A Database of Accurate Electrophoretic Migration Patterns for Human Proteins.

Native molecular weight (MW) is one of the defining features of proteins. Denaturing gel electrophoresis (SDS-PAGE) is a very popular technique for separating proteins and determining their MW. Coupled with antibody-based detection, SDS-PAGE is widely applied for protein identification and quantitation. Yet, electrophoresis is poorly reproducible and the MWs obtained are often inaccurate. This hampers antibody validation and negatively impacts the reliability of western blot data, resulting worldwide in a considerable waste of reagents and labour. We argue that, to alleviate these problems there is a need to establish a database of reference MWs measured by SDS-PAGE. Using mass spectrometry as an orthogonal detection method, we acquired electrophoretic migration patterns for approximately 10'000 human proteins in five commonly used cell lines. We applied a robust internal calibration of migration to determine accurate and reproducible molecular weights. This in turn allows merging replicates to increase accuracy, but also enables comparing different cell lines. Mining of the data obtained highlights structural factors that affect migration of distinct classes of proteins. When combined with peptide coverage, the data produced recapitulates known post-translational modifications and differential splicing and can be used to formulate hypotheses on new or poorly known processing events. The full information is freely accessible as a web resource through a user friendly graphical interface (https://pumba.dcsr.unil.ch/). We anticipate that this database will be useful to investigators worldwide for troubleshooting western blot experiments, but could also contribute to the characterization of human proteoforms.

Robust and sensitive peptidomics workflow for plasma based on specific extraction, lipid removal, capillary LC setup and multinozzle ESI emitter.

We present a new workflow for the LC-MS determination of native peptides in plasma at picomolar levels. Collected whole blood was quickly diluted with an ice-cold solution in order to stop protease activity. Diluted plasma samples were extracted by protein denaturation followed by solid-phase-extraction with a polymeric stationary phase that removed most proteins and lipids. Using a specific LC-MS setup with 3 pumps, 240 µL of extracts were injected without drying-reconstitution, a step known to cause peptide losses. After an 18-fold dilution on-line, peptides were trapped on a 1 × 10 mm C8 column, back-flushed and resolved on a 0.3 × 100 mm C18 column. Extract reproducibility, robustness (column clogging), extraction yields, matrix effects, calibration curves and limits of detection were evaluated with plasma extracts and spiked-in standards. The sensitivity and applicability of 3 electrospray sources were evaluated at capillary flow rates (10 µL/min). We show that ionization sources must have a spray angle with the MS orifice when "real" extracts are injected and that a multinozzle emitter can improve very significantly peptide detection. Finally, using our workflow, we have performed a peptidomics study on dried-blood-spots collected over 65 h in a healthy volunteer and discovered 5 fragments (2.9-3.8 KDa) of the protein statherin showing circadian oscillations. This is the first time that statherin is observed in blood where its role clearly deserves further investigations. Our peptidomic protocol shows low picomolar limits of detection and can be readily applied with or without minor modifications for most peptide determinations in various biomatrices.

Addressing trypsin bias in large scale (phospho)proteome analysis by size exclusion chromatography and secondary digestion of large post-trypsin peptides.

In the vast majority of bottom-up proteomics studies, protein digestion is performed using only mammalian trypsin. Although it is clearly the best enzyme available, the sole use of trypsin rarely leads to complete sequence coverage, even for abundant proteins. It is commonly assumed that this is because many tryptic peptides are either too short or too long to be identified by RPLC-MS/MS. We show through in silico analysis that 20-30% of the total sequence of three proteomes (Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Homo sapiens) is expected to be covered by Large post-Trypsin Peptides (LpTPs) with M(r) above 3000 Da. We then established size exclusion chromatography to fractionate complex yeast tryptic digests into pools of peptides based on size. We found that secondary digestion of LpTPs followed by LC-MS/MS analysis leads to a significant increase in identified proteins and a 32-50% relative increase in average sequence coverage compared to trypsin digestion alone. Application of the developed strategy to analyze the phosphoproteomes of S. pombe and of a human cell line identified a significant fraction of novel phosphosites. Overall our data indicate that specific targeting of LpTPs can complement standard bottom-up workflows to reveal a largely neglected portion of the proteome.

Comprehensive analysis of proteins secreted by Trichophyton rubrum and Trichophyton violaceum under in vitro conditions.

Dermatophytes cause most superficial mycoses in humans and animals. Their pathogenicity is probably linked with the secretion of proteins degrading keratinised structures. Using 2D-PAGE and a shotgun mass spectrometry approach, we identified 80 proteins from Trichophyton rubrum and Trichophyton violaceum secretomes, under conditions mimicking those in the host. Identified proteins included endo- and exoproteases, other hydrolases, and oxidoreductases. Our findings can contribute to a better understanding of the virulence mechanisms of the two species and the different types of infection they cause.

Secreted subtilisin gene family in Trichophyton rubrum.

Secreted proteases constitute potential virulence factors of dermatophytes. A total of seven genes encoding putative serine proteases of the subtilisin family (SUB) were isolated in Trichophyton rubrum. Based on sequence data and intron-exon structure, a phylogenetic analysis of subtilisins from T. rubrum and other fungi revealed a presumed ancestral lineage comprising T. rubrum SUB2 and Aspergillus SUBs. All other SUBs (SUB1, SUB3-7) are dermatophyte-specific and have apparently emerged more recently, through successive gene duplication events. We showed that two subtilisins, Sub3 and Sub4, were detected in culture supernatants of T. rubrum grown in a medium containing soy protein as a sole nitrogen source. Both recombinant enzymes produced in Pichia pastoris are highly active on keratin azure suggesting that these proteases play an important role in invasion of keratinised tissues by the fungus. The set of deduced amino acid sequences of T. rubrum SUB ORFs allowed the identification of orthologous Subs secreted by other dermatophyte species using proteolysis and mass spectrometry.

Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum.

Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in the stratum corneum, nails and hair. In a culture medium containing soy proteins as sole nitrogen source a substantial proteolytic activity was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55-75 % inhibited by o-phenanthroline, attesting that metalloproteases were secreted by all three species. Using a consensus probe constructed on previously characterized genes encoding metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis, a five-member MEP family was isolated from genomic libraries of T. rubrum, T. mentagrophytes and M. canis. A phylogenetic analysis of genomic and protein sequences revealed a robust tree consisting of five main clades, each of them including a MEP sequence type from each dermatophyte species. Each MEP type was remarkably conserved across species (72-97 % amino acid sequence identity). The tree topology clearly indicated that the multiplication of MEP genes in dermatophytes occurred prior to species divergence. In culture medium containing soy proteins as a sole nitrogen source secreted Meps accounted for 19-36 % of total secreted protein extracts; characterization of protein bands by proteolysis and mass spectrometry revealed that the three dermatophyte species secreted two Meps (Mep3 and Mep4) encoded by orthologous genes.

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session.

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.