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.

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

“…In recent years, a different 2D-LC methodology has been developed, based on the RP separation under basic pH (pH 10) conditions in the first dimension and a low-pH RP separation in the second dimension. [26][27][28][29][30][31][32][33][34][35][36][37][38][39] In all reported work using capillary chromatography (e.g., 300 μm ID columns), [26][27][28][29][30][31][32][33][34][35][36] the first LC dimension was operated in an off-line mode using fraction collection, followed by excess organic solvent evaporation mAbs Fig. 2B) from the second dimension (lowpH) separations in four consecutive injections (experiments).…”

“…26,27,30,31 This observation prompted much interest in coupling two RP columns, operated at two pH extremes (pH 10 and pH 2.5), as a 2D chromatographic system for peptide separation, using online or off-line configurations. [26][27][28][29][30][31][32][33][34][35][36][37][38][39] Although the coupling of high-pH RP/low-pH reversed-phase separations was shown to be less orthogonal than the classical SCX/RP multidimensional system for the separation of complex peptide mixtures in proteomic experiments, 30 the separation resolution offered by the high-pH RP in the first chromatographic dimension is far superior to the SCX separation. RP separation elutes peptides almost equally over the entire retention window (trapezoidal distribution of peptides) allowing for a greater spread of peptides across the same number of fractions.…”

The effect of microbial colonization on the host proteome varies by gastrointestinal location

“…For example, we predict that there will be significant colonization-dependent differences in immunoglobulin profiles, given that there is a large difference in serum immunoglobulin profiles between GF and CR mice (Meeuwsen et al, 1989). We expect that application of more nuanced enrichment and fractionation protocols, as deployed in other proteome investigations (Zaia, 2008;Wang et al, 2011), hold the potential to provide even greater insight into the host proteins directing and responding to the commensal microbiota.…”

Ischemia in Tumors Induces Early and Sustained Phosphorylation Changes in Stress Kinase Pathways but Does Not Affect Global Protein Levels

“…Upon fragmentation in the mass spectrometer, reporter ions at defined masses are released, and the ratios of these reporter ions to one another enables relative quantification (21). After iTRAQ labeling, peptide samples were fractionated using basic reverse-phase liquid chromatography to reduce the overall sample complexity per fraction and thus increase the depth of coverage (24). Basic reverse-phase liquid chromatography fractions were combined in a noncontiguous manner into 24 proteome and 12 phosphoproteome fractions, with 5% of the total material contributing to the proteome analysis and 95% to the phosphoproteome analysis.…”

“…Peptides were labeled with 4-plex iTRAQ reagents and separated using an off-line high pH (7.5 or 10) reversed-phase column. Fractions were collected and concatenated (14,24) into 24 fractions, and 5% of each fraction was analyzed via LC-MS/MS for quantitative global proteomics measurement. The remainder (95%) of each of the 24 fractions was further concatenated into 12 fractions, and phosphopeptides were enriched using immobilized metal (Fe 3ϩ ) affinity chromatography prior to LC-MS/MS analysis.…”