20-30% of a trypsinised proteome are constituted of peptides with Mw≥3000 (TReP) Identification of large peptides by shotgun MS is not efficient Isolation of large peptides by SEC and digestion with four cleaving agents yields MS-suitable products, with increase of sequence coverage and identification: Application to human phosphoproteomics : 410 unique (not found with trypsin alone) phosphosites, of which 41% are not annotated in databases Comparison: only 8% non-annotated phosphosites in our trypsin data TReP analysis reveals a «hidden» part of the proteome The developed method uses a second digestion step targeting large tryptic peptides and was successfully employed to discover trypsin-inaccessible phosphorylation sites in the proteome of a human melanoma cell line, significantly enhancing the number of phosphosites. Many of the identified phosphosites and phosphoproteins have not been found in UNIPROT and phosphosite.org databases. This strategy complements regular trypsin-based analysis. Phosphorylation sites: Localization: Ascore ≥ 19  ref 1  Site mapping (custom-made scripts): Compare the phosphosite sets from different proteases Find the new sites not annotated by Uniprot and phosphosite.org databases Improved phosphoproteome analysis by multi-enzymatic digestions targeting the Trypsin-Resistant Proteome (TReP) Bao Tran 1 ; Celine Hernandez 1, 2 ; Alexandra Potts 1 ; Patrice Waridel 1 ; Frederique Lisacek 2 ; Manfredo Quadroni 1 1 University of Lausanne, Lausanne, Switzerland; 2 Swiss Institute of Bioinformatics, Geneva, Switzerland OVERVIEW 1. INTRODUCTION Analysis of complex samples by liquid chromatography- electrospray MS after trypsin cleavage is the most popular approach for bottom-up proteomics. However, comprehensive mapping of post-translational modifications is limited, among other things, by the presence of long sequences without trypsin cleavage sites. We tried to: Complement the trypsin-based workflow with a strategy that specifically targets the fraction of the proteome that cannot be digested by trypsin (the Trypsin-Resistant Proteome, TReP), in order to give compatible masses for shotgun MS Apply the method to characterization of trypsin- resistant protein sequences: Enhance sequence coverage Increase number of phosphosite identifications. Concept : after trypsin digestion, isolation of all polypeptides with Mw ≥ 3000 Da, and digestion with alternative proteases Samples: Saccharomyces cerevisiae vacuolar membrane fraction (SCVMprot) Human melanoma cell line lysate (SKMel28 cells) SEC fractionation: 11 fractions on Äkta purifier 10 system, Superdex Peptide 10/300 GL column RPLC-MS/MS analysis: Agilent nano 1100 HPLC system coupled to LTQ-Orbitrap-XL 2. METHOD Data processing 3. RESULTS 3.1. In silico tryptic peptide analysis Proteome covered by: “small” peptides (≥ 800 Da): 19.3% “medium” peptides (3000>Mw>800Da): 55.7% “large” peptides (≥3000Da): 25.0% Amino acid frequency ratio of in silico large tryptic peptides (69’571) to medium ones (465’160) in human proteome Frequency of S,T,Y residues in large peptides is higher than in medium peptides Fractionation of tryptic digests according to peptide size Total Ion Current (TIC) of SEC fraction 3 of sample SCVMprot analyzed directly and after Glu-C digestion. The inset spectrum was acquired at RT=31.21 min and assigned to one peptide (displayed in red) of protein ID B3LQ90_YEAS1. The corresponding extended tryptic peptide shown is 63 AA long (Mw: 6.5 kDa). Second digestion may lead to the generation of analyzable fragments out of much larger peptides which, in their intact form, would be very difficult to detect and identify by LC- MS/MS. 4. CONCLUSION 3.4. Phosphopeptide analysis 3.3. Improvement of LC-MS/MS mapping of large tryptic peptides Localized: 743 phosphosites with trypsin 410 other phosphosites with 4 second enzymes 225 sites not annotated in Uniprot/Swiss-prot and phophosite.org databases Covered sequence as function of peptide length (1’218’741 peptides of human proteome, Uniprot/Swissprot version 15.13), full trypsin cleavage SCVMprot: 80 µg SKMel: 4 mg Collection of 11 fractions of 1mL Fractions 1-6 containing large peptide were subjected to 2nd digestion with other cleaving agents. Fraction 3 of SCVMprot was used to examine LC-MS behavior of large tryptic peptides before and after second digestion (Glu-C). Fractions of SKMel were used for phosphosite identification experiments. 5. REFERENCE 1. Beausoleil, S. A., Villen, J., Gerber, S. A., Rush, J., and Gygi, S. P. (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotech 24, Time (min) Relative Abundance NL: 2.50E7 SDNEDNDFDEDDEDDAALVAAMTNAPGNSLDIEESVGYGATSAPTSNTNHVVESANAAYYQR Time (min) Relative Abundance Trypsin+ Glu-C NL: 2.50E m/z Relative Abundance Trypsin Example: Tryptic phosphopeptide (24.8 kDa) of protein NFAT5_HUMAN, and the corresponding peptide generated with Glu-C (red, identified in fraction 3). K.QPPPNMIFNPNQNPMANQEQQNQSIFHQQSNMAPMNQEQQPMQFQ SQSTVSSLQNPGPTQSESSQTPLFHSSPQIQLVQGSPSSQEQQVTLFLS PASMSALQTSINQQDMQQSPLYSPQNNMPGIQGATSSPQPQATLFHNT AGGTMNQLQNSPGSSQQTSGMFLFGIQNNCSQLLTSGPATLPDQLMAI SQPGQPQNEGQPPVTTLLSQQMPENpSPLASSINTNQNIEK.I The phosphosite (pS) and the protein phosphorylation were not annotated. Identified: 703 phosphorylated peptides; 440 phosphoproteins (19 not annotated) LogMw mAU ml Calibration curve: logMw = V ____ SCVMprot ____ Human SKMel Peptide Mw ≥ 2.4 kDa Peptide Mw < 2.4 kDa 10kDa 1.0kDa 0.3kDa 3.1kDa 31kDa