Poster Session A

A.1 Labeling of Peptide Fragmented Mass Spectra in Proteomic Studies B. Gerrits1, C. Panse1, B. Bodenmiller2, and R. Schlapbach1 1UZH/ETH Functional Genomics Center, Zurich, Switzerland; 2IMSB/ETH, Zurich, Switzerland Due to the advent of accurate and fast sampling mass spectrometers, proteomic experiments often contain thousands of peptide fragmentation spectra. Although it is commonly accepted that no manual validation of individual spectra in such experiments is feasible, annotated spectra of the peptides assignments with their modifications are required for publication and reviewing purposes. Here we present an algorithm that greatly facilitates the visualisation of peptide fragmentation spectra and aides with the quality assessment of modification sites such as phosphorylation. The application has two inputs: 1) the Mascot dat-file including the Mascot modifications and 2) the assignment list containing the query number and peptide rank. In the first step using Perl, the application retrieves the peptide assignments and computes the theoretical fragments. These are then mapped to the peak list with the error margin specified during the initial search. During the second stage using R, two different heuristics can be chosen to calculate the appropriate text-labels and print the labelled spectra. To demonstrate the usefulness of the peakplot application we built a CGI based world wide web accessible userinterface. Also via this interface several data sets are available for testing. As datfile content based filtering aion score cut-off, peptide query hit selection, and a selection by peptide modification based on the mascot servermodification file are provided. As output on default four different colour schemes areprovided, as well as one multi panel plot, which provides additional graphics and statistics about the assigned peaklist. A.2 Exact Quantification of Complex Protein Mixtures Using MeCAT—Metal Coded Tagging R. Ahrends1, U. Bergmann1, S. Pieper1, B. Neumann2, C. Scheler2, and M. W. Linscheid1 1Humboldt-Universitaet zu Berlin, Germany; 2Proteome Factory AG, Berlin, Germany Quantitative peptide and protein analysis is one of the most promising fields in modern life science. Besides stable isotope coded labeling, metal chelate complexes are an alternative tool for quantification. The development of metal-coded affinity tags (MeCAT) was aimed to provide a robust tool for the quantification of peptides and proteins by utilizing lanthanideharboring metal tags. It was shown that MeCAT is suited for relative quantification of proteins via standard mass spectrometric methods. The approach of tagging biomolecules with MeCAT offers the unique advantage of absolute quantification via inductively coupled plasma mass spectrometry (ICPMS), a well established technique for assessing concentrations down to low attomole ranges. Proteins and peptides are labeled by MeCAT reagents which contain an amino acid residue-reactive labeling group and an element tag loaded with a lanthanide ion. By using different lanthanides such as Lutetium, Holmium, Thulium and Terbium in the MeCAT reagent, multiplex experiments can be performed to analyze several protein samples simultaneously in a proteomic study. After MeCAT labeling peptides and proteins are separated by common chromatography or electrophoresis techniques and quantified by LC/ESI MS or Inductively Coupled Plasma Mass Spectrometry (ICPMS) detecting the amount of MeCAT metal as a measure for quantity of the protein. If required, proteins of interest are identified by nanoLC/ESI MSn. In this work we investigated the compatibility of MeCAT labeling to analysis workflows such as nano liquid chromato-graphy/electrospray ionization tandem mass spectrometry (nano-LC/ESI-MSn) and electrophoresis followed by FIA/ICPMS. Focus was given to the separation behavior of labeled peptides and proteins as well as the dynamic range of detection. Furthermore, we demonstrated that MeCAT complexes are stable under a variety of conditions and that by applying LC/ ESI-MS it is possible to cover a dynamic range of 2 orders of magnitude down to the low femtomole range with an average standard deviation below 15%. Next to the relative quantification pathway applying LC/ ESI-MS we also developed a two dimensional gel based separation system for MeCAT labeled proteins in combination with FIA/ICPMS for absolute quantification of proteinsWith the application of the MeCAT technique to a standard analysis scheme in proteomics, such as the investigation of heat induced expression of recombinant proteins in an Escherichia coli High Cell Density Culture (HCDC), we successfully addressed the suitability to utilize MeCAT on biological samples. Several regulated proteins were identified and quantified including the recombinant Aprotinin::β-galactosidase, heat shock proteins, aconitase, and oligopeptide binding protein. Besides the obtained relative quantification data, we were able to analyze the recombinant expressed pharmacological active protein Aprotinin (Aprotinin::²-galactosidase) on protein level in an absolute fashion by applying MeCAT-tags in combination with FIA/ICPMS and external calibration. For absolute quantification on the peptide level, metal-coded synthetic peptides which are quantified externally by FIA/ICPMS serve as internal standards in complex peptides mixtures obtained from tryptic proteolysis of biological samples. A.3 On the Reproducibility of a Fractionation Procedure for Fish Muscle Proteomics P. Rodrigues1, T. Silva1, F. Jessen2, and J. Dias1 1CCMAR, Universidade do Algarve, FCMA, Centro de Ciências do Mar do Algarve, Campus de Gambelas, Faro, Portugal; 2DTU Aqua, Institut for Akvatiske Ressourcer, Danmarks Tekniske Universitet, Lyngby, Denmark Sub-cellular fractionation procedures for muscle tissue have been used for some time in proteomics, easing analysis by reducing the number of proteins in a given extract (and therefore improving the dynamic range by allowing larger loads per protein). On the other hand, since it implies a greater number of sample processing steps than a whole extract, it is expected that some noise could be introduced by this fractionation procedure. The aim of this study was to assess if the amount of noise introduced by a muscle fractionation procedure was significant by comparing it to the baseline technical noise level inherent to 2DE runs, Indiana, USA order to determine if this fractionation method was valid for proteome analysis, using the gilthead seabream (Sparus aurata) as model. For the experiment, two groups of 5 gilthead seabreams each were subjected to distinct levels of pre-slaughter stress to assess its effects in flesh quality, having obtained three samples of the dorsal muscle from each (post-slaughter, pre-rigor and post-rigor) for a total of 30 samples. These samples were fractionated in five batches, taking care to avoid variable confounding, and separated by 2DE. The results obtained seem to indicate that the use of this fractionation procedure introduces a low amount of noise, due to the fact that samples fractionated in different batches have the same level of variation as two samples that were fractionated in the same batch. In addition, when attempting to cluster the samples using several different metrics, no grouping made a distinction between fractionation batches. This shows that this fractionation method can be useful for some proteomic studies involving muscle tissue, especially in cases where the quantity of low abundance proteins is important. A.4 Optimization of Peak Capacity in One and Two-Dimensional NanoLC S. Eeltink, B. Dolman, R. Swart, and G. Tremintin Dionex Corporation, Sunnyvale, CA To tackle contemporary proteomics samples, different approaches are available for the identification of proteins. In the bottomup approach, proteins are digested and the resulting peptides are separated by high performance liquid chromatography (HPLC). In one-dimensional (1-D) separations, column technology and operating conditions can be optimized to increase peak capacity, but can resolve only relatively simple peptides mixtures. Tryptic digestion of complex proteomics samples containing 1000 proteins can lead up to 50,000 peptides and require powerful separation techniques like multidimensional liquid chromatography (MDLC). To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional liquid che4omatography (LC), column technology and operating condiotions were optimized. The effects of gradient time, flow rate, column temperature and column length were investigated in one-dimensional reverse phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit of time for peptides