Hydrophilic Strong Anion Exchange (hSAX) Chromatography for Highly Orthogonal Peptide Separation of Complex Proteomes

Abstract

Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.

Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

“…We also hypothesized that the differences in the three phosphopeptide enrichment methods investigated in this study would decrease in such a setting, as the aforementioned limitations of data-dependent acquisition would become less pronounced (49). The Trost laboratory has recently shown good orthogonality of hydrophilic strong anion exchange and reversed phase chromatography for ordinary peptides (50). We therefore attempted to determine whether hSAX would show the same desirable characteristics for phosphopeptides (50).…”

“…The Trost laboratory has recently shown good orthogonality of hydrophilic strong anion exchange and reversed phase chromatography for ordinary peptides (50). We therefore attempted to determine whether hSAX would show the same desirable characteristics for phosphopeptides (50). In a first step, we reproduced hSAX performance for the fractionation of full proteome samples by separating 300 g of A431 digest into 24 fractions and measuring each of these via LC-MS/MS using a 110-min reversed phase gradient.…”

What Can Proteomics Tell Us About Platelets?

“…21 Advantages are that, for peptides, (1) separation by chromatography, (2) ionization, and (3) fragmentation are more efficient, (4) their masses are less heterogeneously distributed, and (5) their identification through database searches is more straightforward. Complex peptide mixtures can be either directly analyzed by liquid chromatography-MS using long gradients 22 or fractionated by hyphenation of different separation techniques beforehand 23 to reduce sample complexity and increase depth and coverage of the proteome analysis. Peptide sequences are usually identified by comparing the molecular masses of the peptide (parent) ion and corresponding fragment ions, as determined in MS and MS/MS scans, with the predicted peptide/fragment masses obtained from in silico digestion of a protein database.…”

High‐resolution quantitative proteome analysis reveals substantial differences between phagosomes of RAW 264.7 and bone marrow derived macrophages

“…In recent years, the cell line has also been used extensively in proteomics experiments including a large‐scale proteome 10, phagosome proteomics 4, 8, 11, responses to cytokines 12, 13, and identification of DNA receptors 14.…”