Dataset Information

Analysis of M.xanthus boundary by nano-LC-MS/MS

ABSTRACT: Investigation of different proteins that play role in the formation of boundary between DK1622 and the incompatible mutant strain via proteomics approach.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Myxococcus Xanthus Dk 1622

SUBMITTER: Mian Nabeel Anwar

LAB HEAD: Prof.Yue-zhong Li

PROVIDER: PXD012532 | Pride | 2019-11-13

REPOSITORIES: Pride

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Publications

Omics Studies Revealed the Factors Involved in the Formation of Colony Boundary in Myxococcus xanthus.

Anwar Mian Nabeel MN Li Zhi Feng ZF Gong Ya Y Singh Raghvendra Pratap RP Li Yue-Zhong YZ

Cells 20190603 6

Two unrecognizable strains of the same bacterial species form a distinct colony boundary. During growth as colonies, Myxococcus xanthus uses multiple factors to establish cooperation between recognized strains and prevent interactions with unrecognized strains of the same species. Here, ΔMXAN_0049 is a mutant strain deficient in immunity for the paired nuclease gene, MXAN_0050, that has a function in the colony-merger incompatibility of Myxococcus xanthus DK1622. With ...[more]

PMID: 31163575

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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.

Dataset Information

Analysis of M.xanthus boundary by nano-LC-MS/MS

Publications

Omics Studies Revealed the Factors Involved in the Formation of Colony Boundary in <i>Myxococcus xanthus</i>.

Similar Datasets

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