Comparative Evaluation of Small Molecular Additives and Their Effects on Peptide/Protein Identification

Detergents
and salts are widely used in lysis buffers to enhance
protein extraction from biological samples, facilitating in-depth
proteomic analysis. However, these detergents and salt additives must
be efficiently removed from the digested samples prior to LC-MS/MS
analysis to obtain high-quality mass spectra. Although filter-aided
sample preparation (FASP), acetone precipitation (AP), followed by
in-solution digestion, and strong cation exchange-based centrifugal
proteomic reactors (CPRs) are commonly used for proteomic sample processing,
little is known about their efficiencies at removing detergents and
salt additives. In this study, we (i) developed an integrative workflow
for the quantification of small molecular additives in proteomic samples,
developing a multiple reaction monitoring (MRM)-based LC-MS approach
for the quantification of six additives (i.e., Tris, urea, CHAPS,
SDS, SDC, and Triton X-100) and (ii) systematically evaluated the
relationships between the level of additive remaining in samples following
sample processing and the number of peptides/proteins identified by
mass spectrometry. Although FASP outperformed the other two methods,
the results were complementary in terms of peptide/protein identification,
as well as the GRAVY index and amino acid distributions. This is the
first systematic and quantitative study of the effect of detergents
and salt additives on protein identification. This MRM-based approach
can be used for an unbiased evaluation of the performance of new sample
preparation methods. Data are available via ProteomeXchange under
identifier PXD005405.