Acid/Salt/pH Gradient Improved Resolution and Sensitivity in Proteomics Study Using 2D SCX-RP LC–MS

The
usage of strong cation exchange (SCX) chromatography in proteomics
is limited by its poor resolution and nonspecific hydrophobic interactions
with peptides, which lead to peptide overlap across fractions and
change of peptide retention, respectively. The application of high concentration of salt (up to 1000 mM) in SCX also restricted its use in online 2D SCX-RP LC. In the present
research, we first exploited the chromatographic ability of online
2D SCX-RP LC by combination of acid, salt, and pH gradient, three
relatively independent modes of eluting peptides from SCX column.
50% ACN was added to elution buffer for eliminating hydrophobic interactions
between SCX matrix and peptides, and the concentration of volatile
salt was reduced to 50 mM. Acid/salt/pH gradient showed superior resolution
and sensitivity as well as uniform distribution across fractions,
consequently leading to significant improvements in peptide and protein
identification. 112 191 unique peptides and 7373 proteins were
identified by acid/salt/pH fractionation, while 69 870 unique
peptides and 4536 proteins were identified by salt elution, that is,
62.5 and 60.6% more proteins and unique peptides, respectively, identified
by the former. Fraction overlap was also significantly minimized by
acid/salt/pH approach. Furthermore, acid/salt/pH elution showed more
identification for acidic peptides and hydrophilic peptides.