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Energy-translation coupling limits yeast anaerobic growth

ABSTRACT: This study employs yeast as a model system to thoroughly investigate the differences in regulatory mechanisms under various nutrient limitation conditions, utilizing a systems biology approach integrated with techniques such as multi-omics analysis.To obtain a comprehensive quantitative landscape of protein expression in (human lung adenocarcinoma), we performed TMT 6-plex–based comparative proteomic analysis. Tumour and adjacent non-tumour tissues (n = 6 pairs) were pulverised under liquid nitrogen and total proteins extracted by phenol–ammonium acetate/methanol precipitation. After reduction, alkylation and acetone precipitation, 50 µg proteins per sample were digested in-solution with trypsin (1:50, w/w, 37 °C, 16 h). Peptides were labelled with TMT 6-plex reagents, pooled and fractionated by high-pH reversed-phase chromatography on an Agilent 1100 HPLC (Zorbax Extend-C18, 15 fractions). LC-MS/MS was carried out on a Q Exactive HF-X (Thermo) in DDA mode. Quantitative analysis was performed with Proteome Discoverer 2.4 using a 1 % FDR threshold at PSM and protein levels. The dataset provides relative abundance information for > 6 000 protein groups across tumour and control tissues, serving as a resource for biomarker and pathway studies in lung adenocarcinoma.

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Yingping Zhuang

PROVIDER: PXD078963 | iProX | Thu May 28 00:00:00 BST 2026

REPOSITORIES: iProX

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Project description:All large-scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropanol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through 13CD3-methionine labeling (heavy-methyl SILAC) of S. cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared to target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methyl-PSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-β-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large-scale LC-MS/MS methylation site discovery experiments, and make recommendations on how to reduce methyl-PSM FDRs in samples not amenable to heavy isotope labeling.

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Energy-translation coupling limits yeast anaerobic growth

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