Automated Off-Line Two-Dimensional LC–MS–MS of Complex Proteomic Samples with the UltiMate 3000 System

Multidimensional liquid chromatography (MDLC) techniques are essential for the separation of highly complex proteomic samples. Advantages of off-line MDLC techniques over on-line approaches include high flexibility in choice of column dimensions and mobile-phase compositions, and the ability to reanalyse sample fractions. Here we present a fully automated off-line two-dimensional chromatographic approach for the analysis of proteomic samples using an UltiMate 3000 system optimized for proteomics MDLC.

Instrumentation and Chromatographic Conditions

Off-line two-dimensional (2D) chromatographic experiments were performed with an UltiMate 3000 system (Dionex) equipped with a dual gradient pump with membrane degasser (DGP3600), flow manager module with active flow-split technology (FLM-3100), autosampler with integrated micro-fraction collector (WPS-3000), and two UV detectors (VWD-3400). The system was coupled on-line to an ion-trap mass spectrometer (HCTultra)

The workflow for automated off-line 2D-LC is as follows: (1) a first-dimension LC separation with fraction collection, followed by (2) repeated cycles of injection of the collected fractions (second-dimension), LC separations and detection of peptides by tandem mass spectrometry. Figure 1 shows a schematic diagram of the 2D-LC system. The first-dimension separation was performed on a 15 cm × 300 μm i.d. strong cation-exchange column (SCX). After injection of 10 pmol of tryptic peptides from transferrin, bovine serum albumin, β-galactosidase, alcohol dehydrogenase, lysozyme, and cytochrome c, a salt gradient from 0–600 mM NaCl in 5 mM phosphate buffer pH 3 + 5% ACN was applied in 20 min at a flow-rate of 6 μL/min. Detection was achieved with a 45 nL flow cell at 214 nm. Fractions were collected every minute from 10–30 min. The second dimension separation included on-line sample preconcentration and desalting using a monolithic trap column, and separation of peptides on a PepSwift 5 cm, 200 μm i.d. monolithic column applying an acetonitrile gradient from 0–36% in 0.05% TFA in 10 min. Peptides were detected by UV using a 3 nL flow cell and MS–MS detection.

Figure 1

Automated Off-Line MDLC

To minimize dilution of the peptide fractions, the salt gradient was optimized such that 84% of the peptides eluted in single or adjacent fractions, resulting in the chromatogram shown in Figure 2(a). Excellent retention time precision was observed with RSD values <0.1% for three consecutive 2D-LC runs. Using a polystyrene-divinylbenzene monolithic column (PepSwift) and a steep gradient (3.6% ACN/min), a peak capacity of 125 was obtained within 10 min for the second-dimension separation. Figure 2(b) shows a second-dimension separation of the same fraction (#4) obtained in three consecutive MDLC runs. The chromatograms were obtained 12 h apart, (the time required to complete one MDLC analysis). Retention time precision measured for 16 peptides varied between 0.0 and 0.20% RSD. The protein sequence coverage determined with MASCOT was highly reproducible and varied between 43 and 75% for the samples originating from different proteins.

Figure 2

MDLC Data Visualization

As a result of the complex nature of the sample, a special representation was developed that allows easy comparison of the different samples with Chromeleon software. (See Figure 2.) Figure 2(a) shows the first dimension separation; Figure 2(b) shows the consecutive second-dimension separations, using a visualization approach similar to the gel images obtained after 2D-gel electrophoresis (2D-PAGE). A second-dimension separation of a single fraction sampled from the first dimension separation is shown in Figure 2(c).

Concluding Remarks

The UltiMate 3000 MDLC allows automated off-line 2D-LC of complex proteomic samples. The method provides high peak capacity, high resolution and excellent retention time stability.

Pepswift is a trademark and UltiMate and Chromeleon are registered trademarks of Dionex Corporation, Sunnyvale, California, USA.

HCTultra is a trademark of Bruker Daltonics Incorporated, Billerica, Massachusetts, USA.

Dionex Corporation

1228 Titan Way, Sunnyvale, California 94085, USA

tel. +1 408 737 0700 fax +1 408 730 9403

Website: www.dionex.com

Bas Dolman, Robert van Ling, Evert-Jan Sneekes, Sebastiaan Eeltink and Remco Swart, Dionex, Amsterdam, The Netherlands.