Protein pre-fractionation with a mixed-bed ion exchange column in 3D LC-MS/MS proteome analysis

The fractionation of complex samples at the protein level prior to shotgun proteomics analysis is an efficient means to more comprehensive analysis of samples. A mixed-bed ion-exchange (IEX) column, packed with both weak anion exchange (WAX) and weak cation exchange (WCX) materials, was used for the first dimensional separation of complex samples at the protein level using volatile solvents. The peptides from digestion of each fraction were then identified by 2D SCX-RP-LC-MS/MS. We applied this 3D strategy to mouse mammary tumor 4T1 cell lysate and identified a total of 3084 proteins in a typical experiment. The moderate separation performance of the mixed-bed IEX column facilitated the in-depth identification of the proteins in the complex sample. There were some acceptable inter-fraction overlaps. Nearly half (45.8%) of the proteins were only identified in single fractions, while 82.3% were identified in no more than 3 fractions. The identified proteins covered a broad range of pI, size and grand average hydrophobicity (GRAVY) values. Detailed analysis of proteins identified in each fraction elucidated the separation characteristics of mixed-bed IEX. Retention on mixed-bed IEX was associated, but not restricted to the extreme pI values (pI<5, pI>10) and to the percentage of charged residues of both signs. In conclusion, we have exploited the mixed-bed IEX column to establish an efficient and comprehensive identification method for complex samples.