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DART-ID increases single-cell proteome coverage

ABSTRACT: Analysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS) can identify and quantify thousands of proteins in microgram-level samples, such as those comprised of thousands of cells. Identifying proteins by LC-MS/MS proteomics, however, remains challenging for lowly abundant samples, such as the proteomes of single mammalian cells. To increase the identification rate of peptides in such small samples, we developed DART-ID. This method implements a data-driven, global retention time (RT) alignment process to infer peptide RTs across experiments. DART-ID then incorporates the global RT-estimates within a principled Bayesian framework to increase the confidence in correct peptide-spectrum-matches. Applying DART-ID to hundreds of samples prepared by the Single Cell Proteomics by Mass Spectrometry (SCoPE-MS) design increased the peptide and proteome coverage by 30 - 50% at 1% FDR. The newly identified peptides and proteins were further validated by demonstrating that their quantification is consistent with the quantification of peptides identified from high-quality spectra. DART-ID can be applied to various sets of experimental designs with similar sample complexities and chromatography conditions, and is freely available online.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Nikolai Slavov

PROVIDER: MSV000083149 | MassIVE | Mon Nov 19 15:36:00 GMT 2018

SECONDARY ACCESSION(S): PXD011748

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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DART-ID increases single-cell proteome coverage

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