Off-Line Coupling of High-Resolution Capillary Electrophoresis to MALDI-TOF and TOF/TOF MS Tomas Rejtar,†,‡ Ping Hu,† Peter Juhasz,§ Jennifer M. Campbell,§ Marvin L. Vestal,§ Jan Preisler,†,| and Barry L. Karger*,† Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, and Applied Biosystems, 500 Old Connecticut Path, Framingham, Massachusetts 01701 Received December 21, 2001
High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-µm wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests. Keywords: high-resolution separation • capillary electrophoresis • MALDI-TOF MS • MALDI-TOF/TOF MS
Introduction With the forthcoming completion of the Human Genome Project, much attention has turned to a global examination of the products of the genes, i.e., the proteome.1,2 Currently, most proteome analyses are based on separation of complex protein mixtures by 2D gel electrophoresis, followed by mass spectrometric analysis of proteolytic digests of 2D gel spots to identify individual proteins.3-5 However, this approach has limitations, most importantly, the inability to measure low-abundance, as well as poorly soluble membrane proteins and proteins with extreme MW or pI.6 Recently, new strategies for proteome analysis, e.g., shotgun proteomics,7,8 have been introduced. Shotgun proteomics involves global proteolytic digestion of a cell lysate or protein complex, followed by the analysis of the resulting complex peptide mixture by one or more dimensions of column separations coupled to tandem mass spectrometry. Alternatively, the very accurate mass of a single peptide, measured by FT-ICR MS, has been used as a unique mass tag for protein identification.9,10 Compared to 2D gel-based proteomics, the shotgun approach has the potential for improved proteome coverage, can be fully automated and is readily * To whom correspondence should be addressed. † Northeastern University. ‡ On leave from the Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, Czech Republic. § Applied Biosystems. | On leave from Department of Analytical Chemistry, Masaryk University, Brno, Czech Republic. 10.1021/pr015519o CCC: $22.00
2002 American Chemical Society
applicable to quantitative analysis of protein differential expression using isotopic labeling.11-13 Due to the sample complexity, coupling high-resolution separation to mass spectrometry is necessary for the successful implementation of shotgun proteomics.14 Such separations are required for MS analysis, since both MALDI and ESI analysis of mixtures suffer from ion suppression; i.e., the presence of certain (often major) sample components completely suppresses the signal of other components, resulting in limited dynamic range.15,16 While ESI is routinely coupled to highresolution LC2,8,17 or CE18-20 separations, owing to the solidphase nature of the resultant MALDI sample, coupling highresolution liquid-phase separation to MALDI-MS has been much more problematical. However, MALDI has several distinct features that make separation-MALDI quite attractive. Since separations coupled to ESI-MS are fundamentally online processes, MS/MS spectra must be acquired within the separation window. Precursor ion selection must be performed on-line using data-dependent acquisition, which is often biased toward the analysis of higher abundant species. Although variable-flow LC, using “peak parking”,21 alleviates the time demand on the data acquisition, it is restricted to the characterization of mixtures with limited complexity. On the other hand, off-line separation with continuous deposition of LC or CE effluents on MALDI targets decouples the separation time from MALDI-MS analysis. Database searching and peptide quantitation can be carried out on MALDIJournal of Proteome Research 2002, 1, 171-179
171
Published on Web 02/20/2002
research articles
Rejtar et al.
Figure 1. Diagram of the off-line vacuum deposition chamber coupled to capillary electrophoresis via a liquid junction: (1) Vacuum chamber, (2) standard MALDI target, (3) deposition probe, (4) infusion capillary, (5) liquid junction with grounded electrode, (6) CE separation capillary, (7) anodic reservoir with background electrolyte, (8) scanning lines for automatic determination of the start and end positions of the linear segments. See the Experimental Section for further details.
TOF/MS spectra, and subsequent MS/MS interrogation of the same sample at a different time can then be restricted to the peptides of interest. Such data acquisition schemes reduce redundant spectral acquisition and allow independent focus on such problems as low-intensity peptides or post-translational modifications. Note that the MALDI-deposited samples can be stored under appropriate conditions for months,22 for appropriate archiving. Furthermore, recent advances in MALDITOF/TOF,23 QqTOF,24-26 ion trap/TOF,27 and FTMS28 have the potential to make high-throughput, high-quality MALDI-MS/ MS analysis very powerful. Various efforts have been directed toward coupling liquidphase separation methods to MALDI-MS, either in the online29-31 or off-line modes.22,32-39 Off-line coupling of liquidphase separations is usually achieved with fraction collection, with little need for instrument modification.22,32-34,39 Alternatively, column effluents can be sprayed36 or dispensed35,37,38 on MALDI targets. Although convenient, these devices typically have large dead volumes, generating discrete spots every 2060 s. Loss of separation efficiency is, therefore, encountered, especially for low flow rate, high-resolution techniques such as CE or nano-LC. Our laboratory has previously demonstrated on-line coupling of capillary electrophoresis to MALDI-MS using vacuum deposition on a moving surface.40-42 Rapid evaporation of the solvent resulted in the formation of a narrow (
