Leveraging Immonium Ions for Targeting Acyl‐Lysine Modifications in Proteomic Datasets

Abstract

Acyl modifications vary greatly in terms of elemental composition and site of protein modification. Developing methods to identify acyl modifications more confidently can help to assess the scope of these modifications in large proteomic datasets. The utility of acyl‐lysine immonium ions is analyzed for identifying the modifications in proteomic datasets. It is demonstrated that the cyclized immonium ion is a strong indicator of acyl‐lysine presence when its rank or relative abundance compared to other ions within a spectrum is considered. Utilizing a stepped collision energy method in a shotgun experiment highlights the immonium ion. By implementing an analysis that accounted for features within each MS2 spectrum, the method clearly identifies peptides with short chain acyl‐lysine modifications from complex lysates. Immonium ions can also be used to validate novel acyl modifications; in this study, the first examples of 3‐hydroxylpimelyl‐lysine modifications are reported and they are validated using immonium ions. Overall these results solidify the use of the immonium ion as a marker for acyl‐lysine modifications in complex proteomic datasets.

Discovery of Signature Cyclic Immonium Ion for Lactyllysine Reveals Widespread and Functional Lactylation on Non-histone Proteins

“…This phenomenon suggests the tendency of the linIm ion to lose NH 3 in the gas phase. In agreement, previous research has also noted that the facile immonium ion of acetyllysine at m/z 143.12 is conducive to lose NH 3 and delivers a peak of increased intensity at m/z 126.09 16,19,38 . Following the analysis of lactylated model peptides, the validity of the cycIm ion in signifying lactylation was then con rmed by its prevalence in the MS/MS spectra of antibody-enriched lactylation proteome.…”

“…Consistently, acetylation at lysine residues can generate a facile acetylysine immonium ion at m/z 143.1179 and subsequently produce a secondary immonium ion at m/z 126.0913 15 , which serves as a substitute for the former with improved speci city albeit still suffers from low sensitivity in acetylation recognition 16 . Besides these prevalent PTMs, diagnostic value of immonium ions culminates in con dent identi cation of novel PTMs such as phosphohistidine 17 , arginine rhamnosylation 18 and lysine hydroxypimelylation 19 that have all been rstly and only discovered in bacteria. Collectively, these ndings elicit us to seek for reliable signature ions for lactylation given the depth current proteomics technology can reach even without a nity enrichment.…”

The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationships in Benzoate Degradation

“…Without employing PTM-specific enrichment procedures, we identify a wide range of acylations by targeting acyl-CoAs that are known to be involved in fatty and aromatic acid degradation in this and other microbes (16,21). Using marker ions diagnostic of lysine acylation increases confidence in the post-translational modifications identified (22). Acylations J o u r n a l P r e -p r o o f previously undocumented across the bacterial domain are identified.…”

Small Mass but Strong Information: Diagnostic Ions Provide Crucial Clues to Correctly Identify Histone Lysine Modifications

“…Yet, in the collection of about 5000 synthetic peptides analyzed, less than 1% exhibited an N-terminal modified lysine, whereas such a scheme occurs frequently in histones proteolyzed by trypsin. More recently, a strategy of stepped collisional energy in HCD was suggested to produce both intense diagnostic ions and sequence-determining fragments and allowed identifying sequences containing a few known acylations and pinpointing a new structure in a syntrophic bacterium [ 19 ]. Here, we recapitulate the interest of looking for these immonium and diagnostic ions in MS/MS spectra, to validate the identification of peptides from sequence variants of histone H3 bearing a modified lysine at the first position.…”