The utilization of two SDS-PAGE steps in 3-D SDS-PACGE compared to one single step in 2-D SDS-PAGE has increased the separation between proteins. Three mixtures of multi-colored protein molecular weight standards containing broad, high, and low molecular weight ranges were used to provide a proof-of-concept study. We clearly observed the improvement in the protein separation. Also, the bovine serum albumin was used to demonstrate the improvement of the protein separation. Different steps including making and running of the cube gel, covalently coupling a colored dye to the proteins for visualization, and documenting the cube gel using digital camera, have been optimized to make the 3-D SDS-PACGE technology a reality.

M. R. Pisano¹, M. Saxton¹, K. J. Biederman¹, D. L. Allen¹, R. Nunez²;
¹Proteomic Research Services, Inc, Ann Arbor, MI, United States, ²Invitrogen Corp., Carlsbad, CA, United States.

Two dimensional protein gel separation is an established technique that is still considered to be the best option for protein profiling when one considers resolution. The analysis of a complex mixture of proteins using traditional large format 2D gel electrophoresis is tedious, time consuming, and can be expensive. The ZOOM® IPGRunner™ System (Invitrogen) enables researchers to profile proteins faster and easier. This system utilizes a mini-gel format for 2D separations that makes the first and second dimension gel separation fast, simple and easy. Rat liver tissue extracts were profiled using both traditional large format gels and the mini gel system. Image analysis and mass spectrometric analysis were performed in order to compare the results from these 2 formats. The methods were compared in the context of time, cost, and results of the image and MS analyses. The data acquired on the ZOOM gels at the lower protein loads was found to be superior to that from the large format gels. It is evident from the analysis that the ZOOM® Proteomic System is of great value and complements large format 2D gel profiling.

L. E. Cammish;
NextGen Sciences Ltd, Huntingdon, United Kingdom.

Current technologies used for protein profiling include the use of 2-dimensional electrophoresis (2DE) with subsequent identification via mass spectrometry. The physical process of separating proteins via 2DE has, until now, remained long, multi-step, labour intensive and often results in production of irreproducible data. In this presentation we will describe the design and use of a totally unique, highly flexible, reproducible and fully automated system to resolve these problems and simplify this separation process. It enables solubilised protein samples to be hydrated, focused, equilibrated on conventional IPG strips and finally resolved in the second dimension by SDS-PAGE without user intervention. It provides consistency of processing with thorough and robust methods development capability, eliminating the issues that can result from day-to-day and user-to-user variability. The innovative automation and microfluidic technologies incorporated in the system, which remove the complexities and variation associated with the traditional manual process resulting in the ability to perform protein separation in a highly reproducible fashion, will be described.

N. Laird;
Amersham Biosciences Corporation, San Francisco, CA, United States.

The introduction of immoblized pH gradient gels has significantly improved the quality, simplicity and reproducibility of 2-D electrophoresis. Extended strip lengths and new pH gradient gels have further advanced the technique to provide even higher resolution separation of proteins. IPG separations at basic pH have not always produced optimum results. In this study, proteins extracted from wheat seeds were used to represent a "difficult" sample in order to explore ways to improve IEF separation of basic proteins. Conventional rehydration solutions containing dithiotreitol resulted in significant horizontal streaking of the proteins making identification difficult. Adding a thiol blocking reagent, 2,2´-dithiodiethanol, resulted in superior separations of basic wheat storage proteins.

J. Li, Y. Zhang, T. Osterman, F. Witzmann, F. Witzmann;
IUPUI, Indianapolis, IN, United States.

In our routine proteomic efforts, collaborators and clients often desire access to the 2D images and protein identification information generated by our laboratories. The 2D analysis platform, PDQuest™, is powerful but its output and annotation tools have significant limitations. Even though a free viewer version can be downloaded, for those without a PDQuest™ background or experience, it is difficult to learn. In this poster presentation, we demonstrate a novel method to output PDQuest™ results in html format. With this method, 1) the result can be exported into two files with total size less than 100kb, 2) neither installation nor knowledge of PDQuest™ or the viewer version is needed, 3) the 2D image and highlighted protein spots can be viewed with Internet Explorer or any internet browser, and 4) information about the spot, such as SSP, name, PI, MW, GI access, and additional information from NCBI can be obtained with a mouse click.

