Influence of the Digestion Technique, Protease, and Missed Cleavage Peptides in Protein Quantitation

Quantitative determination
of absolute and relative protein amounts
is an essential requirement for most current bottom-up proteomics
applications, but protein quantitation estimates are affected by several
sources of variability such as sample preparation, mass spectrometric
acquisition, and data analysis. Among them, sample digestion has attracted
much attention from the proteomics community, as protein quantitation
by bottom-up proteomics relies on the efficiency and reproducibility
of protein enzymatic digestion, with the presence of missed cleavages,
nonspecific cleavages, or even the use of different proteases having
been postulated as important sources of variation in protein quantitation.
Here we evaluated both in-solution and filter-aided digestion protocols
and assessed their influence in the estimation of protein abundances
using five E. coli mixtures with known amounts of
spiked proteins. We observed that replicates of trypsin specificity
digestion protocols are highly reproducible in terms of peptide quantitation,
with digestion technique and the chosen proteolytic enzyme being the
major sources of variability in peptide quantitation. Finally, we
also evaluated the result of including peptides with missed cleavages
in protein quantitation and observed no significant differences in
precision, accuracy, specificity, and sensitivity compared with the
use of fully tryptic peptides.