separations. A.5 Improving the Utility of Electron-Transfer Dissociation K. F. Medzihradszky, S. P. Salas-Castillo, and A. L. Burlingame Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA Captured electron-triggered fragmentation produces almost exclusively peptide backbone cleavages, Indiana, USA form of c and z˙ fragments. Such “limited” fragmentation may be more beneficial for the characterization of longer sequences than CID. In addition, electron-transfer dissociation (ETD) may hold the key for the large scale, reliable site-assignment of such “fragile” post-translational modifications as phosphorylation and O-glycosylation. Evidence has been presented in the recent literature[1] that suggests that charge states equal to 3 or greater provide higher quality ETD spectra of peptides. In addition, it also has been reported that precursor ions below ∼ m/z 850 deliver the best results. In the present study we compare the ETD-based information obtained from digests generated with different sequence cleavage specificities. In addition, we report improved ETD results in conjuction with the charge-increasing derivatization of Cys-side-chains as well as carboxyl-groups. Support for this work was provided by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A. L. Burlingame, Director) through the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH NCRR P41RR001614. References 1. Good, D. M., Wirtala, M., McAlister, G. C., and Coon, J. J. (2007) Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 6, 1942–1951. A.6 Enrichment and Characterization of Secreted Glycopeptides Bearing SA1-0Galβ1-3GalNAcα Structures Z. Darula1, and K. F. Medzihradszky1,2 1Proteomics Research Group, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary; 2Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments. In addition, O-linked structures also undergo a gas-phase rearrangement reaction that eliminates the sugar without leaving a telltale sign at its former attachment site. In the present study we used electron-transfer dissociation for the characterization of intact glycopeptides affinity-enriched from bovine serum. Some glycopeptide-containing fractions were analyzed also after exoglycosidase treatment. Reducing the size of the carbohydrate chain aided the identification of multiply modified species. We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the current methods. This work was supported by Hungarian Science Foundation grants OTKA T60283 (to KFM) and by NIH grant NCRR P41RR001614 to the UCSF MS Facility (Director, A. L. Burlingame). A.7 Enrichment of O-GlcNAc Modified Proteins by the Periodate Oxidation – Hydrazide Resin Capture Approach E. Klement1, Z. Lipinszky2, Z. Kupihar3, A. Udvardy2, and K. F. Medzihradszky1,4 1Proteomics Research Group, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary; 2Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary; 3Department of Medical Chemistry, University of Szeged, Szeged, Hungary; 4Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA O-GlcNAc is a post-translational modification found on serine and threonine residues of cytosolic and nuclear proteins. The modification is dynamic and occurs at substoichiometric levels, therefore enrichment is essential. The different strategies so far include chemoenzymatic labeling, beta-elimination of the sugar moiety and lectin weak affinity chromatography. Here, we present a novel enrichment strategy that is based on the periodate oxidation – hydrazide capture approach developed for N-linked glycoproteins. Because of the differences between the two types of carbohydrate modifications the oxidation and elution steps had to be modified. The enrichment protocol was optimized on a mixture of alpha-crystallin and BSA. Then the method was applied to the proteasome complex previously reported as O-GlcNAc modified [1]. Novel modification sites on proteins co-purifying with the proteasome complex will be presented. This work was supported by Hungarian Science Foundation grant OTKA T60283 (to KFM) and by NIH grant NCRR P41RR001614 to the UCSF MS Facility (Director, A. L. Burlingame). References 1. Suümegi, M., Hunyadi-Gulyás, E., Medzihradszky, K. F., and Udvardy, A. (2003) 26S proteasome subunits are O-linked N-acetylglucosaminemodified in Drosophila melanogaster. Biochem. Biophys. Res. Commun. 312, 1284–1289. A.8 Proteome Survey Using Affinity Proteomics and Mass Spectrometry N. Olsson1, C. Wingren1, M. Mattsson2, P. James1, F. Nilsson2, and C. A. K. Borrebaeck1 1Department of Immunotechnology, Lund University, Lund, Sweden; 2BioInvent International AB, Lund, Sweden Affinity proteomic methodologies, such as antibody-based microarrays, have shown great promise in several proteome expression profiling applications. The resolution of such proteome analyses is, however, directly related to the number of antibodies included on the array, which currently is a key bottleneck. Here, we present a conceptually new method, denoted Global Proteome Survey (GPS), based on combining the premium features of affinity proteomics and mass spectrometry. The approach will provide novel possibilities for targeting a significant fraction of a proteome in a specie independent manner still using a limited set of antibodies. To this end, we have designed a new class of antibodies, denoted context-independent motif specific (CIMS) antibodies. We have defined sets of short peptide motifs, 4 or 6 amino acids long, where each motif was present in up to a few hundred different proteins (using the human proteome as model system). In this manner, 200 antibodies, binding 50 different motifs commonly distributed among different proteins, would potentially target a protein cluster of 10000 individual molecules, i.e. around 50% of the nonredundant human proteome. To date, we have successfully selected 91 CIMS antibodies against 27 motifs, using our human recombinant scFv antibody library, composed of 2 × 1010 members and microarray adapted by molecular design, as a renewable probe source. Next, the binders were immobilized in an array format, multiplexed plate format or column format and used to capture and enrich motifcarrying peptides from the digested (trypsinated) proteome(s). The captured peptides were then detected, identified and in some cases even quantified (label-free) using MS and tandem-MS based readout. In this study, we profiled human colon tissue extracts and mouse liver homogenates to demonstrate proof-of-concept of the method. The results showed that the CIMS antibodies were capable of binding and enriching numerous peptides (proteins) harboring the corresponding selection motif, even when targeting crude digests originating from different species. The CIMS antibodies were found to recognize a linear epitope composed of two to four conserved residues, while the identity of the neighboring residues was more flexible. Taken together, the GPS platform has the potential to become a key discovery technology for highthroughput analysis of complex proteomes in health and disease in a specie independent manner. A.9 Sampling the N-terminal Proteome of Human Serum and Plasma P. Wildes1 and J. A. Wells1,2 Departments of 1Pharmaceutical Chemistry and 2Cellular and Molecular Pharmacology, University of California, San Francisco, CA The N-terminal proteomes of serum and plasma are complex and diverse, due to the importance of limited proteolysis in many extracellular signaling pathways. We have developed a method for labeling and enrichment of N-terminal peptides based on specific biotinylation of N-terminal alpha amines using subtiligase, an engineered enzyme. We have employed this method to identify nearly 800 N-terminal peptides in over 200 proteins in human serum and plasma, ranging down to low nM concentrations. While many of these N-termini correspond to known proteolytic processing events (e.g. signal peptide removal, prohormone processing, coagulation or complement activation), nearly 75 percent correspond to exo- or endo-proteolytic cleavages that have not been reported previously. In addition to identifying previously unknown sites of proteolytic processing, N-terminal isolation also allows us to sample one or a few representative peptides from proteins in serum, dramatically reducing the complexity of the sample. The N-terminal peptides can serve as markers of proteolytic events, Oregon, USA surrogates of the intact protein abundance. We are currently developing methods for label-free quantitation of N-terminal peptides to investigate their potential utility as biomarkers. A.10 Profiling Cell Surface and Secreted Glycoproteins Isolated from Human Thyroid Cancer Cell Lines T.