Z. Lin, D. Crockett, M. Lim, K. Elenitoba-Johnson;
ARUP Laboratories, Salt Lake City, UT, United States.

Background: The identification of the interacting proteins within complexes is key to understanding the regulation of cell signaling pathways, and discovery of novel disease markers. Immunoprecipitation of the protein complex followed by LC-MS/MS has been used to identify targets that bind to a protein of interest. Here, we report a high-throughput NSI-LC-MS/MS method for analysis of protein/peptide complexes.
Approach: To overcome the large dwell volume from connecting lines and the injector when using an autosampler in microcapillary LC-MS/MS, we employed a valve-controlled variable flow method with a peptide trap. This method enables fast trapping of peptides from samples injected by an auto-sampler within minutes followed by a split-controlled nanoflow of acetonitrile gradient through a microcapillary C18 column to separate and elute peptides into the ion-trap MS/MS. Over 40 proteins/peptides samples at fmol levels can be analyzed continuously using the same in-house packed microcapillary C18 column.
The p38 MAP kinase is a signaling protein that is involved in regulation of apoptosis, cell cycle and tumorigenesis. Immuno-complex of monoclonal anti-p38 antibodies from a 2 mg total lysates of lymphoma cells was resolved in 1-D-PAGE gel followed by silver staining of the gel. Protein bands were excised and digested with trypsin. Peptides were extracted and analyzed by the automated LC-MS/MS method.
Results and Conclusions: From 37 excised protein bands in the 1-D-page gel, we identified more than 50 proteins, including cytoskeletal proteins, ribosomal proteins, transcription factors and KIAA potential signaling proteins. These proteins are the potential targets that may interact directly or indirectly with p38 MAP kinase. Our studies demonstrate the utility of automated nanoflow LC-MS/MS for sensitive and high-throughput analysis of protein/peptide complexes.

P. T. Jedrzejewski, G. Ganshaw, A. Gaertner;
Genencor International, Inc., Palo Alto, CA, United States.

The study of the proteome is a critical step towards the functional annotation of the genome. In contrast to genomic analyses, i.e. gene arrays, robust, sensitive, and comprehensive methods for proteome separation and identification are not yet available. Current proteomic methodologies, such as 2D-PAGE, ICAT suffer from limitations (e.g., difficult to automate, sample throughput, sensitivity, dynamic range, not comprehensive). In order to overcome these limitations, we have developed alternate methods, specifically, multidimensional chromatographic, for proteome analyses. These methods allow for high degree of automation in addition to other advantages (e.g., higher sample capacity, dynamic range).
In this presentation, we will show the application of multidimensional methods for the analysis of complex biological samples. Several multidimensional approaches were evaluated. As a model biological system, we have selected the exoproteome of a cellulase producing strain of the filamentous fungus Trichoderma reesei. We have evaluated the effectiveness of multidimensional chromatography (ion-exchange chromatography followed by reversed-phase chromatography) on the protein level. In order to improve throughput, we also investigated single dimension chromatography followed by solid phase extraction in a microtiter format. Following separation, fractions were processed by a liquid handler and analyzed on nanoLC-MS/MS system. Protein identification was performed using the database search software.
Using these approaches we successfully identified over 35 proteins from this fungal exoproteome. In less than a third of the time of a typical 2D-PAGE analysis (ca 26hrs), approximately 4-fold greater number of proteins were identified. The advantages and limitations of these techniques for the characterization of proteomes as compared to other methods, such as 2D-PAGE will be presented.

K. Meyer-Arendt^1,2, A. Mendoza², L. Aveline^1,2, N. Ahn^1,2, K. Resing²;
¹Howard Hughes Medical Institute, Boulder, CO, United States, ²University of Colorado, Boulder, CO, United States.

