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.