Quantitation of Reactive Acyl-CoA Species Mediated Protein Acylation by HPLC–MS/MS

Abstract

Recently discovered acylation by reactive acyl-CoA species is considered a novel regulatory mechanism in epigenetics and metabolism. Established analytical methods like Western blotting and proteomics fail to detect the plethora of acylation structures in a single analysis and lack the ability of absolute quantitation. In this paper, we developed an HPLC–MS/MS method for the simultaneous detection and quantitation of 14 acylated lysine species in biological samples. Extensive effort was invested into method validation resulting in recovery rates between 75 and 93% and levels of detection in the nanomolar range. Thus, we were able to quantitate 8 acylation structures in mouse liver, kidney, heart, and brain. Further enrichment by repetitive HPLC fractionation resulted in the quantitation of 6 additional acylation structures including 4 novel modifications: N 6-acetoacetyl lysine, N 6-isovaleryl lysine, N 6-(2-methylbutyryl) lysine, and N 6-tiglyl lysine.

Comprehensive analysis of posttranslational protein modifications in aging of subcellular compartments

“…N 6 -formyl lysine, N 6 -acetyl lysine, furosine, methionine sulfoxide, methionine sulfone, o-tyrosine, o,o-dityrosine, and citrulline were purchased from Sigma-Aldrich. Synthesis and structure elucidation of commercially unavailable authentic reference standards was described previously 9,13,19,20 . Structural formulas of analytes are included in the supporting information ( Figure S2).…”

“…Enzymatic hydrolysis. Enzymatic hydrolysis was performed as described previously 20 . A small crystal of thymol was added to aliquots (250 µL) of protein solutions.…”

“…To our surprise, no comprehensive data of the various PTMs exists. We previously identified AGEs as excellent markers of aging and disease in cytosolic liver proteins 19 and identified liver as a local hotspot of protein acylation 20 . Hence, we quantitated 20 different PTMs including 6 lysine acylation modifications, 7 AGEs, 6 oxidative stress markers, and citrulline in histone, mitochondrial, and cytosolic proteins extracted from mice liver using a novel HPLC-MS/MS approach.…”

Pathways of Non-enzymatic Lysine Acylation

“…Several hydrophobic derivatives of lysine acetylation ( Figure 1 ) like aliphatic propionylation and butyrylation ( Chen et al, 2007 ), unsaturated crotonylation and tiglylation ( Tan et al, 2011 ; Baldensperger et al, 2019 ), branched-chain isobutyrylation, 2-methylbutyrylation, and isovalerylation ( Baldensperger et al, 2019 ; Zhu et al, 2021 ), as well as aromatic benzoylation ( Huang et al, 2018 ) have been reported in literature previously. Due to structural similarity, propionylation and butyrylation have effects comparable to acetylation in remodeling chromatin structure and activation of transcription ( Kebede et al, 2017 ).…”

Quantification of N6‐formylated lysine in bacterial protein digests using liquid chromatography/tandem mass spectrometry despite spontaneous formation and matrix effects

“…Posttranslational modifications (PTMs) of proteins, including acetylation and formylation, are important in regulating biological systems 1,2 . Measuring acetyl and formyl PTMs employs liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis via a proteomic approach (analyzing peptides), 2,3 or measuring modifications after digesting proteins to single amino acids 4–6 . Measuring N 6 ‐formyl lysine in proteins is of increasing interest because this modified amino acid forms at locations in proteins that interfere with other PTMs (acetylation and methylation).…”