Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage

Abstract

We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.

Identifying Proteins in Zebrafish Embryos Using Spectral Libraries Generated from Dissected Adult Organs and Tissues

“…Reducing protein or peptide complexity by multiple fractionation techniques (e.g., by SDS-PAGE and/or LC separation) is an efficient tool to increase the number of identifications 39 and to learn more about the proteins themselves. In this work, we built rich spectral libraries from fractionated organs and tissues.…”

Towards comprehensive and quantitative proteomics for diagnosis and therapy of human disease

“…This rationale was indeed successfully applied to increase the number of targeted mass spectrometry assays scheduled in a single experiment [68]. Improved chromatographic resolution can also be achieved by multi-dimensional and orthogonal separation techniques [69,70], which also provide a means to improve mass spectral sampling, and detection and quantification of low abundance ions, thereby increasing the exposure of specific proteome subsets such as 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 F o r P e e r R e v i e w 10 post-translationally modified peptides [7,[71][72][73]. However, most offline sample fractionation workflows are potentially hindered by sample losses that limit their overall robustness and reproducibility [74].…”

Towards comprehensive and quantitative proteomics for diagnosis and therapy of human disease

“…This rationale was indeed successfully applied to increase the number of targeted MS assays scheduled in a single experiment . Improved chromatographic resolution can also be achieved by multidimensional and orthogonal separation techniques , which also provide a means to improve mass spectral sampling, and detection and quantification of low abundance ions, thereby increasing the exposure of specific proteome subsets such as posttranslationally modified peptides . However, most offline sample fractionation workflows are potentially hindered by sample losses that limit their overall robustness and reproducibility .…”