Expression profiling of the proteome by proteolysis of cell lysates and mass spectrometric (MS) identification of the peptides (shotgun proteomics) requires multidimensional chromatographic separation of the peptides. As multidimensional protein identification technology (MudPIT) MS analysis moves into a high-throughput domain, automated protein identification becomes increasingly important. Identification based on database search methods as done by Sequest and Mascot has become the de facto standard. Repeated analyses of peptides from a mammalian proteome show that approximately 80% of MS/MS spectra are identified incorrectly. We have implemented software which bases our peptide identification on a combination of database search programs and our own algorithms. A preprocessing step removes +2/+3 decoy dtas, recognizes implausible results by database search programs, and corrects elution time values across samples. Our software then groups and scores dtas based on spectral similarity. Finally, we combine our analyses with validated peptide identification obtained from the database search programs in order to provide evidence for protein identification. The program also supports visualization of comparative protein profiles from different experimental samples. Evaluation of our software results in fewer, but far more believable, identified proteins.

C. Shieh, R. Kiyonami, R. Shen, B. Wu;
ThermoFinnigan corp., San Jose, CA, United States.

Protein identification using peptide mapping has become an important technique for proteomic studies. Proteins were digested with a site-specific enzyme and analyzed by mass spectrometry. By comparison of the measured fragment ions of the peptides (e.g. in tandem MS or ion trap) to those predicated by the sequence, one could elucidate the corresponding protein identification through a database search. Previous analysis of proteins relies on 1D- or 2D-gel separation, followed by MS analysis. In this paper, we reported the analysis of protein mixture using an automated 2D-LC/MS/MS system equipped with a new orthogonal source in the LCQ ion trap mass spectrometer to increase the sensitivity of analysis.
Protein mixture from the human plasma is digested and loaded onto a strong cation exchange column and then gradually released to a 0.18 mm diameter C18 column by stepwise elution with salt steps of increasing molarity. After reversed phase HPLC separation, the peptides are analyzed by a LCQ^TM Deca XP mass spectrometer with a microflow electro-spray interface. Using Sequest™ software, more than 600 proteins were identified in one experiment. Compared to the current two dimensional electrophoresis method, this on-line 2D LC-MS/MS system has the advantages of higher identification capacity, higher sensitivity, higher throughput and a higher degree of automation.

R. Van Soest¹, R. Swart², M. Smoluch², G. Mitulovic², J. Chervet², H. Schwartz³, D. Patterson⁴;
¹LC Packings USA, San Francisco, CA, United States, ²LC Packings, a Dionex Company, Amsterdam, Netherlands, ³Dionex Corporation, Sunnyvale, CA, United States, ⁴Applied Biosystems, Framingham, MA, United States.

Here we present the use of a high precision X-Y-Z robotic system (Probot™) allowing direct interfacing of Capillary/Nano LC separation with MALDI/MS. By moving only the collection table of the robotic system and not the collection needle, precision of ±2 µm is achieved routinely. The use of a static needle allows for the collection of extremely small, nanoliter volumes (impossible with a moving needle due to capillary forces). For optimal crystallization the needle set-up allows for co-axial addition of matrix solution. This can be done during or prior to fraction collection and generates a perfect sweet spot. For high-throughput using MALDI/TOF/TOF MS, up to 6 high density targets can be placed on the table deck. With Nano LC (~200 nl/min), collection times as small as 2 sec are possible resulting in collection (spotting) volumes down to 7 nl. Under these conditions no chromatographic dispersion is observed resulting in the highest MALDI/MS sensitivity. Another advantage of the interfacing of Capillary/Nano LC with MALDI/MS is the storage capability of targets (up to several months, thus permitting optimization of work flows and sample re-analysis).
PROBOT is a versatile instrument, allowing the use of MALDI targets from different vendors including MALDI/TOF/TOF high-throughput analyzers. In this poster data will be shown using the PROBOT Micro Fraction Collector as the perfect interface between Capillary/Nano LC and MALDI/MS for high-throughput Proteomics.

S. J. Berger¹, K. Millea², A. Chakraborty¹, I. S. Krull², S. A. Cohen¹;
¹Waters Corporation, Milford, MA, United States, ²Department of Chemistry, Northeastern University, Boston, MA, United States.

