Lee, Hankyul Ju, Shinyeong Lee, Seonjeong Lee, So Ha Kim, Sang-Yoon Park, Narae Hwang, Cheol-Sang Park, Kang-Sik Lee, Cheolju 2025-08-20T03:09:34Z 2025-08-20T03:09:34Z 2025-08-20 2025-09 1535-3893 https://pubs.kist.re.kr/handle/201004/152962 alpha-N-Terminal methylation is an understudied post-translational modification involved in protein-protein or protein-DNA interactions. Its global profiling by mass spectrometry is challenging due to low abundance and interference from near-isobaric modifications like Nt-acetylation, even after N-terminome enrichment. To address this problem, we assume that a-, b-, and y-ions will exhibit different mass error distributions in MS2 spectra if falsely assigned to a near-isobaric Nt-modification. We exploit this statistically to correct the Nt-modification, a procedure we name the mass error test (MET). We confirmed the effectiveness of MET by manual inspection of chemically methylated BSA peptides. MET was further confirmed by comparing a- and b-ion proportions and predicted retention times between Nt-methylation and Nt-acetylation in chemically modified cell lysates. We applied MET to potentially Nt-methylated spectra from a repurposed dataset and reassigned the correct Nt-modification. By implementing MET on the HCT116 N-terminome, we were able to reassign Nt-modified PSMs with a net change of similar to 17.1% reduction in falsely assigned Nt-trimethyl PSMs. These results indicate that MET is a useful tool for the detection of Nt-methylated proteins in complex proteomes. English American Chemical Society Distinguishing N-Terminal Methylation from Near-Isobaric Modifications by Statistical Analysis of Mass Error Distributions of Fragment Ions Article 10.1021/acs.jproteome.4c01045 1 Journal of Proteome Research, v.24, no.9, pp.4804 - 4815 Journal of Proteome Research 24 9 4804 4815 N scie scopus 001541348600001 Biochemical Research Methods Biochemistry & Molecular Biology Article PROTEIN METHYLATION PEPTIDES IDENTIFICATION SPECTROMETRY YEAST ACID RCC1 LC-MS/MS N-terminal methylation mass error test post-translational modification mass error distribution near isobaric modification