-Y. Yen, N. Haste, A. Castanieto, A. Arcinas, and B. Macher San Francisco State University, Department of Chemistry & Biochemistry, San Francisco, CA We have obtained proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using 2-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), Hürthle cell carcinoma (XTC-1), and metastases of follicular thyroid cancer (FTC-133, FTC-236 and FTC-238) were evaluated. On average more than 100 glycoproteins were identified per cell line, of which about 60 percent are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g. CD44 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in a specific thyroid cancer cell line. Based on the results obtained by mass spectrometry, a set of glycoprotein biomarker candidates for thyroid cancer is proposed. We are currently quantitatively comparing the relative abundance of a subset of the glycoproteins identified using a label-free quantification method. These results are being compared with quantifications done using antibodies to the glycoproteins. A.11 Chemical Cross-linking in Complex Mixtures M. J. Trnka and A. L. Burlingame Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA Many fundamental physiological processes are catalyzed by stable protein complexes (e.g. histone remodelling, mRNA splicing by the spliceosome, protein degradation by the proteosome, transit across the nuclear envelope by the nuclear pore complex, initiation of apoptosis). The compositions of these complexes have been intensively studied by immunoprecipitation and affinity purification methodologies combined with mass spectrometry. However, these methods remove all information concerning the spatial arrangement of the constituent proteins within a complex. Furthermore, x-ray structures of larger complexes are difficult to obtain. Chemical cross-linking reagents are used to preserve topographic information by inserting new covalent bonds between neighboring proteins of a complex. After tryptic digestion, the covalently joined peptides are identified by tandem mass spectrometry, and the sites of modification are used to infer proximity of the original proteins. Chemical cross-linking methodologies have suffered due to the low yields of true interpeptide cross-links relative to unmodified peptides and so called “dead-end” modified peptides, where only one of the two reactive moieties of the cross-linking reagent reacts with protein. This problem is inherent to all cross-linking reagents that employ activated esters, such as N-hydroxy succinimide esters, as reactive moieties, because succesful cross-linking must compete with hydrolysis. Here we present a cross-linking reaction and enrichment strategy that allows discrimination between “dead-end” modified peptides, cross-linked peptides, and unmodified peptides. We have synthesized a trifunctional cross-linking reagent which contains two electrophilic formyl groups and an alkyne moiety. The formyl groups react with lysine residues in the presence of a reducing agent via a reductive amination mechanism. “Dead-end” modified peptides therefore contain an aldehyde moiety that can be used as a chemical handle to deplete these peptides. Cross-linked peptides can then be enriched by Copper catalyzed Huisgen cylcoaddition of the alkyne handle with a cleavable azido-biotin reagent. This poster presents optimization of reaction conditions to effect this discrimination in both model proteins and in E. coli cell lysates. Research support was provided by the Bio-Organic Biomedical Mass Spectrometry Resource (A. L. Burlingame, Director) through the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH NCRR P41RR001614. A.12 Rapid MRM Assay Development Strategies — Intelligent Software and Acquisition Strategies for Highest Productivity S. Mollah, M. M. Champion, and C. L. Hunter Applied Biosystems, Foster City, CA Targeted peptide quantitation is a rapidly growing application within proteomics mass spectrometry due to its widespread utility in biomarker verification, protein/peptide confirmation and characterization, as well as pathway mapping. As more extensive protein panels need to be monitored in a targeted way across multiple samples, higher throughput is becoming essential. The need for rapid assay development, higher multiplexing and more robust assays are some of the key challenges. In this work, the combination of unique workflows on the hybrid triple quadrupole linear ion trap mass spectrometer and automated softwares, MRMPilot™ Software and MultiQuant™ Software, have been used to automate and simplify creation of highly multiplexed MRM assays. The complete workflow of taking proteomics discovery data, to refinement of MRM transitions, resulting in creation of a final MRM assay of >1000 MRM transitions can now take just a matter of days. In this study, E. coli samples grown in various growth conditions were used for analysis to illustrate the efficiency of the MRM assay development process. Labeling strategy using mTRAQ reagents Δ 0, Δ 4, and Δ 8 were used with one of the mTRAQ® reagent labeled sample acting as the internal standard (GIS). This provides an added improvement in the robustness of the assay development. This efficient workflow resulted in developing an MRM assay at a rate of ∼2 peptide/hr (∼48 peptide/day). This has reduced the time required for developing a large MRM assay from weeks to just a matter of days. All assay development/MRM refinement was done from biological matrix, no synthetic peptides are required for this assay development strategy, reducing overall project cost. A.13 Structural Proteins in the Complex Phage 201phi2-1 S. Weintraub, J. A. Thomas, K. Hakala, P. Serwer, and S. C. Hardies University of Texas Health Science Center, San Antonio, TX Tailed phages are bacterial viruses with a DNA genome in a protein head that is connected to a tail used to inject the DNA into a host cell. The wide variety of phage types and infective mechanisms are of interest for use as antibacterial agents. We isolated and sequenced the 316,674-bp genome of the unusual Pseudomonas chlororaphis phage 201phi2-1. Since the function of most of the 460 predicted encoded proteins could not be assigned by comparative methods, proteins identified by MS were used to supplement the informatics. An unprecedented number of virion proteins (88) were identified, with several of them (18) having been cleaved to more than one polypeptide. With the high sequence coverage and large number of semitryptic peptides that were found, we could define many of the polypeptide end points, and hence the cleavage motif of the prohead protease responsible for these cleavages. Most phages cleave the major capsid protein to enable capsid expansion during DNA packaging, accounting for one of the detected 201phi2-1 cleavages. The cleavage patterns were combined with informatic analysis to hypothesize what other maturation processes may be occurring in this phage. One of the cleaved proteins was found by customized profile building methods to be a distant homolog of the beta chain of RNA polymerase. After cleavage, the propeptide as well as the polymerase chain remained in the head. We hypothesize that the propeptide determines capsid localization and that cleavage releases the polymerase chain for injection into the cell. For one of the three RNAP subunits found in the virion, Mississippi, USA showed that there had been a selfsplicing intron. Most phages encode a scaffold protein around which the capsid assembles and is then cleaved during DNA entry and leaves the virion. Scaffold-like sequences were found in the N-terminal propeptides of a family of 6 capsid proteins for which the C-terminal domains were homologous and retained in the mature virion. The stoichiometry of the C-terminal domains within the virion was estimated by spectrum counting, and the mass of propeptides that had been released from the virion was subsequently estimated to be appropriate for the scaffold of a virion of this size. The retained C-terminal domains were presumed to compose the novel inner head body reported for this virus. Finally we sought to resolve a discrepancy by which plots of spectrum counts indicated substantial downgel smears inconsistent with the Coomassie profile. The peptide coverage in the downgel smears was consistent with a degree of nonspecific protein degradation. By comparison of spectrum counts with known stoichiometry of several virion proteins, a saturation effect was found which overestimates the amount of rare degraded species in each gel slice. Efforts are underway to include precursor ion intensities in these assessments as a way to more accurately determine the relative copy number of the 201phi2-1 structural proteins. A.14 A