The complexity of proteomic samples is highly conducive to the use of chromatographic prefractionation strategies at the intact protein level. A primary benefit derived from prefractionation is that simplified mixtures of analytes can be presented to a detection system (e.g. gel or LC/MS) or analysis technique (e.g. ICAT). In many cases, prefractionation also addresses dynamic range issues with proteomic samples, by resolving lower abundance from higher abundance components. When using mass spectrometry directly for intact protein analysis, another important benefit arises from grouping proteins of similar characteristics. The capabilities of ion exchange chromatography (IEX) to enrich proteins of similar charge density (basic/neutral/acidic proteins), and denaturing size exclusion chromatography (SEC) to enrich proteins of similar MW, creates protein subpopulations less likely to be obscured by competitive ionization effects encountered during ESI and MALDI MS experiments. Reversed phase separation conditions can also be optimized for the individual resolved fractions, leading to more effective LC/ESI-MS and LC/MALDI-MS analyses.
While affinity selection typically exhibits the greatest selectivity of prefractionation techniques, it is often used only to answer very focused biological questions. More typically, a scientist requires a generalized methodology to accomplish a global proteomic analysis. In this work, we will demonstrate a general approach to developing strategies for proteomic sample prefractionation, using two basic modes of biochromatography. In particular, the implementation of the AutoBlend™ (Warren et.al. (1989) Amer. Biotechnol Lab 6:120-6) technique for systematic ion exchange method development will be presented, and the capabilities of denaturing versus nondenaturing SEC will be described with regard to multiple global proteomic samples.

M. L. Shen¹, D. A. Sarracino², P. Kowalski¹, R. van Soest³;
¹Bruker Daltonics, Inc., Billerica, MA, United States, ²Matritech, Newton, MA, United States, ³LC Packings USA/Dionex, San Francisco, CA, United States.

Breast cancer is one of the leading causes in cancer deaths in women. Alternative methods that could improve the early detection and diagnosis are being explored. With recent developments in nanoscale separation technology and mass spectrometry, this task can be achieved with relative ease. We report a technology which could be employed to discover novel disease markers from human tissues, which could be extended to screen for breast cancer development at an earlier stage.
Specifically, human serum samples were collected from patients in various stages of breast cancer development. Proteins in the serum were extensively prefractionated with cation exchange chromatography to enrich and concentrate potential biomarkers which are present at very low abundance. Fractions were collected from the cation exchange chromatography, and reduced, alkylated and digested. The digestion products were then subjected to two-dimensional chromatography with an LC Packings/Dionex UltiMate™ system followed by MS/MS analyses on mass spectrometers. The data collected from the two-dimensional analyses were automatically processed, combined and searched against human protein database. The results obtained from cancerous serum samples were directly compared with those from normal samples to identify potential biomarkers.
We demonstrate here that protein markers could be identified in cancerous serum while they were not observed in normal serum. The experimental approach that we used in this pilot study has proved to be indispensable in the detection of disease markers which are present in extremely low concentration. The strategy of combining extensive prefractionation with multidimensional LC-MS/MS can dramatically enhance the detectability of biomarkers which were, otherwise, masked by hugely abundant serum proteins.

B. Glatz¹, P. Hoerth¹, M. Vollmer¹, P. Goodley²;
¹R+D, Waldbronn, Germany, ²R+D, Palo Alto, CA, United States.

The major limitations in the analysis of low abundance proteins from complex mixtures of complex biological fluids can be attributed to an over abundance of co-eluting peptides in a limited time domain. Over crowding and "stacking" of peptides inhibits the effective MS/MS identification even with data dependent MS/MS detection. This is especially true for low-level peptides, which are over whelmed by the larger abundance peptides. In order to reduce the sheer number of co-eluting peptides from the biological extracts, both gel-bands and fluids, multi-dimensional separations have been preformed using shallow gradients, longer columns. One current approach is to use salt steps to elute the peptides from strong cat ion exchanger columns (SCX) however the salt step process limits the separation and the selectivity. This work will demonstrate the effective separation, collection, and reinjection to achieve additional peptides and protein identifications. Optimized linear SCX salt gradients were used with micro-fraction collection and re-injection onto high performance reverse phase analytical columns, which significantly increased the observed number of low level peptides. These additional peptides allow for increase in the number of proteins from trypsin digest of Plastids. An about 30% increase in the number of protein identifi