Search

The HUPO Human Proteome Project (HPP) has two overall goals: (1) stepwise completion of the protein parts list-the draft human proteome including confidently identifying and characterizing at least one protein product from each protein-coding gene, with increasing emphasis on sequence variants, post-translational modifications (PTMs), and splice isoforms of those proteins; and (2) making proteomics an integrated counterpart to genomics throughout the biomedical and life sciences community. PeptideAtlas and GPMDB reanalyze all major human mass spectrometry data sets available through ProteomeXchange with standardized protocols and stringent quality filters; neXtProt curates and integrates mass spectrometry and other findings to present the most up to date authorative compendium of the human proteome. The HPP Guidelines for Mass Spectrometry Data Interpretation version 2.1 were applied to manuscripts submitted for this 2016 C-HPP-led special issue [ www.thehpp.org/guidelines ]. The Human Proteome presented as neXtProt version 2016-02 has 16,518 confident protein identifications (Protein Existence [PE] Level 1), up from 13,664 at 2012-12, 15,646 at 2013-09, and 16,491 at 2014-10. There are 485 proteins that would have been PE1 under the Guidelines v1.0 from 2012 but now have insufficient evidence due to the agreed-upon more stringent Guidelines v2.0 to reduce false positives. neXtProt and PeptideAtlas now both require two non-nested, uniquely mapping (proteotypic) peptides of at least 9 aa in length. There are 2,949 missing proteins (PE2+3+4) as the baseline for submissions for this fourth annual C-HPP special issue of Journal of Proteome Research. PeptideAtlas has 14,629 canonical (plus 1187 uncertain and 1755 redundant) entries. GPMDB has 16,190 EC4 entries, and the Human Protein Atlas has 10,475 entries with supportive evidence. neXtProt, PeptideAtlas, and GPMDB are rich resources of information about post-translational modifications (PTMs), single amino acid variants (SAAVSs), and splice isoforms. Meanwhile, the Biology- and Disease-driven (B/D)-HPP has created comprehensive SRM resources, generated popular protein lists to guide targeted proteomics assays for specific diseases, and launched an Early Career Researchers initiative.

The growing complexity of proteomics samples and the desire for deeper analysis drive the development of both better MS instrument and advanced multidimensional separation schemes. We applied 1D, 2D, and 3D LC-MS/MS separation protocols (all of reversed-phase C18 functionality) to a tryptic digest of whole Jurkat cell lysate to estimate the depth of proteome coverage and to collect high-quality peptide retention information. We varied pH of the eluent and hydrophobicity of ion-pairing modifier to achieve good separation orthogonality (utilization of MS instrument time). All separation modes employed identical LC settings with formic-acid-based eluents in the last dimension. The 2D protocol used a high pH-low pH scheme with 21 concatenated fractions. In the 3D protocol, six concatenated fractions from the first dimension (C18, heptafluorobutyric acid) were analyzed using the identical 2D LC-MS procedure. This approach permitted a detailed evaluation of the analysis output consuming 21× and 126× the analysis time and sample load compared to 1D. Acquisition over 189 h of instrument time in 3D mode resulted in the identification of ∼14 000 proteins and ∼250 000 unique peptides. We estimated the dynamic range via peak intensity at the MS(2) level as approximately 10(4.2), 10(5.6), and 10(6.2) for the 1D, 2D, and 3D protocols, respectively. The uniform distribution of the number of acquired MS/MS, protein, and peptide identifications across all 126 fractions and through the chromatographic time scale in the last LC-MS stage indicates good separation orthogonality. The protocol is scalable and is amenable to the use of peptide retention prediction in all dimensions. All these features make it a very good candidate for large-scale bottom-up proteomic runs, which target both protein identification as well as the collection of peptide retention data sets for targeted quantitative applications.

The honey bee is a key pollinator in agricultural operations as well as a model organism for studying the genetics and evolution of social behavior. The Apis mellifera genome has been sequenced and annotated twice over, enabling proteomics and functional genomics methods for probing relevant aspects of their biology. One troubling trend that emerged from proteomic analyses is that honey bee peptide samples consistently result in lower peptide identification rates compared with other organisms. This suggests that the genome annotation can be improved, or atypical biological processes are interfering with the mass spectrometry workflow. First, we tested whether high levels of polymorphisms could explain some of the missed identifications by searching spectra against the reference proteome (OGSv3.2) versus a customized proteome of a single honey bee, but our results indicate that this contribution was minor. Likewise, error-tolerant peptide searches lead us to eliminate unexpected post-translational modifications as a major factor in missed identifications. We then used a proteogenomic approach with ~1500 raw files to search for missing genes and new exons, to revive discarded annotations and to identify over 2000 new coding regions. These results will contribute to a more comprehensive genome annotation and facilitate continued research on this important insect.

