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Identification of the ACTL8 interactome using immunoprecipitation-mass spectrometry

ABSTRACT: Immunoprecipitation coupled with mass spectrometry (IP-MS) was performed to identify proteins interacting with ACTL8 in human cells. Cell lysates were immunoprecipitated using an anti-ACTL8 antibody or IgG control, followed by LC-MS/MS analysis on a Q Exactive HF Orbitrap mass spectrometer. Protein abundance was estimated based on peptide-spectrum matches (PSMs). Proteins enriched more than two-fold in ACTL8 immunoprecipitates compared with IgG controls and with p < 0.05 were considered high-confidence ACTL8-interacting proteins.

ORGANISM(S): Homo Sapiens

SUBMITTER: Lei Ming

PROVIDER: PXD080644 | iProX | Mon Jul 06 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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Identification of the ACTL8 interactome using immunoprecipitation-mass spectrometry

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