Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jan 1;80(1):294-302.
doi: 10.1021/ac701727r. Epub 2007 Nov 29.

Fully Automated Four-Column Capillary LC-MS System for Maximizing Throughput in Proteomic Analyses

Affiliations
Free PMC article

Fully Automated Four-Column Capillary LC-MS System for Maximizing Throughput in Proteomic Analyses

Eric A Livesay et al. Anal Chem. .
Free PMC article

Abstract

We describe a four-column, high-pressure capillary liquid chromatography (LC) system for robust, high-throughput liquid chromatography-mass spectrometry (LC-MS(/MS)) analyses. This system performs multiple LC separations in parallel, but staggers each of them such that the data-rich region of each separation is sampled sequentially. By allowing nearly continuous data acquisition, this design maximizes the use of the mass spectrometer. Each analytical column is connected to a corresponding ESI emitter in order to avoid the use of postcolumn switching and associated dead volume issues. Encoding translation stages are employed to sequentially position the emitters at the MS inlet. The high reproducibility of this system is demonstrated using consecutive analyses of global tryptic digest of the microbe Shewanella oneidensis.

Figures

Figure 1
Figure 1
Schematic of the 4-column, 2-mixer LC system. The components of subsystems 1 and 2 are: (1,2) mixers, (3,4) ‘tee’ flow split points, (5,6) injection valves, (7,8) mobile phase solvent selection valves, (9,10) column selection valves, (11,12) mixer purge valves. Other components include: (13) syringe pump controller, (14) mobile phase A syringe pump for subsystem 1, (15) mobile phase A syringe pump for subsystem 2 (16) mobile phase B syringe pump for both subsystems, (17) control valve for sub-system 1 pump A, (18) control valve for sub-system 2 pump A, (19) control valve for pump B, (20) refill reservoir for sub-system 1 pump A, (21) refill reservoir for sub-system 2 pump A, (22) refill reservoir for pump B, (23) autosampler that uses a single syringe, (24) encoding translation stages for positioning ESI emitters, (25-28) ESI emitters, (29) motion controller for encoding translation stages (30) laptop computer (31) USB HUB. For simplification, several components have not been shown, including electronic connections between the valves and the USB HUB, an on-line degasser and a 3-drawer cool-stack sample holder accessed by the autosampler. The valves of sub-system 1 are shown in positions for sample loading onto the column connected to emitter 26 and the valves of sub-system 2 are shown in positions for a gradient elution on the column connected to emitter 28. The direction of flow through each connection is illustrated. In the configuration shown there is no flow through the sample loop of sub-system 2 nor through any of the refill reservoirs.
Figure 2
Figure 2
Duty cycle comparison of various single- and multi-column LC systems with all times drawn to scale for LC configuration 3 in Table 1 except for the data rich region and data poor region which are estimates for the chromatogram which is shown. (a) Schematic description of the steps which must be performed for each sample processed on an analytical column. The steps in consecutive analyses on systems that use (b) 1 column and 1 mixer, (c) 2 columns and 1 mixer, (d) 4 columns and 2 mixers with no elution hopping and (e) the same system shown in (d) but with the dead time at the beginning of the gradient elution bypassed or ‘hopped over’ and without acquiring data during the ‘data poor’ region at the end of the gradient elution.
Figure 3
Figure 3
Comparison of (a) absolute and (b) relative elution times for all 20 MS analyses which are numbered according to the order in which they were run (absolute run order) and the column used for analysis (Column #). The method for calculating relative elution times is described in the text. The elution times for peaks were obtained by analyzing the selected ion chromatogram for the 10 peaks simultaneously.
Figure 4
Figure 4
Comparison between normalized chromatograms for the first cycle of analyses (i.e., consecutive analyses on columns 1, 2, 3 and 4). Elution times have been linearly normalized based on 2 peaks common to all analyses. Peak intensities have been linearly normalized with the maximum peak intensity set to 1.
Figure 5
Figure 5
Duty cycle comparison for all of the LC configurations listed in Table 1. Duty cycles are based on theoretical calculations given in Table 2 and empirical times for chromatographic steps (e.g., load, etc.).

Similar articles

See all similar articles

Cited by 84 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback