High-field asymmetric waveform ion mobility spectrometry (FAIMS) improves proteome-wide N-homocysteinylation mapping in mouse liver and brain proteins

Abstract N -Homocysteinylation induces immunogenic, thrombogenic, and amyloidogenic properties of proteins. Although important to gain insight into the mechanisms of homocysteine (Hcy) toxicity, proteome-wide studies of the effects of Hcy-thiolactone (HTL) protein modification remain challenging due to the low abundance of N -Hcy-proteins. High-field asymmetric waveform ion mobility spectrometry (FAIMS), as a robust online fractionation method, improves the identification of other PTMs, we therefore expected it to facilitate the detection of N -homocysteinylated proteins ( N -Hcy-proteins) and help gain insight into their role in human disease. After extensive measurement optimization, we compared the yield of N -Hcy-protein/peptide identification across mouse liver and brain samples, either native or modified in vitro with HTL. Additionally, we examined the influence of different thiol reduction and blocking agents, namely dithiothreitol (DTT)/iodoacetamide (IAA) and tris(2-carboxyethyl)phosphine (TCEP)/methyl methanethiosulfonate (MMTS), on the number of identified N -Hcy-sites. FAIMS increased the number of N -Hcy-Lys-peptides and N -Hcy-proteins by 1.3–7-fold and 1.1–7-fold, respectively, and was on average higher with the usage of TCEP/MMTS thiol reduction/blocking method. We have identified 69 and 1198 in vivo and in vitro N -Hcy-proteins, respectively. We conclude that FAIMS is a valuable addition to N -Hcy-proteome analysis workflow and facilitates the mapping of N -Hcy-sites. Data are available via ProteomeXchange with identifier PXD062860.