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Multidimensional separation of tryptic peptides from human serum proteins using reversed‐phase, strong cation exchange, weak anion exchange, and fused‐core fluorinated stationary phases
Author(s) -
Boichenko Alexander P.,
Govorukhitalia,
Zee Ate G. J.,
Bischoff Rainer
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201300750
Subject(s) - chemistry , chromatography , proteome , peptide , fractionation , ion exchange , ion mobility spectrometry , mass spectrometry , analytical chemistry (journal) , ion , biochemistry , organic chemistry
Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high‐abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C 18 at pH 2.5 (low pH), fused‐core fluorinated at pH 2.5, and RP C 18 at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip‐LC–MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low‐pH RP chromatography. A 3D separation scheme combining ERLIC, high‐pH RP, and low‐pH nanoChip‐LC–MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.