Quick Method for Differential PTM Analysis of Hypermodified Proteins by FTICR/ECD/MS/MS and Bioinformatics F. Li1, S. Guan1, F. Chu2, R. Talroze1, and A. L. Burlingame1 1Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA; 2College of Life Sciences and Agriculture, University of New Hampshire, Durham, NH Mass Spectrometry has evolved to be the most powerful method for protein identification and characterization of protein covalent modifications. The nature and position of many protein post-translational modifications (PTM) can be identified and even quantified by recently developed mass spectrometry methods. Moreover, different from other methods, mass spectrometry can be employed to discover new proteins and new modifications on proteins. Despite the impressive progress of mass spectrometry in biological applications in the past decade, determination of post-translational modifications on proteins is far from being routine. Usually labor-intensive and time-consuming sample preparation step is required to enrich or isolate the modified species and success may not be guaranteed. Quick, easy and economical methods for differential PTM analysis without identifying each PTM on multiple samples for comparison are highly desired in many biological research fields. However, there is no such method reported in literature. Here, we present a method to address this unmet need. Histones are biologically important proteins with many modifications. Characterizing the modifications on histones is even more challenging than a regular PTM determination. Two Histone H4 samples from the wild type and a mutant of mouse embryonic stem cells were used to illustrate this method. Each of the two histone H4 samples was first directly infused to a FTICR mass spectrometer respectively as intact proteins. MS molecular profiles of all modified species can be obtained for each sample. All isomers with the same M/Z were further isolated from other species and subjected to MS/MS fragmentation by ECD. Multiple MS/MS spectra were accumulated to improve the signal to noise ratio of ECD spectra. A “search all possibility” algorithm was developed to match theoretical MS/MS isotope profiles of all the possible PTM combinations on the sequence to the experimental MS/MS peaks. All the matched experimental peaks are mapped on the sequence indicating numbers of PTMs and the mapping clearly illustrates the “segmental” PTM assignments. In addition, all the modified sequences were ranked in the order of the degree how well the theoretical peaks were matched with the experimental data. Such a list of the best matched modified sequences, combined with the segmental PTM assignments, was used to detect the differences between two samples without having to identify the individual PTM on each sample. The final results show that the nature of PTMs was the same for both samples while the abundances of some certain modified species were different in the detectable dynamic range. This method requires minimal sample preparation. Neither enzymatic digestion nor analytical separation of PTM variants is required. The difficulty of handling sample complexity is shifted from sample preparation to the MS/MS data analysis stage. This method can serve as a general screening method for detecting difference among biologically related samples such as (1) wild type and mutant, (2) normal and degraded, (3) healthy and disease samples and it is especially useful for those hypermodified proteins such as histones in a fast and high throughput way. This work is supported by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A. L. Burlingame, Director) through the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH Grants NCRR P41RR001614 and NCRR RR019934. A.15 Improved Data Mining by Using TPP-based Analysis Workflows for Searching MS/MS Data A. Quandt1, L. Malstroem1, H. Lam2, D. Shteynberg3, and R. Aebersold1 1Institute for Molecular Systems Biology, ETH Zurich, Switzerland; 2Department of Chemical and Biomolcular Engineering, The Hong Kong University of Science and Technology, Kowloon, Hong Kong; 3Institute for Systems Biology, Seattle, WA The identification and characterisation of peptides from tandem mass spectrometry (MS/MS) data represents a critical aspect of proteomics. In the past, many software packages have been developed to tackle this problem. Beside the development of new analysis tools, recent publications describe also the pipelining of different search programs to increase the identification rate. Unfortunately, it remains still unclear for the practical user when to apply which software or parameter set to retrieve optimal results. Most people rely on the usage of a identification software what consequently often leads to a significant proportion of the experimental spectra which are not going to be identified. Hence, the usage of different tools and parameter combinations is crucial but seldom approached in reality. Among other reasons such as manual result validation and the handling of various data formats, the main problem still remains in the automated combination of different identification tools. Here, we present a workflow approach which is based on the TransProteomicPipeline (Keller et al., 2005; Pedrioli et al., 2004) and combines multiple search tools and strategies with the result to retrieve a more complete list of peptide identifications and a higher protein coverage. For us, a workflow is the combination of various identification tools and search strategies in parallel and/or in sequential order. In the example workflow, we are going to present here, two classical search engines (X!Tandem and OMSSA) have been combined with a spectral library search search (SpectraST). In fact, we use the output of the first two search tools to dynamically create a spectral library which is searched afterwards. To compare the performance of our workflow with the results of the individual search engines, we used the 18-protein-mix dataset, which has been especially created to benchmark different search tools and pipelines. On these initial dataset, we are able to show that the combination of various identification tools in so-called workflows leads to an increased trust into the results by lowering the level of accepted false positives identifications and a significant increased protein coverage leading to a more reliable search results. A.16 Microwave-assisted Phosphoproteomics P. Liu and W. Sandoval Genentech, Inc., S. San Francisco, CA Reversible protein phosphorylation controls a multitude of important biological functions. Elucidation of the exact site of phosphorylation is often necessary to further understand the intricate mechanisms involved in intracellular sites. We have investigated the use of microwave enhanced tryptic digestions on phosphopeptide recovery. We have incorporated the use of stable isotopically labeled peptides to quantitate differences observed. In addition we have quantitatively evaluated the use of stabilizing chemical modification of phosphopeptides and phosphoproteins for subsequent analysis by mass spectrometry or Edman degradation. Finally we have applied our findings to a global phosphoproteomic analysis of peroxide treated cell lysate. A.17 Proteome Analysis of Apoplastic Proteins in Rice Shoot Respond to Salt Stress Y. Song1, A. L. Burlingame2, and Y. Guo1,2 1Institute of Molecular and Cell Biology, Hebei Normal University, Shijiazhuang, China; 2Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA Plants have evolved sophisticated systems to cope with the adverse environmental conditions such as cold, drought and salinity due to their sessile nature. Although a lot of stress response networks have been proposed, the roles of plant apoplast have been obviously ignored in plant stress response. To investigate the role of apoplastic proteins in the salt-stress response, 10-day-old rice plants were treated with 200 mM NaCl for 1, 6 or 12 hours, and the soluble apoplast proteins were extracted and for differential analysis compared to untreated controls by 2-D DIGE saturation labeling techniques. 