Shotgun proteomic analysis of the microsomal fraction of eukaryotic cells using a two‐dimensional reversed‐phase×ion‐pair reversed‐phase HPLC setup

Abstract

A RPxIP-RP HPLC separation scheme was combined with on-line ESI-IT tandem MS or off-line MALDI tandem TOF MS and applied to the analysis of eukaryotic subcellular proteomes. Previous proteomic studies [1] were complemented by the approval of the approach to eukaryotic proteomes using the fission yeast Schizosaccharomyces pombe. The major focus was set to the analysis of primary human hepatocyte microsomes, representing a compartment of high interest due to its involvement in xenobiotic detoxification and cholesterol homeostasis. Of the 588 proteins identified from two donors, 24% are involved in cholesterol homeostasis or xenobiotic/lipid metabolism. Up to 50% of the identified proteins belong to the group of membrane proteins, difficult to investigate using gel-based proteomic approaches. We further demonstrated the reproducibility and comparability of the approach and reduced the amount of sample load by almost 70% with only minor loss of information about the proteins identified in the samples. The presented study clearly demonstrates the good applicability of the experimental setup to the analysis of subcellular proteomes including large membrane fractions, where only low amounts of sample material are available.

RP‐LC×RP‐LC analysis of a tryptic digest using a combination of totally porous and partially porous stationary phases

“…A large number of separation modes have been exploited to deliver a wide range of selectivities in the first dimension, employing ion exchange chromatography 25, hydrophilic interaction LC 26, 27, affinity LC 28–31, size‐exclusion chromatography 32–35, capillary IEF 36–39, RP‐LC operated at different pH 40–48, while the second dimension usually consists of RP‐LC, due to its high compatibility to linkage with MS detection. Among these, the use of strong cation exchange coupled to either RP‐ or ion‐pair‐LC turned out to be by far the most used combination of chromatographic separation modes, delivering high orthogonality in the separation of peptides, according to their charge and hydrophobicity 49–53. Anyway, 2‐D separation set‐ups based on RP‐LC are characterized by superior resolution and peak capacity, more homogeneous distribution of peptides eluting in the separation window, robustness, and easy handling 54, 55.…”