In-source fragmentation and the sources of partially tryptic peptides in shotgun proteomics

Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, their sources have been controversial. Herein, we investigate the impact of in-source fragmentation on shotgun proteomics profiling of three biological samples: a standard protein mixture, a mouse brain tissue homogenate, and mouse plasma. Because the in-source fragments of peptide ions have the same LC elution time as their parental peptides, partially tryptic peptide ions from in-source fragmentation can be distinguished from other partially tryptic peptides based on their elution time differences from those computationally predicted data. The percentage of partially tryptic peptide identifications resulting from in-source fragmentation in a standard protein digest was observed to be ∼60%. In more complex mouse brain or plasma samples, in-source fragmentation contributed to a lesser degree of 1-3% of all identified peptides due to the limited dynamic range of LC-MS/MS measurements. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic cleavage by endogenous proteases in the samples. Our work also provides a method to identify such proteolytic-derived partially tryptic peptides due to endogenous proteases in the samples by removing in-source fragmentation artifacts from the identified peptides.