This work has been supported by the US NIH BD2K grant U54 GM114833 and a National Natural Science Foundation of China grant (61501071). A.I.N. is supported by US National Institute of Health grant (R01-GM-094231). Y.P.-R. is supported by BBSRC ‘PROCESS’ grant (BB/K01997X/1). M.B. is supported by Projects of International Cooperation and Exchanges grant (2014DFB30010). M.W. is supported by an NIH grant (5P41GM103484-07). J.A.V. and N.d.-T. are supported by the Wellcome Trust (grant WT101477MA). T.T. is supported by the BBSRC ‘ProteoGenomics’ grant (BB/L024225/1). E.W.D. and D.S.C. are supported in part by grant (U24 AI117966- 02S1). S.-A.S. is supported in part by US NIH BD2K grant (1U24AI117966-01). M.W. and N.Bandeira were supported by NIH grant (5P41GM103484-07). N.Bandeira was also partially supported as an Alfred P. Sloan Fellow. S.Subramaniam is supported by NIH grants U01 DK097430 and U01 CA198941

Summary: The Global Proteome Machine and Database (GPMDB) representational state transfer (REST) service was designed to provide simplified access to the proteomics information in GPMDB using a stable set of methods and parameters. Version 1 of this interface gives access to 25 methods for retrieving experimental information about protein post-translational modifications, amino acid variants, alternate splicing variants and protein cleavage patterns. Availability and implementation: GPMDB data and database tables are freely available for commercial and non-commercial use. All software is also freely available, under the Artistic License. http://rest.thegpm.org/1 (GPMDB REST Service), http://wiki.thegpm.org/wiki/GPMDB_REST (Service description and help), and http://www.thegpm.org (GPM main project description and documentation). The code for the interface and an example REST client is available at ftp://ftp.thegpm.org/repos/gpmdb_rest Contact: rbeavis@thegpm.org or david@fenyolab.org Supplementary information: Supplementary data are available at Bioinformatics online.

We report progress assembling the parts list for chromosome 17 and illustrate the various processes that we have developed to integrate available data from diverse genomic and proteomic knowledge bases. As primary resources, we have used GPMDB, neXtProt, PeptideAtlas, Human Protein Atlas (HPA), and GeneCards. All sites share the common resource of Ensembl for the genome modeling information. We have defined the chromosome 17 parts list with the following information: 1169 protein-coding genes, the numbers of proteins confidently identified by various experimental approaches as documented in GPMDB, neXtProt, PeptideAtlas, and HPA, examples of typical data sets obtained by RNASeq and proteomic studies of epithelial derived tumor cell lines (disease proteome) and a normal proteome (peripheral mononuclear cells), reported evidence of post-translational modifications, and examples of alternative splice variants (ASVs). We have constructed a list of the 59 "missing" proteins as well as 201 proteins that have inconclusive mass spectrometric (MS) identifications. In this report we have defined a process to establish a baseline for the incorporation of new evidence on protein identification and characterization as well as related information from transcriptome analyses. This initial list of "missing" proteins that will guide the selection of appropriate samples for discovery studies as well as antibody reagents. Also we have illustrated the significant diversity of protein variants (including post-translational modifications, PTMs) using regions on chromosome 17 that contain important oncogenes. We emphasize the need for mandated deposition of proteomics data in public databases, the further development of improved PTM, ASV, and single nucleotide variant (SNV) databases, and the construction of Web sites that can integrate and regularly update such information. In addition, we describe the distribution of both clustered and scattered sets of protein families on the chromosome. Since chromosome 17 is rich in cancer-associated genes, we have focused the clustering of cancer-associated genes in such genomic regions and have used the ERBB2 amplicon as an example of the value of a proteogenomic approach in which one integrates transcriptomic with proteomic information and captures evidence of coexpression through coordinated regulation.

Biomedical data, in particular omics datasets are being generated at an unprecedented rate. This is due to the falling costs of generating experimental data, improved accuracy and better accessibility to different omics platforms such as genomics, proteomics and metabolomics1,2. As a result, the number of deposited datasets in public repositories originating from various omics approaches has increased dramatically in recent years. With strong support from scientific journals and funders, public data sharing is increasingly considered to be a good scientific practice, facilitating the confirmation of original results, increasing the reproducibility of the analyses, enabling the exploration of new or related hypotheses, and fostering the identification of potential errors, discouraging fraud3. This increase in public data deposition of omics results is a good starting point, but opens up a series of new challenges. For example the research community must now find more efficient ways for storing, organizing and providing access to biomedical data across platforms. These challenges range from achieving a common representation framework for the datasets and the associated metadata from different omics fields, to the availability of efficient methods, protocols and file formats for data exchange between multiple repositories. Therefore, there is a great need for development of new platforms and applications to make possible to search datasets across different omics fields, making such information accessible to the end-user. The FAIR paradigm describes a set of guiding principles to address many of these issues, and aims to make data Findable, Accessible, Interoperable and Re-usable(https://www.force11.org/group/fairgroup/fairprinciples).