122 significant changed (1-ANOVA p-value <0.05) spots are identification by LC-MS/MS, and 117 spots representing 69 proteins have been identified. Of these, 37 proteins are apoplastic proteins according to bioinformatic analysis. These proteins are mostly involved in carbohydrate metabolism, oxido-reduction, protein processing and degradation. According to the results of functional categories and cluster analysis, a stress response model of apoplastic proteins has been proposed. These dates indicate that apoplast is an important portion in plant stress signal reception and response. LC-MS/MS data was provided by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A. L. Burlingame, Director) supported by the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH NCRR P41RR001614 and NIH NCRR RR019934. A.18 Investigation into the Use of Lys-N Combined with Electron Transfer Dissociation on a Quadrupole Time-of-Flight Mass Spectrometer for Peptide Sequencing J. Langridge1, J. Brown1, S. Mohammed2, N. Taouatas2, Iain D G Campuzano1, and A. J. R. Heck2 1Waters Corporation, MS Technologies Center, Manchester, United Kingdom; 2Department of Biomolecular Mass Spectrometry, Utrecht University, Utrecht, The Netherlands Recently, Lys-N was introduced as a near ideal enzyme for proteomics workflows that involve electron transfer dissociation (ETD) or MALDI-CID. Lys-N peptides with a single basic residue, Indiana, USA combination with ETD, have a clear advantage for spectral interpretation due to peptide tandem mass spectra consisting of almost exclusively c-type fragment ions. Furthermore, the combination of Lys-N and SCX enables facile fractionation/enrichment of acetylated, phosphorylated peptides with a single basic residue and non-modified peptides with a single basic residue. Recently, we described the implementation of ETD on a Q-Tof instrument and we describe here the evaluation of the Lys-N strategy on this modified instrument. BSA was reduced with 45 mM dithiothreitol (50 °C, 15 min), followed by alkylation using 110 mM Iodoacetamide (dark, RT, 15min). Lys-N was added at a ratio of 1:85 (w/w) and incubated over night at 37 °C. Metalloendopeptidase from Grifola Frondosa (Lys-N) was obtained from Seikagaku Corporation (Tokyo, Japan). Doubly or triply charged precursor cations were selected by the quadrupole and allowed to mix and react with either fluoranthene or azobenzene reagent anions previously collected in the travelling wave ion guide (TWAVE) collision cell of the QTOF. Product ions spectra were recorded by the TOF mass analyzer. We have acquired preliminary data using electron transfer dissociation (ETD) on a modified hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-Tof). Several peptides from a Lys-N digest of bovine serum albumin (BSA) have initially been investigated. A variety of the peptides from the digest were selected with the quadrupole, prior to dissociation via electron transfer from the reagent anion. For the doubly charged precursors the MS/MS data acquired show almost exclusively c-ions, these being N-terminal sequence related ions. Cleavage was observed at almost every amide bond in the peptide backbone, yielding easy-to-interpret sequence ladders. This coupled with the inherent mass measurement accuracy and resolution of the oa-TOF mass analyser makes the data easily amenable to de novo sequencing. The signal intensity of the fragment ions seemed to diminish with decreasing m/z, as previously reported. In addition to the ETD experiments the mass spectrometer can acquire alternate scans in CID, and as such data will be compared and contrasted between ETD and CID on a variety of peptides produced from Lys-N digestion and separated by nanoscale LC.

Due to the advent of accurate and fast sampling mass spectrometers, proteomic experiments often contain thousands of peptide fragmentation spectra. Although it is commonly accepted that no manual validation of individual spectra in such experiments is feasible, annotated spectra of the peptides assignments with their modifications are required for publication and reviewing purposes. Here we present an algorithm that greatly facilitates the visualisation of peptide fragmentation spectra and aides with the quality assessment of modification sites such as phosphorylation.
The application has two inputs: 1) the Mascot dat-file including the Mascot modifications and 2) the assignment list containing the query number and peptide rank. In the first step using Perl, the application retrieves the peptide assignments and computes the theoretical fragments. These are then mapped to the peak list with the error margin specified during the initial search. During the second stage using R, two different heuristics can be chosen to calculate the appropriate text-labels and print the labelled spectra.
To demonstrate the usefulness of the peakplot application we built a CGI based world wide web accessible userinterface. Also via this interface several data sets are available for testing. As datfile content based filtering aion score cut-off, peptide query hit selection, and a selection by peptide modification based on the mascot servermodification file are provided.
As output on default four different colour schemes areprovided, as well as one multi panel plot, which provides additional graphics and statistics about the assigned peaklist.

Exact Quantification of Complex Protein Mixtures Using MeCAT-Metal Coded Tagging
R. Ahrends 1 , U. Bergmann 1 , S. Pieper 1 , B. Neumann 2 , C. Scheler 2 , and M. W. Linscheid 1 Quantitative peptide and protein analysis is one of the most promising fields in modern life science. Besides stable isotope coded labeling, metal chelate complexes are an alternative tool for quantification. The development of metal-coded affinity tags (MeCAT) was aimed to provide a robust tool for the quantification of peptides and proteins by utilizing lanthanideharboring metal tags. It was shown that MeCAT is suited for relative quantification of proteins via standard mass spectrometric methods. The approach of tagging biomolecules with MeCAT offers the unique advantage of absolute quantification via inductively coupled plasma mass spectrometry (ICPMS), a well established technique for assessing concentrations down to low attomole ranges. Proteins and peptides are labeled by MeCAT reagents which contain an amino acid residue-reactive labeling group and an element tag loaded with a lanthanide ion. By using different lanthanides such as Lutetium, Holmium, Thulium and Terbium in the MeCAT reagent, multiplex experiments can be performed to analyze several protein samples simultaneously in a proteomic study.
After MeCAT labeling peptides and proteins are separated by common chromatography or electrophoresis techniques and quantified by LC/ESI MS or Inductively Coupled Plasma Mass Spectrometry (ICPMS) detecting the amount of MeCAT metal as a measure for quantity of the protein. If required, proteins of interest are identified by nanoLC/ESI MSn.
In this work we investigated the compatibility of MeCAT labeling to analysis workflows such as nano liquid chromato-graphy/electrospray ionization tandem mass spectrometry (nano-LC/ESI-MSn) and electrophoresis followed by FIA/ICPMS. Focus was given to the separation behavior of labeled peptides and proteins as well as the dynamic range of detection. Furthermore, we demonstrated that MeCAT complexes are stable under a variety of conditions and that by applying LC/ ESI-MS it is possible to cover a dynamic range of 2 orders of magnitude down to the low femtomole range with an average standard deviation below 15%.
Next to the relative quantification pathway applying LC/ ESI-MS we also developed a two dimensional gel based separation system for MeCAT labeled proteins in combination with FIA/ICPMS for absolute quantification of proteinsWith the application of the MeCAT technique to a standard analysis scheme in proteomics, such as the investigation of heat induced expression of recombinant proteins in an Escherichia coli High Cell Density Culture (HCDC), we successfully addressed the suitability to utilize MeCAT on biological samples. Several regulated proteins were identified and quantified including the recombinant Aprotinin::␤-galactosidase, heat shock proteins, aconitase, and oligopeptide binding protein.
Besides the obtained relative quantification data, we were able to analyze the recombinant expressed pharmacological active protein Aprotinin (Aprotinin::ß-galactosidase) on protein level in an absolute fashion by applying MeCAT-tags in combination with FIA/ICPMS and external calibration. For absolute quantification on the peptide level, metal-coded synthetic peptides which are quantified externally by FIA/ICPMS serve as internal standards in complex peptides mixtures obtained from tryptic proteolysis of biological samples.
Sub-cellular fractionation procedures for muscle tissue have been used for some time in proteomics, easing analysis by reducing the number of proteins in a given extract (and therefore improving the dynamic range by allowing larger loads per protein). On the other hand, since it implies a greater number of sample processing steps than a whole extract, it is expected that some noise could be introduced by this fractionation procedure.
