Recent development of multi-dimensional chromatography strategies in proteome research

Abstract

As a complementary approach to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), multi-dimensional chromatography separation methods have been widely applied in all kinds of biological sample investigations. Multi-dimensional liquid chromatography (MDLC) coupled with bio-mass spectrometry (MS) is playing important roles in proteome research due to its high speed, high resolution and high sensitivity. Proteome analysis strategies mainly include bottom-up and top-down approaches which carry out biological sample separation based on peptide and protein levels, respectively. Electrophoretic methods combined with liquid chromatography like IEF-HPLC and HPLC-SDS-PAGE have been successful applied for protein separations. As for MDLC strategy, ion-exchange chromatography (IEX) together with reversed phase liquid chromatography (RPLC) is still a most widely used chromatography in proteome analysis, other chromatographic methods are also frequently used in protein pre-fractionations, while affinity chromatography is usually adopted for specific functional protein analysis. Recent MDLC technologies and applications to variety of proteome analysis have been achieved great development. A digest peptide-based approach as so-called "bottom-up" and intact protein-based approach "top-down" analysis of proteome samples were briefly reviewed in this paper. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as high-abundance protein depletion and chromatography array were also included in this review.

Fig. 1

MudPIT electrospray interface including a biphasic micro-capillary column, packed with SCX and RP packing material connected to a micro-cross .

Fig. 2

Diagram of SEC–SCX–RPLC approach. The flow of experimental information starting from cell lysate through data analysis is shown. Two NHL global protein samples were prepared, either digested or non-digested, and subjected to different SEC, removing urea, tryptic digestion of each protein fraction, and strong cation-exchange chromatography. The subsequent 240 peptide fractions were then analyzed via RPLC-MS/MS, resulting in spectra that were searched against IPI database using MASCOT .

Fig. 3

Schematic outline of the separation of the tissue lysate proteins .

Fig. 4

(a) Separation chromatogram of rat liver tissue lysate proteins by 1D SCX. Fractions were collected every 2 min automatically from 3 to 127 min, and collection time of fraction was demonstrated in the magnified illustration. (b) The RPLC elution profile of SCX fraction 3. (c) 3D display of SCX–RPLC separation of rat liver proteins sample. In total, 62 fractions were obtained from the first dimensional separation of SCX, which were separated by RPLC further. Positions of part high-abundance proteins were as labeled .

Fig. 5

(A) Schematic diagram of column array-based 2D-LC system, (B) post-column micro-valve was open, and (C) post-column micro-valve were closed .

Fig. 6

Schematic diagram of the array-based 2D-LC–MS/MS system .