Distinguishing N-Terminal Methylation from Near-Isobaric Modifications by Statistical Analysis of Mass Error Distributions of Fragment Ions

α-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 ∼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.