The aim of this study was to assess if the amount of noise introduced by a muscle fractionation procedure was significant by comparing it to the baseline technical noise level inherent to 2DE runs, Indiana, USA order to determine if this fractionation method was valid for proteome analysis, using the gilthead seabream (Sparus aurata) as model.
For the experiment, two groups of 5 gilthead seabreams each were subjected to distinct levels of pre-slaughter stress to assess its effects in flesh quality, having obtained three samples of the dorsal muscle from each (post-slaughter, pre-rigor and post-rigor) for a total of 30 samples. These samples were fractionated in five batches, taking care to avoid variable confounding, and separated by 2DE.
The results obtained seem to indicate that the use of this fractionation procedure introduces a low amount of noise, due to the fact that samples fractionated in different batches have the same level of variation as two samples that were fractionated in the same batch. In addition, when attempting to cluster the samples using several different metrics, no grouping made a distinction between fractionation batches.
This shows that this fractionation method can be useful for some proteomic studies involving muscle tissue, especially in cases where the quantity of low abundance proteins is important.

A.4
Optimization of Peak Capacity in One and Two-Dimensional NanoLC S. Eeltink, B. Dolman, R. Swart, and G. Tremintin

Dionex Corporation, Sunnyvale, CA
To tackle contemporary proteomics samples, different approaches are available for the identification of proteins. In the bottomup approach, proteins are digested and the resulting peptides are separated by high performance liquid chromatography (HPLC). In one-dimensional (1-D) separations, column technology and operating conditions can be optimized to increase peak capacity, but can resolve only relatively simple peptides mixtures. Tryptic digestion of complex proteomics samples containing 1000 proteins can lead up to 50,000 peptides and require powerful separation techniques like multidimensional liquid chromatography (MDLC).
To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional liquid che4omatography (LC), column technology and operating condiotions were optimized. The effects of gradient time, flow rate, column temperature and column length were investigated in one-dimensional reverse phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit of time for peptides separations.

A.5
Improving the Utility of Electron-Transfer Dissociation K. F. Medzihradszky, S. P. Salas-Castillo, and A. L. Burlingame Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA Captured electron-triggered fragmentation produces almost exclusively peptide backbone cleavages, Indiana, USA form of c and z . fragments. Such "limited" fragmentation may be more beneficial for the characterization of longer sequences than CID. In addition, electron-transfer dissociation (ETD) may hold the key for the large scale, reliable site-assignment of such "fragile" post-translational modifications as phosphorylation and O-glycosylation. Evidence has been presented in the recent literature[1] that suggests that charge states equal to 3 or greater provide higher quality ETD spectra of peptides. In addition, it also has been reported that precursor ions below ϳ m/z 850 deliver the best results. In the present study we compare the ETD-based information obtained from digests generated with different sequence cleavage specificities. In addition, we report improved ETD results in conjuction with the charge-increasing derivatization of Cys-side-chains as well as carboxyl-groups. The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments. In addition, O-linked structures also undergo a gas-phase rearrangement reaction that eliminates the sugar without leaving a telltale sign at its former attachment site.
In the present study we used electron-transfer dissociation for the characterization of intact glycopeptides affinity-enriched from bovine serum. Some glycopeptide-containing fractions were analyzed also after exoglycosidase treatment. Reducing the size of the carbohydrate chain aided the identification of multiply modified species.
We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the current methods.
This work was supported by Hungarian Science Foundation grants OTKA T60283 (to KFM) and by NIH grant NCRR P41RR001614 to the UCSF MS Facility (Director, A. L. Burlingame). O-GlcNAc is a post-translational modification found on serine and threonine residues of cytosolic and nuclear proteins. The modification is dynamic and occurs at substoichiometric levels, therefore enrichment is essential. The different strategies so far include chemoenzymatic labeling, beta-elimination of the sugar moiety and lectin weak affinity chromatography. Here, we present a novel enrichment strategy that is based on the periodate oxidation -hydrazide capture approach developed for N-linked glycoproteins. Because of the differences between the two types of carbohydrate modifications the oxidation and elution steps had to be modified. The enrichment protocol was optimized on a mixture of alphacrystallin and BSA. Then the method was applied to the proteasome complex previously reported as O-GlcNAc modified [1]. Novel modification sites on proteins co-purifying with the proteasome complex will be presented. Affinity proteomic methodologies, such as antibody-based microarrays, have shown great promise in several proteome expression profiling applications. The resolution of such proteome analyses is, however, directly related to the number of antibodies included on the array, which currently is a key bottleneck. Here, we present a conceptually new method, denoted Global Proteome Survey (GPS), based on combining the premium features of affinity proteomics and mass spectrometry. The approach will provide novel possibilities for targeting a significant fraction of a proteome in a specie independent manner still using a limited set of antibodies. To this end, we have designed a new class of antibodies, denoted contextindependent motif specific (CIMS) antibodies. We have defined sets of short peptide motifs, 4 or 6 amino acids long, where each motif was present in up to a few hundred different proteins (using the human proteome as model system). In this manner, 200 antibodies, binding 50 different motifs commonly distributed among different proteins, would potentially target a protein cluster of 10000 individual molecules, i.e. around 50% of the nonredundant human proteome. To date, we have successfully selected 91 CIMS antibodies against 27 motifs, using our human recombinant scFv antibody library, composed of 2 x 10 10 members and microarray adapted by molecular design, as a renewable probe source. Next, the binders were immobilized in an array format, multiplexed plate format or column format and used to capture and enrich motifcarrying peptides from the digested (trypsinated) proteome(s). The captured peptides were then detected, identified and in some cases even quantified (label-free) using MS and tandem-MS based readout. In this study, we profiled human colon tissue extracts and mouse liver homogenates to demonstrate proof-of-concept of the method. The results showed that the CIMS antibodies were capable of binding and enriching numerous peptides (proteins) harboring the corresponding selection motif, even when targeting crude digests originating from different species. The CIMS antibodies were found to recognize a linear epitope composed of two to four conserved residues, while the identity of the neighboring residues was more flexible. Taken together, the GPS platform has the potential to become a key discovery technology for highthroughput analysis of complex proteomes in health and disease in a specie independent manner.