Large scale proteomics have made it possible to broadly screen samples for the presence of many types of post-translational modifications, such as phosphorylation, acetylation, and ubiquitination. This type of data has allowed the localization of these modifications to either a specific site on a proteolytically generated peptide or to within a small domain on the peptide. The resulting modification acceptor sites can then be mapped onto the appropriate protein sequences and the information archived. This paper describes the usage of a very large archive of experimental observations of human post-translational modifications to create a map of the most reproducible modification observations onto the complete set of human protein sequences. This set of modification acceptor sites was then directly translated into the genomic coordinates for the codons for the residues at those sites. We constructed the database g2pDB using this protein-to-codon site mapping information. The information in g2pDB has been made available through a RESTful-style API, allowing researchers to determine which specific protein modifications would be perturbed by a set of observed nucleotide variants determined by high throughput DNA or RNA sequencing.

Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junction in cell permeability control but also vital in organizing cell contact signaling by protein-protein interactions. How this family of protein is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation.

Experiments to probe for protein-protein interactions are the focus of functional proteomic studies, thus proteomic data repositories are increasingly likely to contain a large cross-section of such information. Here, we use the Global Proteome Machine database (GPMDB), which is the largest curated and publicly available proteomic data repository derived from tandem mass spectrometry, to develop an in silico protein interaction analysis tool. Using a human histone protein for method development, we positively identified an interaction partner from each histone protein family that forms the histone octameric complex. Moreover, this method, applied to the α subunits of the human proteasome, identified all of the subunits in the 20S core particle. Furthermore, we applied this approach to human integrin αIIb and integrin β3, a major receptor involved in the activation of platelets. We identified 28 proteins, including a protein network for integrin and platelet activation. In addition, proteins interacting with integrin β1 obtained using this method were validated by comparing them to those identified in a formaldehyde-supported coimmunoprecipitation experiment, protein-protein interaction databases and the literature. Our results demonstrate that in silico protein interaction analysis is a novel tool for identifying known/candidate protein-protein interactions and proteins with shared functions in a protein network.

This paper described a simple heuristic method for determining the merit of a set of peptide sequence assignments made using tandem mass spectra. The method involved comparing a prediction based on the known stochastic behavior of a sequence assignment algorithm with the assignments generated from a particular data set. A particular formulation of this comparison was defined through the construction of a plot of the data, the rho-diagram, as well as a parameter derived from this plot, the rho-score. This plot and parameter were shown to be able to readily characterize the relative quality of a set of peptide sequence assignments and to allow the straightforward determination of probability threshold values for the interpretation of proteomics data. This plot is independent of the algorithm or scoring scheme used to estimate the statistical significance of a set of experimental results; rather, it can be used as an objective test of the correctness of those estimates. The rho-score can also be used as a parameter to evaluate the relative merit of protein identifications, such as those made across proteome species taxonomic categories.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been successfully applied to elucidating biological questions trough the analysis of proteins, peptides, and nucleic acids. Here, we review the different approaches for analyzing the data that is generated by MALDI-MS. The first step in the analysis is the processing of the raw data to find peaks that correspond to the analytes. The peaks are characterized by their areas (or heights) and their centroids. The peak area can be used as a measure of the quantity of the analyte, and the centroid can be used to determine the mass of the analyte. The masses are then compared to models of the analyte, and these models are ranked according to how well they fit the data and their significance is calculated. This allows the determination of the identity (sequence and modifications) of the analytes. We show how this general data analysis workflow is applied to protein and nucleic acid chemistry as well as proteomics.

V-erb-b2 erythroblastic leukemia viral oncogene homologue 2, known as ERBB2, is an important oncogene in the development of certain cancers. It can form a heterodimer with other epidermal growth factor receptor family members and activate kinase-mediated downstream signaling pathways. ERBB2 gene is located on chromosome 17 and is amplified in a subset of cancers, such as breast, gastric, and colon cancer. Of particular interest to the Chromosome-Centric Human Proteome Project (C-HPP) initiative is the amplification mechanism that typically results in overexpression of a set of genes adjacent to ERBB2, which provides evidence of a linkage between gene location and expression. In this report we studied patient samples from ERBB2-positive together with adjacent control nontumor tissues. In addition, non-ERBB2-expressing patient samples were selected as comparison to study the effect of expression of this oncogene. We detected 196 proteins in ERBB2-positive patient tumor samples that had minimal overlap (29 proteins) with the non-ERBB2 tumor samples. Interaction and pathway analysis identified extracellular signal regulated kinase (ERK) cascade and actin polymerization and actinmyosin assembly contraction as pathways of importance in ERBB2+ and ERBB2- gastric cancer samples, respectively. The raw data files are deposited at ProteomeXchange (identifier: PXD002674) as well as GPMDB.

