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HUPO HPP Phosphopeptide Challenge

ABSTRACT: The Phosphopeptide Challenge of the MS Resource Pillar of the Human Proteome Project (HPP) provides a unique opportunity for the HUPO membership to evaluate and compare methods for peptide sequence analysis by mass spectrometry, phosphosite localization, phosphopeptide enrichment, and data processing. Each participant is to apply their own methods and chosen bioinformatic pipeline to fully characterize the provided Phosphopeptide Challenge samples. As a result of this collaborative endeavor, multiple purification schemes, analytical protocols and data processing strategies will be evaluated, making it possible to determine the approach(es) that provide the highest coverage of the phosphopeptides in the mixture. Each participating laboratory will receive two sample vials. The vial labeled 'Phosphopeptide' contains a set of synthesized phosphorylated (Ser, Thr, or Tyr) peptides of human sequence origin at various concentrations, mixed with their non-phosphorylated counterparts. For some peptides, there is more than one phosphorylated form. The peptide sequences are included in the attached Excel file. The second vial labeled 'Phosphopeptide-Yeast' contains the same peptides in a background matrix consisting of 6 ug of trypsin-digested yeast lysate. Each vial is provided dry. Resuspension in 100 uL will result in synthetic peptide concentrations of 3 fmol/uL to 30 fmol/uL. The goal for the study is for you to provide your best method(s) to: 1. Identify the peptide sequences in the vial and determine the number and location of phosphorylation sites on each peptide (Phosphopeptide). 2. Determine the relative abundance of phosphorylation at each modified site by comparison with its non-phosphorylated counterpart (Phosphopeptide). 3. Enrich for phosphorylated peptides from the sample containing the yeast background matrix and re-analyze by MS (Phosphopeptide-Yeast).

INSTRUMENT(S): Orbitrap Fusion Lumos, maXis, 6550 iFunnel Q-TOF LC/MS (Agilent instrument model), impact, TripleTOF 5600, amaZon ETD, LTQ Orbitrap XL, Orbitrap Fusion, Q Exactive Plus, Q Exactive HF, Q Exactive HF-X (Thermo Scientific instrument model), Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Michael Hoopmann Susan Weintraub Robert L. Moritz Ulrike Kusebauch

PROVIDER: MSV000085932 | MassIVE | Sat Aug 08 15:08:00 BST 2020

SECONDARY ACCESSION(S): PXD020801

REPOSITORIES: MassIVE

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Project description:The mechanism of action of antimicrobial peptides (AMPs) was initially correlated with peptide membrane permeation properties. However, recent evidences indicate that action of a number of AMPs is more complex and involves specific interactions at cell envelopes or with intracellular targets. In this study, a genomic approach was undertaken on the model yeast Saccharomyces cerevisiae to characterize the antifungal effect of two unrelated AMPs. Two differentiated peptides were used: the synthetic cell-penetrating PAF26 and the natural cytolytic melittin. Transcriptomic analyses demonstrated distinctive gene expression changes for each peptide. Quantitative RT-PCR confirmed differential expression of selected genes. Gene onthology (GO) annotation of differential gene lists showed that the unique significant terms shared by treatment with both peptides were related to the cell wall (CW). Assays with mutants lacking CW related genes, including those of MAPK signaling pathways, revealed genes having influence on sensitivity to peptides. Fluorescence microscopy and flow cytometry demonstrated PAF26 interaction with cells and internalization that correlated with cell killing in sensitive CW-defective mutants such as ∆ecm33 or ∆ssd1. GO annotation also showed differential responses between peptides, which included ribosomal biogenesis, ARG genes from the metabolism of amino groups (specifically induced by PAF26), or the reaction to unfolded protein stress. Susceptibility of deletion mutants confirmed the involvement of these processes. Specifically, mutants lacking ARG genes from the metabolism of arginine pathway were markedly more resistant to PAF26 and had a functional CW. In the deletant in the arginosuccinate synthetase (ARG1) gene, PAF26 interaction occurred normally, thus uncoupling peptide interaction from cell killing. The previously described involvement of the glycosphingolipid gene IPT1 was extended to the peptides studied here. Reinforcement of CW is a general response common after exposure to distinct AMPs, and likely contributes to shield cells from peptide interaction. However, a weakened CW is not necessarily indicative of a higher sensitivity to AMPs. Additional processes modulate susceptibility to specific peptides, exemplified in the involvement of the metabolism of amino groups in the case of PAF26. The relevance of the response to unfolded protein stress or the sphingolipid biosynthesis, previously reported for other unrelated AMPs, was also independently confirmed. We carried out the characterization of the transcriptome of S. cerevisiae after exposure to PAF26 and melittin peptides. The global transcriptome response to peptides was undertaken by treating S. cerevisiae FY1679 cells in the logarithmic growth phase to sub-lethal concentrations (5 µM) of either PAF26 (PAF samples) or melittin (MEL samples) for 3 hours. Control treatment (CTR samples) consisted in cells exposed to buffer in the absence of peptide. Three biological replicates were conducted for each treatment. DNA macroarrays representing 6,020 yeast genes were hybridized with labelled cDNAs from cells.

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HUPO HPP Phosphopeptide Challenge

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