A.9
Sampling the N-terminal Proteome of Human Serum and Plasma P. Wildes 1 and J. A. Wells 1,2

Departments of 1 Pharmaceutical Chemistry and 2 Cellular and Molecular Pharmacology, University of California, San Francisco, CA
The N-terminal proteomes of serum and plasma are complex and diverse, due to the importance of limited proteolysis in many extracellular signaling pathways. We have developed a method for labeling and enrichment of N-terminal peptides based on specific biotinylation of N-terminal alpha amines using subtiligase, an engineered enzyme. We have employed this method to identify nearly 800 N-terminal peptides in over 200 proteins in human serum and plasma, ranging down to low nM concentrations. While many of these N-termini correspond to known proteolytic processing events (e.g. signal peptide removal, prohormone processing, coagulation or complement activation), nearly 75 percent correspond to exo-or endoproteolytic cleavages that have not been reported previously. In addition to identifying previously unknown sites of proteolytic processing, N-terminal isolation also allows us to sample one or a few representative peptides from proteins in serum, dramatically reducing the complexity of the sample. The N-terminal peptides can serve as markers of proteolytic events, Oregon, USA surrogates of the intact protein abundance. We are currently developing methods for label-free quantitation of N-terminal peptides to investigate their potential utility as biomarkers. We have obtained proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using 2-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), Hü rthle cell carcinoma (XTC-1), and metastases of follicular thyroid cancer (FTC-133, FTC-236 and FTC-238) were evaluated. On average more than 100 glycoproteins were identified per cell line, of which about 60 percent are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g. CD44 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in a specific thyroid cancer cell line. Based on the results obtained by mass spectrometry, a set of glycoprotein biomarker candidates for thyroid cancer is proposed. We are currently quantitatively comparing the relative abundance of a subset of the glycoproteins identified using a label-free quantification method. These results are being compared with quantifications done using antibodies to the glycoproteins.

Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA
Many fundamental physiological processes are catalyzed by stable protein complexes (e.g. histone remodelling, mRNA splicing by the spliceosome, protein degradation by the proteosome, transit across the nuclear envelope by the nuclear pore complex, initiation of apoptosis). The compositions of these complexes have been intensively studied by immunoprecipitation and affinity purification methodologies combined with mass spectrometry. However, these methods remove all information concerning the spatial arrangement of the constituent proteins within a complex. Furthermore, x-ray structures of larger complexes are difficult to obtain.
Chemical cross-linking reagents are used to preserve topographic information by inserting new covalent bonds between neighboring proteins of a complex. After tryptic digestion, the covalently joined peptides are identified by tandem mass spectrometry, and the sites of modification are used to infer proximity of the original proteins.
Chemical cross-linking methodologies have suffered due to the low yields of true interpeptide cross-links relative to unmodified peptides and so called "dead-end" modified peptides, where only one of the two reactive moieties of the cross-linking reagent reacts with protein. This problem is inherent to all cross-linking reagents that employ activated esters, such as N-hydroxy succinimide esters, as reactive moieties, because succesful cross-linking must compete with hydrolysis.
Here we present a cross-linking reaction and enrichment strategy that allows discrimination between "dead-end" modified peptides, crosslinked peptides, and unmodified peptides. We have synthesized a trifunctional cross-linking reagent which contains two electrophilic formyl groups and an alkyne moiety. The formyl groups react with lysine residues in the presence of a reducing agent via a reductive amination mechanism. "Dead-end" modified peptides therefore contain an aldehyde moiety that can be used as a chemical handle to deplete these peptides.
Cross-linked peptides can then be enriched by Copper catalyzed Huisgen cylcoaddition of the alkyne handle with a cleavable azido-biotin reagent.
This poster presents optimization of reaction conditions to effect this discrimination in both model proteins and in E. coli cell lysates.
Research support was provided by the Bio-Organic Biomedical Mass Spectrometry Resource (A. L. Burlingame, Director) through the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH NCRR P41RR001614.

S. Mollah, M. M. Champion, and C. L. Hunter Applied Biosystems, Foster City, CA
Targeted peptide quantitation is a rapidly growing application within proteomics mass spectrometry due to its widespread utility in biomarker verification, protein/peptide confirmation and characterization, as well as pathway mapping. As more extensive protein panels need to be monitored in a targeted way across multiple samples, higher throughput is becoming essential. The need for rapid assay development, higher multiplexing and more robust assays are some of the key challenges. In this work, the combination of unique workflows on the hybrid triple quadrupole linear ion trap mass spectrometer and automated softwares, MRMPilot TM Software and MultiQuant TM Software, have been used to automate and simplify creation of highly multiplexed MRM assays. The complete workflow of taking proteomics discovery data, to refinement of MRM transitions, resulting in creation of a final MRM assay of Ͼ1000 MRM transitions can now take just a matter of days.
In this study, E. coli samples grown in various growth conditions were used for analysis to illustrate the efficiency of the MRM assay development process. Labeling strategy using mTRAQ reagents ⌬ 0, ⌬ 4, and ⌬ 8 were used with one of the mTRAQ® reagent labeled sample acting as the internal standard (GIS). This provides an added improvement in the robustness of the assay development. This efficient workflow resulted in developing an MRM assay at a rate of ϳ2 peptide/hr (ϳ48 peptide/day). This has reduced the time required for developing a large MRM assay from weeks to just a matter of days. All assay development/MRM refinement was done from biological matrix, no synthetic peptides are required for this assay development strategy, reducing overall project cost.

University of Texas Health Science Center, San Antonio, TX
Tailed phages are bacterial viruses with a DNA genome in a protein head that is connected to a tail used to inject the DNA into a host cell. The wide variety of phage types and infective mechanisms are of interest for use as antibacterial agents. We isolated and sequenced the 316,674-bp genome of the unusual Pseudomonas chlororaphis phage 201phi2-1. Since the function of most of the 460 predicted encoded proteins could not be assigned by comparative methods, proteins identified by MS were used to supplement the informatics. An unprecedented number of virion proteins (88) were identified, with several of them (18) having been cleaved to more than one polypeptide. With the high sequence coverage and large number of semitryptic peptides that were found, we could define many of the polypeptide end points, and hence the cleavage motif of the prohead protease responsible for these cleavages. Most phages cleave the major capsid protein to enable capsid expansion during DNA packaging, accounting for one of the detected 201phi2-1 cleavages. The cleavage patterns were combined with informatic analysis to hypothesize what other maturation processes may be occurring in this phage. One of the cleaved proteins was found by customized profile building methods to be a distant homolog of the beta chain of RNA polymerase. After cleavage, the propeptide as well as the polymerase chain remained in the head. We hypothesize that the propeptide determines capsid localization and that cleavage releases the polymerase chain for injection into the cell. For one of the three RNAP subunits found in the virion, Mississippi, USA showed that there had been a selfsplicing intron. Most phages encode a scaffold protein around which the capsid assembles and is then cleaved during DNA entry and leaves the virion. Scaffold-like sequences were found in the N-terminal propeptides of a family of 6 capsid proteins for which the C-terminal domains were homologous and retained in the mature virion. The stoichiometry of the C-terminal domains within the virion was estimated by spectrum counting, and the mass of propeptides that had been released from the virion was subsequently estimated to be appropriate for the scaffold of a virion of this size. The retained C-terminal domains were presumed to compose the novel inner head body reported for this virus. Finally we sought to resolve a discrepancy by which plots of spectrum counts indicated substantial downgel smears inconsistent with the Coomassie profile. The peptide coverage in the downgel smears was consistent with a degree of nonspecific protein degradation. By comparison of spectrum counts with known stoichiometry of several virion proteins, a saturation effect was found which overestimates the amount of rare degraded species in each gel slice. Efforts are underway to include precursor ion intensities in these assessments as a way to more accurately determine the relative copy number of the 201phi2-1 structural proteins. Mass Spectrometry has evolved to be the most powerful method for protein identification and characterization of protein covalent modifications. The nature and position of many protein post-translational modifications (PTM) can be identified and even quantified by recently developed mass spectrometry methods. Moreover, different from other methods, mass spectrometry can be employed to discover new proteins and new modifications on proteins.