Remarkable progress continues on the annotation of the proteins identified in the Human Proteome and on finding credible proteomic evidence for the expression of "missing proteins". Missing proteins are those with no previous protein-level evidence or insufficient evidence to make a confident identification upon reanalysis in PeptideAtlas and curation in neXtProt. Enhanced with several major new data sets published in 2014, the human proteome presented as neXtProt, version 2014-09-19, has 16 491 unique confident proteins (PE level I), up from 13 664 at 2012-12 and 15 646 at 2013-09. That leaves 2948 missing proteins from genes classified having protein existence level PE 2, 3, or 4, as well as 616 dubious proteins at PE 5. Here, we document the progress of the HPP and discuss the importance of assessing the quality of evidence, confirming automated findings and considering alternative protein matches for spectra and peptides. We provide guidelines for proteomics investigators to apply in reporting newly identified proteins. QC 20151022

A new algorithm, sequence-specific retention calculator, was developed to predict retention time of tryptic peptides during RP HPLC fractionation on C18, 300-A pore size columns. Correlations of up to approximately 0.98 R2 value were obtained for a test library of approximately 2000 peptides and approximately 0.95-0.97 for a variety of real samples. The algorithm was applied in conjunction with an exclusion protocol based on mass (15 ppm tolerance) and retention time (2-min tolerance for 0.66% acetonitrile/min gradient), MART criteria to significantly reduce the instrument time required for complete MS/MS analysis of a digest separated by RP HPLC. This was confirmed by reanalyzing the set of HPLC-MALDI MS/MS data with no loss in protein identifications, despite the number of virtually executed MS/MS analyses being decreased by 57%.

A system for creating a library of tandem mass spectra annotated with corresponding peptide sequences was described. This system was based on the annotated spectra currently available in the Global Proteome Machine Database (GPMDB). The library spectra were created by averaging together spectra that were annotated with the same peptide sequence, sequence modifications, and parent ion charge. The library was constructed so that experimental peptide tandem mass spectra could be compared with those in the library, resulting in a peptide sequence identification based on scoring the similarity of the experimental spectrum with the contents of the library. A software implementation that performs this type of library search was constructed and successfully used to obtain sequence identifications. The annotated tandem mass spectrum libraries for the Homo sapiens, Mus musculus, and Saccharomyces cerevisiae proteomes and search software were made available for download and use by other groups.

This paper describes an algorithm to apply proteotypic peptide sequence libraries to protein identifications performed using tandem mass spectrometry (MS/MS). Proteotypic peptides are those peptides in a protein sequence that are most likely to be confidently observed by current MS-based proteomics methods. Libraries of proteotypic peptide sequences were compiled from the Global Proteome Machine Database for Homo sapiens and Saccharomyces cerevisiae model species proteomes. These libraries were used to scan through collections of tandem mass spectra to discover which proteins were represented by the data sets, followed by detailed analysis of the spectra with the full protein sequences corresponding to the discovered proteotypic peptides. This algorithm (Proteotypic Peptide Profiling, or P3) resulted in sequence-to-spectrum matches comparable to those obtained by conventional protein identification algorithms using only full protein sequences, with a 20-fold reduction in the time required to perform the identification calculations. The proteotypic peptide libraries, the open source code for the implementation of the search algorithm and a website for using the software have been made freely available. Approximately 4% of the residues in the H. sapiens proteome were required in the proteotypic peptide library to successfully identify proteins.

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-A pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.

An algorithm for reducing the time necessary to match a large set of peptide tandem mass spectra with a list of protein sequences is described. This algorithm breaks the process into multiple steps. A rapid survey step identifies all protein sequences that are reasonable candidates for a match with a set of tandem mass spectra. These candidates are then used as models, which are refined by detailed analysis of the set of tandem mass spectra for evidence of incomplete enzymatic hydrolysis, non-specific hydrolysis and chemical modifications of amino acid residues resulting from either post-translational modifications or sample handling. Compared with current one-step methods for matching proteins to mass spectra, this multiple-step method can decrease the time required for the calculation by several orders of magnitude.

Selenocysteine is a naturally occurring proteogenic amino acid that is encoded in the genomic sequence of relatively abundant proteins in many of the model species commonly used for biomedical research. On the basis of an analysis of publicly available proteomics information, it was discovered that peptides containing selenocysteine were not being identified in tandem mass spectrometry proteomics data. Once the chemical basis for this exclusion was understood, a simple alteration in search parameters led to the confident identification of selenocysteine containing peptides from existing proteomics data, with no change in experimental protocols required.

RADARS, a rapid, automated, data archiving and retrieval software system for high-throughput proteomic mass spectral data processing and storage, is described. The majority of mass spectrometer data files are compatible with RADARS, for consistent processing. The system automatically takes unprocessed data files, identifies proteins via in silico database searching, then stores the processed data and search results in a relational database suitable for customized reporting. The system is robust, used in 24/7 operation, accessible to multiple users of an intranet through a web browser, may be monitored by Virtual Private Network, and is secure. RADARS is scalable for use on one or many computers, and is suited to multiple processor systems. It can incorporate any local database in FASTA format, and can search protein and DNA databases online. A key feature is a suite of visualisation tools (many available gratis), allowing facile manipulation of spectra, by hand annotation, reanalysis, and access to all procedures. We also described the use of Sonar MS/MS, a novel, rapid search engine requiring 40 MB RAM per process for searches against a genomic or EST database translated in all six reading frames. RADARS reduces the cost of analysis by its efficient algorithms: Sonar MS/MS can identifiy proteins without accurate knowledge of the parent ion mass and without protein tags. Statistical scoring methods provide close-to-expert accuracy and brings robust data analysis to the non-expert user.

