Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells

Abstract

In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed-phase liquid chromatography strategy as a first dimension for two-dimensional liquid chromatography tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed-phase liquid chromatography as a first-dimension fractionation strategy resulted in 1.8- and 1.6-fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed-phased strategy is an attractive alternative to strong cation exchange for two-dimensional shotgun proteomic analysis.

Figure 1

Scheme of concatenation strategy applied to the first-dimensional low-pH RP and high-pH RP fractionation (A), second-dimensional LC/MS/MS base peak chromatography of low-pH RPLC (B) and high-pH RPLC (C) at postconcatenation fraction 10.

Figure 2

Venn diagrams showing the overlap of proteins identified using high-pH RPLC with proteins identified using SCX (A) and Low-pH-RPLC (B).

Figure 3

Two-dimensional displays of separated species, illustrating the orthogonality of the first-dimension separation (i.e. low-pH RPLC, high-pH RPLC, or SCX) with the second-dimension (low pH) RPLC separation. The heat maps illustrate the distinct number of unique peptides identified in each bin, with the X-axis representing the postconcatenation fraction number (1-D) and the Y-axis, the NET of the second-dimensional RPLC (2-D). The number of peptides (low to high) is indicated by the blue–white–red color scale.