A Quick Method for Differential PTM Analysis of Hypermodified Proteins by FTICR/ECD/MS/MS and Bioinformatics
Despite the impressive progress of mass spectrometry in biological applications in the past decade, determination of post-translational modifications on proteins is far from being routine. Usually labor-intensive and time-consuming sample preparation step is required to enrich or isolate the modified species and success may not be guaranteed. Quick, easy and economical methods for differential PTM analysis without identifying each PTM on multiple samples for comparison are highly desired in many biological research fields. However, there is no such method reported in literature. Here, we present a method to address this unmet need.
Histones are biologically important proteins with many modifications. Characterizing the modifications on histones is even more challenging than a regular PTM determination. Two Histone H4 samples from the wild type and a mutant of mouse embryonic stem cells were used to illustrate this method. Each of the two histone H4 samples was first directly infused to a FTICR mass spectrometer respectively as intact proteins. MS molecular profiles of all modified species can be obtained for each sample. All isomers with the same M/Z were further isolated from other species and subjected to MS/MS fragmentation by ECD. Multiple MS/MS spectra were accumulated to improve the signal to noise ratio of ECD spectra.
A "search all possibility" algorithm was developed to match theoretical MS/MS isotope profiles of all the possible PTM combinations on the sequence to the experimental MS/MS peaks. All the matched experimental peaks are mapped on the sequence indicating numbers of PTMs and the mapping clearly illustrates the "segmental" PTM assignments. In addition, all the modified sequences were ranked in the order of the degree how well the theoretical peaks were matched with the experimental data. Such a list of the best matched modified sequences, combined with the segmental PTM assignments, was used to detect the differences between two samples without having to identify the individual PTM on each sample. The final results show that the nature of PTMs was the same for both samples while the abundances of some certain modified species were different in the detectable dynamic range.
This method requires minimal sample preparation. Neither enzymatic digestion nor analytical separation of PTM variants is required. The difficulty of handling sample complexity is shifted from sample preparation to the MS/MS data analysis stage.
This method can serve as a general screening method for detecting difference among biologically related samples such as (1) wild type and mutant, (2) normal and degraded, (3) healthy and disease samples and it is especially useful for those hypermodified proteins such as histones in a fast and high throughput way. This work is supported by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A. L. Burlingame, Director) through the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH Grants NCRR P41RR001614 and NCRR RR019934.
The identification and characterisation of peptides from tandem mass spectrometry (MS/MS) data represents a critical aspect of proteomics. In the past, many software packages have been developed to tackle this problem. Beside the development of new analysis tools, recent publications describe also the pipelining of different search programs to increase the identification rate. Unfortunately, it remains still unclear for the practical user when to apply which software or parameter set to retrieve optimal results. Most people rely on the usage of a identification software what consequently often leads to a significant proportion of the experimental spectra which are not going to be identified. Hence, the usage of different tools and parameter combinations is crucial but seldom approached in reality. Among other reasons such as manual result validation and the handling of various data formats, the main problem still remains in the automated combination of different identification tools. Here, we present a workflow approach which is based on the TransProteomicPipeline (Keller et al., 2005;Pedrioli et al., 2004) and combines multiple search tools and strategies with the result to retrieve a more complete list of peptide identifications and a higher protein coverage. For us, a workflow is the combination of various identification tools and search strategies in parallel and/or in sequential order. In the example workflow, we are going to present here, two classical search engines (X!Tandem and OMSSA) have been combined with a spectral library search search (SpectraST). In fact, we use the output of the first two search tools to dynamically create a spectral library which is searched afterwards. To compare the performance of our workflow with the results of the individual search engines, we used the 18-protein-mix dataset, which has been especially created to benchmark different search tools and pipelines. On these initial dataset, we are able to show that the combination of various identification tools in so-called workflows leads to an increased trust into the results by lowering the level of accepted false positives identifications and a significant increased protein coverage leading to a more reliable search results.

A.16
Microwave-assisted Phosphoproteomics P. Liu and W. Sandoval Genentech, Inc., S. San Francisco, CA Reversible protein phosphorylation controls a multitude of important biological functions. Elucidation of the exact site of phosphorylation is often necessary to further understand the intricate mechanisms involved in intracellular sites. We have investigated the use of microwave enhanced tryptic digestions on phosphopeptide recovery. We have incorporated the use of stable isotopically labeled peptides to quantitate differences observed. In addition we have quantitatively evaluated the use of stabilizing chemical modification of phosphopeptides and phosphoproteins for subsequent analysis by mass spectrometry or Edman degradation. Finally we have applied our findings to a global phosphoproteomic analysis of peroxide treated cell lysate.

A.17
Proteome Analysis of Apoplastic Proteins in Rice Shoot Respond to Salt Stress Y. Song 1 , A. L. Burlingame 2 , and Y. Guo 1,2 Recently, Lys-N was introduced as a near ideal enzyme for proteomics workflows that involve electron transfer dissociation (ETD) or MALDI-CID. Lys-N peptides with a single basic residue, Indiana, USA combination with ETD, have a clear advantage for spectral interpretation due to peptide tandem mass spectra consisting of almost exclusively c-type fragment ions. Furthermore, the combination of Lys-N and SCX enables facile fractionation/enrichment of acetylated, phosphorylated peptides with a single basic residue and non-modified peptides with a single basic residue. Recently, we described the implementation of ETD on a Q-Tof instrument and we describe here the evaluation of the Lys-N strategy on this modified instrument.
BSA was reduced with 45 mM dithiothreitol (50°C, 15 min), followed by alkylation using 110 mM Iodoacetamide (dark, RT, 15min). Lys-N was added at a ratio of 1:85 (w/w) and incubated over night at 37°C. Metalloendopeptidase from Grifola Frondosa (Lys-N) was obtained from Seikagaku Corporation (Tokyo, Japan). Doubly or triply charged precursor cations were selected by the quadrupole and allowed to mix and react with either fluoranthene or azobenzene reagent anions previously collected in the travelling wave ion guide (TWAVE) collision cell of the QTOF. Product ions spectra were recorded by the TOF mass analyzer.
We have acquired preliminary data using electron transfer dissociation (ETD) on a modified hybrid quadrupole orthogonal acceleration time-offlight mass spectrometer (Q-Tof). Several peptides from a Lys-N digest of bovine serum albumin (BSA) have initially been investigated. A variety of the peptides from the digest were selected with the quadrupole, prior to dissociation via electron transfer from the reagent anion. For the doubly charged precursors the MS/MS data acquired show almost exclusively c-ions, these being N-terminal sequence related ions. Cleavage was observed at almost every amide bond in the peptide backbone, yielding easy-to-interpret sequence ladders. This coupled with the inherent mass measurement accuracy and resolution of the oa-TOF mass analyser makes the data easily amenable to de novo sequencing. The signal intensity of the fragment ions seemed to diminish with decreasing m/z, as previously reported. In addition to the ETD experiments the mass spectrometer can acquire alternate scans in CID, and as such data will be compared and contrasted between ETD and CID on a variety of peptides produced from Lys-N digestion and separated by nanoscale LC.