One year ago the Human Proteome Project (HPP) leadership designated the baseline metrics for the Human Proteome Project to be based on neXtProt with a total of 13,664 proteins validated at protein evidence level 1 (PE1) by mass spectrometry, antibody-capture, Edman sequencing, or 3D structures. Corresponding chromosome-specific data were provided from PeptideAtlas, GPMdb, and Human Protein Atlas. This year, the neXtProt total is 15,646 and the other resources, which are inputs to neXtProt, have high-quality identifications and additional annotations for 14,012 in PeptideAtlas, 14,869 in GPMdb, and 10,976 in HPA. We propose to remove 638 genes from the denominator that are "uncertain" or "dubious" in Ensembl, UniProt/SwissProt, and neXtProt. That leaves 3844 "missing proteins", currently having no or inadequate documentation, to be found from a new denominator of 19,490 protein-coding genes. We present those tabulations and web links and discuss current strategies to find the missing proteins.

Database searching using molecular-mass information has become a popular method for determining the sequence of a protein, a key step in proteomics. This article discusses the various database-search algorithms that are available for protein identification and the issues involved in interpreting the results.

The post-translational methylation of the N-terminally extended or high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) has been shown to affect the nuclear accumulation of the growth factor. In this study, we determined the extent and position of methyl groups in HMW FGF-2. Using mass spectrometry and amino acid sequence analysis, we have shown that the 22- and 22.5-kDa forms of HMW FGF-2 contain five dimethylated arginines located at positions -22, -24, -26, -36, and -38 using the methionine residue normally used to initiate the 18-kDa form as position 0. The 24-kDa form of HMW FGF-2 contains seven to eight dimethylated arginines located at positions -48, -50, and -52, in addition to positions -22, -24, -26, -36, and -38. In vitro methylation reactions demonstrate that the N-terminal extension of HMW FGF-2 acts as a specific substrate for yeast Hmt1p and human HRMT1L2 arginine methyltransferases. These findings indicate that HMW FGF-2, with the presence of five or more dimethylated Gly-Arg-Gly repeats, contains an RGG box-like domain, which may be important for protein-protein and/or protein-RNA interactions.

The synthesis of virulence factors and other extracellular proteins responsible for pathogenicity in Staphylococcus aureus is under the control of the agr locus. A secreted agr -encoded peptide, AgrD, processed from the AgrD gene product, is known to be an effector of self-strain activation and cross-strain inhibition of the agr response. Biochemical analysis of AgrD peptides isolated from culture supernatants has suggested that they contain an unusual thiol ester-linked cyclic structure. In the present work, chemical synthesis is used to confirm that the mature AgrD peptides contain a thiolactone structure and that this feature is absolutely necessary for full biological activity. The AgrD synthetic thiolactone peptides exhibited biological activity in vivo in a mouse protection test. Structure-activity studies have allowed key aspects of the peptide structure involved in the differential activation and inhibition functions to be identified. Accordingly, we propose a model for activation and inhibition of the agr response in which the former, but not the latter, involves specific acylation of the agr transmembrane receptor, AgrC.

International audience; A cystatin C variant with L68Q substitution and a truncation of 10 NH2-terminal residues is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). Variant and wild type cystatin C production, processing, secretion, and clearance were studied in human cell lines stably overexpressing the cystatin C genes. Immunoblot and mass spectrometry analyses demonstrated monomeric cystatin C in cell homogenates and culture media. While cystatin C formed concentration-dependent dimers, the HCHWA-I variant dimerized at lower concentrations than the wild type protein. Amino-terminal sequence analysis revealed that the variant and normal proteins produced and secreted are the full-length cystatin C. Pulse-chase experiments demonstrated similar levels of normal and variant cystatin C production and secretion. However, the secreted variant cystatin C exhibited an increased susceptibility to a serine protease in conditioned media and in human cerebrospinal fluid, explaining its depletion from the cerebrospinal fluid of HCHWA-I patients. Thus, the amino acid substitution may induce unstable cystatin C with intact inhibitory activity and predisposition to self-aggregation and amyloid fibril formation.

alpha-Latrotoxin is a potent stimulator of neurosecretion. Its action requires extracellular binding to high affinity presynaptic receptors. Neurexin I alpha was previously described as a high affinity alpha-latrotoxin receptor that binds the toxin only in the presence of calcium ions. Therefore, the interaction of alpha-latrotoxin with neurexin I alpha cannot explain how alpha-latrotoxin stimulates neurotransmitter release in the absence of calcium. We describe molecular cloning and functional expression of the calcium-independent receptor of alpha-latrotoxin (CIRL), which is a second high affinity alpha-latrotoxin receptor that may be the major mediator of alpha-latrotoxin's effects. CIRL appears to be a novel orphan G-protein-coupled receptor, a member of the secretin receptor family. In contrast with other known serpentine receptors, CIRL has two subunits of the 120 and 85 kDa that are the result of endogenous proteolytic cleavage of a precursor polypeptide. CIRL is found in brain where it is enriched in the striatum and cortex. Expression of CIRL in chromaffin cells increases the sensitivity of the cells to the effects of alpha-latrotoxin, demonstrating that this protein is functional in coupling to secretion. Syntaxin, a component of the fusion complex, copurifies with CIRL on an alpha-latrotoxin affinity column and forms stable complexes with this receptor in vitro. Interaction of CIRL with a specific presynaptic neurotoxin and with a component of the docking-fusion machinery suggests its role in regulation of neurosecretion.

