Hydrophilic strong anion exchange (hSAX) chromatography enables deep fractionation of tissue proteomes

The bottom-up proteomic analysis of cell line and tissue samples to a depth > 10,000 proteins still represents a considerable challenge because of the sheer number of peptides generated by proteolytic digestions and the high dynamic range of protein expression. As a result, comprehensive protein coverage requires multidimensional peptide separation. Recently, off-line hydrophilic strong cation exchange (hSAX) chromatography has proven its merits for high resolution separation of peptides due to its high degree of orthogonality to reversed-phase liquid chromatography. Here we describe the use of hSAX for the deep analysis of tissue proteomes. The protocol includes optimized sample preparation steps (lysis with the aid of mechanical disruption, one-step disulfide bridge reduction and alkylation), setup and operation of hSAX columns and gradients, desalting of hSAX fractions prior to LC-MS/MS analysis, and suggestions for the choice of data acquisition parameters and data analysis using MaxQuant. Application of the protocol to the fractionation of 300 μg human brain tissue digest led to the identification of more than 100,000 unique peptide sequences representing over 10,195 proteins and 9,500 genes in 3 days of measurement time on a Q Exactive Plus mass spectrometer.