Automated Off-Line Multidimensional LC-MS-MS of Complex Peptide Samples
by Bas Dolman, Evert-Jan Sneekes and Remco Swart, Dionex Corporation
Abstract
Multidimensional liquid chromatography is a valuable technique for bottom-up and top-down workflows in proteomics, greatly enhancing the resolution of peptide and protein separations. Various 2-D LC techniques have been described for proteomic studies. Off-line 2-D LC has several advantages over on-line approaches: higher flexibility in column dimensions and mobile phase selection, easier method development, and the ability to reanalyze the sample fractions. However, off-line 2-D LC methods are labor intensive and not automated. We present a novel solution to optimize off-line 2-D LC that combines the flexibility of traditional off-line 2-D LC techniques with a fully automated workflow. The UltiMate 3000 nano LC system with microfraction collection used for this application demonstrated strong analytical performance for both separation dimensions.
System and Method for Automated Off-Line 2-D LC of Peptides
The workflow for automated off-line 2-D LC involves of the following steps: sample injection, a first dimension LC separation with fraction collection followed by repeated cycles of injection of the collected fractions, second dimension LC separation, and detection of peptides by tandem MS. The method was evaluated by performing consecutive 2-D LC experiments on a complex tryptic peptide sample.
Figure 1. Schematic set-up for automated off-line 2-D LC of peptides.Click to enlarge.
Figure 1 represents a schematic diagram of the 2-D LC system. The UltiMate 3000 nano LC System used in this study consists of a dual gradient pump with a membrane degasser, a flow manager module with UltiFlow technology, two UV detectors and a WPS 3000 autosampler modified with a micro fraction collection option. The UltiMate 3000 was coupled on-line to an ion-trap mass spectrometer (Bruker, HCT ultra).
The first dimension separation was performed on a 1.0-mm ID 15-cm polysulfoethyl aspartamide strong cation-exchange (SCX) column. Approximately 10 pmol of tryptic peptides from transferrin, bovine serum albumin, -galactosidase, alcohol dehydrogenase, lysozyme and cytochrome C were injected onto the SCX column. Peptides were eluted by a linear salt gradient from 0 to 600 mM NaCl in 10-mM phosphate buffer pH 3 + 5% acetonitrile in 20 min at a flow rate of 50 L/min. Detection was performed with a 180-nL UV flow cell at 214 nm. A total of 24 fractions were collected every 1 min from 2 to 26 min. The second dimension separation was performed on a capillary column switching set-up for online sample preconcentration and desalting. PepSwift polystyrene-divinylbenzene (PS-DVB) monolithic columns with an ID of 200 m were used for the reversed-phase (RP) separation with lengths of 5 and 50 mm for the trap- and separation column respectively. One-fifth of the SCX fractions corresponding to 10 L were injected onto the reversed-phase trap column. After desalting the SCX fractions, peptides were eluted with an acetonitrile gradient from 0% to 36% in 0.05% trifluoroacetic acid in 10 min. Peptides were detected by UV at 214 nm (3-nL flow cell) and tandem MS.
Injection Performance
The injection performance of the autosampler with an eight-port valve was evaluated by repetitive analysis of a mixture of parabenes in RP-HPLC mode using a 2.1-mm ID narrow bore column. The injection precision was better than 0.3% RSD for full loop injections. The carry-over of the modified autosampler was measured for parabenes and peptides in RP and ion-exchange chromatography respectively. For both types of analytes the carry-over was below 0.02%. This performance is acceptable for the off-line 2-D LC application.
First Dimension Strong Cation-Exchange Separation
Figure 2. Overlay of three first-dimension separations of tryptic peptides.Click to enlarge.
An overlay of three first dimension separations of the sample on the SCX column is shown in Figure 2. The retention time precision of peptides separated on this microbore SCX column in the 2-D LC setup was better than 0.1% RSD.
The peptides are well retained on the column using a mobile phase of pH 3. From a chromatographic point of view a peptide is ideally eluted in one single fraction. However, some peak splitting as a result of the fractionation process is inevitable. Distribution of the peptides over the fractions was examined by applying tandem MS detection. It appeared that 84% of the peptides eluted in single or adjacent fractions. This result assures that minimal redundant MS data is obtained and that the peptide detection is not hampered as a result of dilution over multiple fractions as is sometimes seen with less efficient salt step 2-D LC methods. Extracolumn band broadening contribution from the fractionation tubing and needle was determined to be smaller than 5%.
Second Dimension RP Separation
The use of PepSwift monolithic capillary columns in the preconcentration set-up resulted in fast, highly efficient peptide separations. The applied steep LC gradient (3.6% acetonitrile/min) yielded a peak capacity of approximately 125 for peptides. Even with these fast RP separations the 2-D LC analysis of one sample, comprising 24 SCX fractions continues for approximately 12 h. This analysis time must be multiplied if one wants to compare different samples. Retention time fluctuations must be very small in order to allow comparison of two chromatograms from two samples that are obtained with a time shift corresponding to the entire 2-D LC analysis time. Figure 3 shows three 2-D RP separations of SCX fraction 13 taken from consecutive 2-D LC runs. The chromatograms were obtained 12 h after each other, the time that was needed to complete the 2-D LC analysis. Retention time precision measured for 20 peptides in different SCX fractions was between 0.0 and 0.20% RSD.
Figure 3. Second dimension RP separation of SCX fraction #13 on capillary PS-DVB monolithic columns. Click to enlarge.
Peptides eluting from the monolithic capillary RP column were detected and sequenced by tandem MS. The sample was analyzed in triplicate to examine the precision of peptide sequencing and protein identification. Peptide tandem MS spectra were submitted to MASCOT and searched against an in-house protein database. Protein sequence coverages were found to be highly reproducible between the consecutive 2-D LC-MS-MS experiments and varied between 43% and 75% for the different proteins in the sample.
Visualization of 2-D LC Data
Chromatographic data from 2-D LC experiments require a special representation to accomodate the complexity of the sample and to allow comparison of different samples. The two chromatographic separation dimensions and the peak intensity can be shown by a contour plot or a 2-D retention map. Chromeleon software is capable of generating 2-D retention maps as shown in Figure 4. The top panel shows the first dimension SCX separation, the middle panel shows the consecutive second dimension separations in a 2-D format. The lower panel is actively linked with the first dimension separation; after selecting a fraction, the chromatogram of the second dimension separation appears. This visualization tool is similar to gel images obtained using 2-D gel electrophoresis and offers a technique to directly compare complex peptides or proteins samples.
Conclusion
High-resolution LC methods are required for increased sample complexity in proteomics analysis.
Figure 4. Visualization of 2-D LC data in Chromeleon. Top panel first dimension separation, middle panel 2-D retention map, lower panel second dimension separation. Click to enlarge.
Automated off-line 2-D LC of peptides is a novel technique that combines excellent chromatography, flexible methods, and automated operation. Retention time precision is excellent for both separation dimensions. The same workflow can be applied for 2-D LC separation of intact proteins for top-down proteomics applications after selection of appropriate column chemistries.
For more information, contact Remco Swart, manager HPLC applications, Dionex Corporation, at remco.swart
dionex.com or by phone at +31 20 683 9768. Laboratory Equipment Advantage Business Media