Soluble A beta (Sa beta) is normally present at a low concentration in human plasma and cerebrospinal fluid. Although the factors involved in the regulation of Sa beta plasma levels are still unknown, we have explored its excretion in the urine as one of the possible homeostatic mechanisms. The presence of Sa beta in the urine was investigated via immunoprecipitation experiments with anti-A beta antibodies followed by detection and identification by immunoblot, MALDI mass spectrometry and sequence analysis. Soluble A beta (4.3 kDa) immunoreactivity was present in the urine of normal donors, Down's syndrome individuals as well as in patients with renal disorders exhibiting glomerular or mixed proteinuria. Edman degradation of the immunoprecipitated material yielded the intact A beta N-terminus and mass spectra analysis indicated the existence of a major component at mlz 4327, corresponding to the molecular mass of A beta1-40. Semiquantitative data obtained from the immunoprecipitation experiments indicate that under normal conditions the daily excretion of intact Sa beta in the urine represents less than 1% of the circulating pool.

The malaria circumsporozoite (CS) protein binds to glycosaminoglycans from heparan sulfate proteoglycans on the cell surface of hepatocytes and is specifically cleared from the bloodstream by the liver. We show here that the two conserved regions, I and II-plus, of the CS protein, in a concerted action, preferentially bind to highly sulfated heparin-like oligosaccharides in heparan sulfate. In a concentration-dependent manner, peptides representing region I and region II-plus inhibited the binding of recombinant CS protein to HepG2 cells by 62 and 84%, respectively. Furthermore, the action of endoproteinase Arg-C, which cleaves the recombinant CS constructs CS27IVC and CSFZ(Cys) predominantly at the conserved region I, was inhibited by heparin in a concentration-dependent fashion. CSFZ(Cys), which has a higher affinity to HSPGs than CS27IVC, was stabilized by heparin at a w/w ratio (CS protein:glycosaminoglycan) of 20/1, whereas full protection of CS27IVC required more heparin (5/1). Heparan sulfate provided full protection of CSFZ(Cys) only at a ratio of 1/10. Native fucoidan as well as normally sulfated fuco-oligosaccharides (0.76 mol sulfate/mol fucose) inhibited Plasmodium berghei development in HepG2 cells by 84 and 66%, respectively, in a concentration-dependent manner and sporozoite invasion into CHO cells by 80%. Desulfated fucoidan oligosaccharides were inactive. These results may explain the selective interaction between the CS protein and the unique heparan sulfate from liver, which is noted for its unusually high degree of sulfation, and may provide a plausible explanation for the selective targeting of the malaria CS protein to the liver.

In hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), a genetic variant (E22Q) of amyloid beta (Abeta) accumulates predominantly in the small vessels of leptomeninges and cerebral cortex, leading to fatal strokes in the fifth or sixth decade of life. Abeta deposition in the neuropil occurs mainly in the form of preamyloid, Congo red negative deposits, while mature neuritic plaques and neurofibrillary tangles, hallmark lesions in Alzheimer's disease (AD), are characteristically absent. A recent hypothesis regarding the pathogenesis of AD states that Abeta extending to residues 42-43 (as opposed to shorter species) can seed amyloid formation and trigger the development of neuritic plaques followed by neuronal damage in AD. We characterized biochemically and immunohistochemically Abeta from three cases of HCHWA-D to determine its length in vascular and parenchymal deposits. Mass spectrometry of formic acid-soluble amyloid, purified by size-exclusion gel chromatography, showed that Abeta 1-40 and its carboxyl-terminal truncated derivatives were the predominant forms in leptomeningeal and cortical vessels. Abeta 1-42 was a minor component in these amyloid extracts. Immunohistochemistry with antibodies S40 and S42, specific for Abeta ending at Val-40 or Ala-42, respectively, were consistent with the biochemical data from vascular amyloid. In addition, parenchymal preamyloid lesions were specifically stained with S42 and were not labeled by S40, in agreement with the pattern reported for AD, Down's syndrome, and aged dogs. Our results suggest that in HCHWA-D the carboxyl-terminal Abeta heterogeneity is due to limited proteolysis in vivo. Moreover, they suggest that Abeta species ending at Ala-42 may not be critical for the seeding of amyloid formation and the development of AD-like neuritic changes.

