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Stability assessment of the human proteotypic peptides for proteomics quantification

ABSTRACT: Mass spectrometry coupled to liquid chromatography is one of the most powerful technologies for proteome quantification in a variety of biomedical samples, including cells, tissues and liquid biopsies. In peptide-centric workflows protein mixtures proteins are enzymatically digested to peptides prior their analysis. Experimental spectra are used for peptide identification, and extracted peptide areas, peak heights, or spectral counts are used to infer protein quantities using different heuristics. However, proteome-wide quantification studies rarely identify all potential peptides for any given protein while targeted proteomics experiments focus on a set of peptides for the proteins of interest. Consequently, proteomics methods rely on the use of a limited subset of all possible peptides as proxies for protein quantitation, which are specified either prior or after data acquisition. Several studies have evaluated the quantitative response of tryptic peptides and have described rules to guide peptide selection for protein quantification. However, beyond the effect of tryptic digestion and missed cleavages, short and mid-term peptide stability can also have a profound effect on the measurement reproducibility, specially when analysing proteomics samples from large cohorts of patients that expand for several days or weeks of measurement. In this project we assessed the stability of more than 100,000 peptides from the proteotypic peptide set of the Proteome Tools collection, and we used the obtained experimental data to train and validate a new peptide stability predictor for proteomics quantification.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Cristina Chiva

LAB HEAD: Eduard Sabidó

PROVIDER: PXD035198 | Pride | 2025-02-24

REPOSITORIES: pride

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			Action	DRS
	180426_S_ProteotypicP1_A10_pool10_01.raw	Raw
	180426_S_ProteotypicP1_A10_pool10_02.raw	Raw
	180426_S_ProteotypicP1_A10_pool10_03.raw	Raw
	180426_S_ProteotypicP1_A10_pool10_04.raw	Raw
	180426_S_ProteotypicP1_A10_pool10_05.raw	Raw

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Project description:Understanding how pathogens respond to antimicrobial peptides, and how this compares to currently available antibiotics, is crucial to optimizing antibiotic therapy. Staphylococcus aureus has several known resistance mechanisms against human cationic antimicrobial peptides (CAMPs). We aim to determine how S. aureus responds to sheep and frog CAMPs, and whether this response is associated with resistance. Gene expression changes in Staphylococcus aureus Newman cells exposed to linear CAMPs were analyzed by DNA microarray. Three antimicrobial peptides were used in the analysis, two of them are derived from frog, temporin L and dermaseptin K4-S4(1-16), one is from sheep, ovispirin-1. The peptides induced the VraSR cell-wall regulon and several other genes which are also upregulated in cells treated with vancomycin and other cell wall-active antibiotics. In addition to this similarity, three genes/operons were particularly strongly induced by the peptides: vraDE, SA0205 and SAS016, encoding an ABC transporter, a putative membrane-bound lysostaphin-like peptidase and a small functionally unknown protein, respectively. Ovispirin-1 and dermaseptin K4-S4(1-16), which disrupt lipid bilayers by the carpet mechanism, were strong inducers of the vraDE operon. We show that high level induction by ovispirin-1 was dependent on the amide modification of the peptide C-terminus. This suggests that the amide group has a crucial role in the activation of the Aps sensory system, the regulator of vraDE. In contrast, temporin L, which disrupts lipid bilayers by forming pores, was clearly a weaker inducer of vraDE despite the C-terminal amide modification. Sensitivity testing with CAMPs and other antimicrobials suggested that VraDE is a transporter dedicated to resist bacitracin. We also showed that SA0205 belongs to the VraSR regulon. Furthermore, VraSR was shown to be important for resistance against a wide range of cell wall-active antibiotics and other antimicrobial agents including the amide-modified ovispirin-1, bacitracin, teicoplanin, cefotaxime and 10 other β-lactam antibiotics, chlorpromazine, thioridazine and EGTA. The effects of the three different antimicrobial peptides on gene expression of S. aureus Newman were studied by using whole genome oligo-DNA microarrays. Bacteria were grown in BHI medium to the early exponential phase (OD600=0.6) and antimicrobial peptides were added at sublethal concentrations. Samples were taken for RNA isolations after treating the cultures with the peptides for 10 minutes. Control cultures without peptide additions were treated similarly and in parallel.

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Stability assessment of the human proteotypic peptides for proteomics quantification

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