Web technology can be used to manipulate analytical data and facilitate the exchange of scientific information

This paper describes the application of matrix convolution filters to time-of-flight matrix-assisted laser desorption mass spectra to improve the appearance of a particular spectrum and to extract peaks from poorly resolved signals. These filters are commonly used in image enhancement to sharpen blurry images and remove noise because of their general applicability and their modest requirements for calculation speed. The theory of these filters is discussed, as applied to mass spectra, and examples of spectra processed using a variety of filters are shown to demonstrate the improvements and artifacts that can be generated.

Some bacterial pathogens elaborate and secrete virulence factors in response to environmental signals, others in response to a specific host product, and still others in response to no discernible cue. In this study, we have demonstrated that the synthesis of Staphylococcus aureus virulence factors is controlled by a density-sensing system that utilizes an octapeptide produced by the organism itself. The octapeptide activates expression of the agr locus, a global regulator of the virulence response. This response involves the reciprocal regulation of genes encoding surface proteins and those encoding secreted virulence factors. As cells enter the postexponential phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The intracellular agr effector is a regulatory RNA, RNAIII, whose transcription is activated by an agr-encoded signal transduction system for which the octapeptide is the ligand.

Synthetic peptides containing the sequence of Alzheimer's amyloid-beta peptide (A beta) spontaneously form amyloid-like fibrils in vitro, and have been extensively used to study the factors that modulate fibrillogenesis. Contradictory observations have been reported regarding the neurotoxicity of A beta and the influence of some A beta-binding proteins on in vitro A beta amyloid formation. In this study, we show that A beta 1-40 synthetic peptides obtained from different suppliers, have significantly distinct fibrillogenic properties. No differences were detected in the chemical structure or in the initial assembly state by mass spectroscopy, reverse-phase high performance liquid chromatography and denaturing or non-denaturing gel electrophoresis. However, there was a direct correlation between the ability of soluble peptides to form amyloid and their percentage of beta-sheet structure, as determined by electron microscopy, fluorescence associated to thioflavine T bound to amyloid, and circular dichroism. The data suggest that the determinant factor of A beta fibrillogenesis is the secondary structure adopted by the peptide in its soluble state.

This paper describes the fabrication and use of thin polycrystalline films in matrix-assisted laser desorption experiments. These films consist of microcrystals of protein-doped matrix (crystal dimensions ⩽ 1 μm). The films produce intense protein-ion currents and can be grown in the presence of high concentrations of involatile solvents (e.g., glycerol, 6 M urea) without any purification. They strongly adhere to the substrate, allowing easier washing of the film, compared to dried-droplet deposits. The films are also more uniform than dried-droplet deposits, with respect to ion production. It is suggested that matrix-assisted laser desorption/ionization samples are composed of nonlinear optical devices that function as polymer ion-current sources. These devices (protein-doped matrix crystals) can be designed and fabricated in many forms to serve the special functions required by the analytical scientist.

This paper describes several protocols for growing large, protein-doped 3,5-dimethoxy-4-hydroxy-trans-cinnamic acid crystals. Examination of these crystals using laser desorption shows that the mass spectra obtained from the crystals can be useful for biochemical analysis. One particular crystal growing protocol allowed a non-covalently bound heme group of horse muscle myoglobin to remain attached to the polypeptide following laser ablation and ionization. Crystals could be grown in solutions that contained involatile solvents that normally inhibit polypeptide ion production, such as glycerol. These crystals were protein doped and produced acceptable analytical mass spectra. The results suggest that some problems associated with the frequently used droplet-drying method of sample preparation are caused by the changing concentration conditions present in drying solutions.

A new approach to protein sequencing is described. It consists of two steps: (i) ladder-generating chemistry, the controlled generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted laser-desorption mass spectrometry. Each amino acid was identified from the mass difference between successive peaks, and the position in the data set defined the sequence of the original peptide chain. This method was used to directly locate a phosphoserine residue in a phosphopeptide. The protein ladder sequencing method lends itself to very high sample throughput at very low per cycle cost.

Protein identification by mass spectrometry is widely used in biological research. Here, we describe how the Global Proteome Machine (GPM) can be used for protein identification and for validation of the results. We cover identification by searching protein sequence collections and spectral libraries, as well as validation of the results using expectation values, rho-diagrams, and spectrum databases.

We report the first molecular characterization of a precursor sequence for a small, Ca2+ channel blocking, peptide spider toxin, omega-agatoxin IA. By integrating information generated from a molecular genetic approach using agatoxin cDNAs with data provided from mass spectrometry of the mature toxin, we were able to deduce the likely mechanisms by which the toxin precursor peptide is processed to its mature heterodimeric form. A particularly interesting feature of the prepropeptide is the occurrence of two glutamate-rich sequences interposed between the signal sequences, the major peptide toxin, and the minor toxin peptide. Excision of the more distal glutamate-rich region appears to be signaled by flanking arginine residues but likely occurs only after a disulfide linkage has formed between the major and minor chains of the mature toxin. Our molecular genetic approach toward characterizing this toxin will allow us to quickly generate a series of spider sequences from which mature toxin structures can be deduced and eventually expressed. Additionally, this approach will provide insights into the evolutionary divergence observed among spider peptide toxins.

Phenomenological models were proposed to explain the experimentally observed dependence of protein ion yields with laser fluence in matrix-assisted laser desorption. Assuming that the illuminating laser had a Gaussian intensity profile at the sample being examined, it was possible to fit the experimental points with a model that only assumes a fluence threshold for ion production. No additional dependence of protein ion yield on illuminating fluence above the threshold value was necessary to explain the data.

Acetylation is a well-studied posttranslational modification that has been associated with a broad spectrum of biological processes, notably gene regulation. Many studies have contributed to our knowledge of the enzymology underlying acetylation, including efforts to understand the molecular mechanism of substrate recognition by several acetyltransferases, but traditional experiments to determine intrinsic features of substrate site specificity have proven challenging. Here, we combine experimental methods with clustering analysis of protein sequences to predict protein acetylation based on the sequence characteristics of acetylated lysines within histones with our unique prediction tool PredMod. We define a local amino acid sequence composition that represents potential acetylation sites by implementing a clustering analysis of histone and nonhistone sequences. We show that this sequence composition has predictive power on 2 independent experimental datasets of acetylation marks. Finally, we detect acetylation for selected putative substrates using mass spectrometry, and report several nonhistone acetylated substrates in budding yeast. Our approach, combined with more traditional experimental methods, may be useful for identifying acetylated substrates proteome-wide.

Policies supporting the rapid and open sharing of genomic data have directly fueled the accelerated pace of discovery in large-scale genomics research. The proteomics community is starting to implement analogous policies and infrastructure for making large-scale proteomics data widely available on a precompetitive basis. On August 14, 2008, the National Cancer Institute (NCI) convened the "International Summit on Proteomics Data Release and Sharing Policy" in Amsterdam, The Netherlands, to identify and address potential roadblocks to rapid and open access to data. The six principles agreed upon by key stakeholders at the summit addressed issues surrounding (1) timing, (2) comprehensiveness, (3) format, (4) deposition to repositories, (5) quality metrics, and (6) responsibility for proteomics data release. This summit report explores various approaches to develop a framework of data release and sharing principles that will most effectively fulfill the needs of the funding agencies and the research community.

The velocity distributions of polypeptide molecule ions (molecular mass 1000–15600 u) produced by matrix assisted ultraviolet laser desorption were measured using a modified time-of-flight mass spectrometer. The data demonstrate that polypeptide molecule ions produced by matrix assisted laser desorption at the ion production threshold irradiance have similar velocity distributions, with an average velocity of approximately 750 m/s. This result has important implications for the design of mass spectrometers that use the effect to generate polypeptide molecule ions and for the theoretical treatment of the laser desorption process. A jet expansion model for the desorption of high mass polymers is proposed to explain the results.

Human hemoglobin A has been crosslinked by diisothiocyanatobenzenesulfonate to give a limited number of products in a yield of approximately 70%. The predominant product was crosslinked between subunits within a tetramer and had a Mr of 64,000; no higher Mr species were formed. This product had one crosslink per tetramer located between the NH2 termini of its alpha chains, as established by HPLC analysis, amino acid analysis, Edman degradation, and mass spectrometry. This crosslinked derivative had a slightly increased oxygen affinity [P50 = 9 mmHg (1 mmHg = 133 Pa); P50 for unmodified hemoglobin = 11 mmHg], and the retention time of this derivative in the circulation of rats was 2.9 and 3.3 hr at two hemoglobin concentrations (7 g/dl and 14 g/dl, respectively). The half-life of an uncrosslinked carboxymethylated derivative, which has a low oxygen affinity (P50 = 28 mmHg), was 0.6 and 0.7 hr under the same conditions. Therefore, prolongation of the plasma-retention time of infused hemoglobin is dependent on the crosslinking of the tetramer but independent of the oxygen affinity of the derivative.

Multiple reaction monitoring (MRM), commonly employed for the mass spectrometric detection of small molecules, is rapidly gaining ground in proteomics. Its high sensitivity and specificity makes this targeted approach particularly useful when sample throughput or proteome coverage limits global studies. Existing tools to design MRM assays rely exclusively on theoretical predictions, or combine them with previous observations on the same type of sample. The additional mass spectrometric experimentation this requires can pose significant demands on time and material. To overcome these challenges, a new MRM worksheet was introduced into The Global Proteome Machine database (GPMDB) that provided all of the information needed to design MRM transitions based solely on archived observations made by other researchers in previous experiments. This required replacing the precursor ion intensity by the number of peptide observations, which proved to be an adequate substitute if peptides did not occur in multiple forms. While the absence of collision energy information proved largely inconsequential, successful prediction of unique transitions depended on the type of fragment ion involved. The design of MRM assays for iTRAQ-labeled tryptic peptides obtained from human platelet proteins demonstrated the usefulness of the MRM worksheet also for quantitative applications. This workflow, which relies exclusively on experimental observations stored in data repositories, therefore represents an attractive alternative for the prediction of MRM transitions prior to experimental validation and optimization.

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