Multi-dimensional liquid chromatography in proteomics—A review (original) (raw)
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Journal of Chromatography B, 2009
Multidimensional liquid-based separation techniques are described for maximizing the resolution of the enormous number of peptides generated upon tryptic digestion of proteomes, and hence, reduce the spatial and temporal complexity of the sample to a level that allows successful mass spectrometric analysis. This review complements the previous contribution on unidimensional high performance liquid chromatography (HPLC). Both chromatography and electrophoresis will be discussed albeit with reversed-phase HPLC (RPLC) as the final separation dimension prior to MS analysis.
Reference map for liquid chromatography–mass spectrometry-based quantitative proteomics
Analytical Biochemistry, 2009
The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC-tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.
2010
A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications. Molecular & Cellular Proteomics 9:225-241, 2010. LC-MS/MS provides the most widely used technology platform for proteomics analyses of purified proteins, simple mixtures, and complex proteomes. In a typical analysis, protein mixtures are proteolytically digested, the peptide digest is fractionated, and the resulting peptide fractions then are analyzed by LC-MS/MS (1, 2). Database searches of the MS/MS spectra yield peptide identifications and, by inference and assembly, protein identifications. Depending on protein sample load and the extent of peptide fractionation used, LC-MS/MS analytical systems can generate from hundreds to thousands of peptide and protein identifications (3). Many variations of LC-MS/MS analytical platforms have been described, and the performance of these systems is influenced by a number of experimental design factors (4). Comparison of data sets obtained by LC-MS/MS analyses provides a means to evaluate the proteomic basis for biologically significant states or phenotypes. For example, data-dependent LC-MS/MS analyses of tumor and normal tissues enabled unbiased discovery of proteins whose expression is enhanced in cancer (5-7). Comparison of data-dependent LC-MS/MS data sets from phosphotyrosine peptides in drugresponsive and-resistant cell lines identified differentially regulated phosphoprotein signaling networks (8, 9). Similarly, activity-based probes and data-dependent LC-MS/MS analysis were used to identify differentially regulated enzymes in normal and tumor tissues (10). All of these approaches assume that the observed differences reflect differences in the proteomic composition of the samples analyzed rather than analytical system variability. The validity of this assumption is From the
Review of Three-Dimensional Liquid Chromatography Platforms for Bottom-Up Proteomics
Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.
Anal Chem, 2003
The ability to manipulate and effectively utilize small proteomic samples is important for analyses using liquid chromatography (LC) in combination with mass spectrometry (MS) and becomes more challenging for very low flow rates due to extra column volume effects on separation quality. Here we report on the use of commercial switching valves (150-μm channels) for implementing the on-line coupling of capillary LC columns operated at 10 000 psi with relatively large solid-phase extraction (SPE) columns. With the use of optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained demonstrating peak capacities of ∼1000 for capillaries having inner diameters between 15 and 150 μm. The on-line coupled SPE columns increased the sample processing capacity by ∼400-fold for sample solution volume and ∼10-fold for sample mass. The proteomic applications of this on-line SPEcapillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Using an ion trap tandem MS it was typically feasible to identify 1100-1500 unique peptides in a 5-h analysis. Peptides extracted from the SPE column and then eluted from the LC column covered a hydrophilicity/hydrophobicity range that included an estimated ∼98% of all tryptic peptides. The SPE-capillary LC implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for automated proteomic analyses.
Analytical Chemistry, 2001
We report on the design and application of a highefficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10 000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of ∼650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of >6 × 10 7 components. For yeast cytosolic tryptic digests >100 000 polypeptides were detected, and ∼1000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (∼1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification. The "postgenomic era" presents the challenge of analyzing the complex array of proteins (i.e., the proteome) expressed by an organism, tissue, or cell to aid in the understanding of the operation of complex cellular pathways, networks, and "modules" under various physiological conditions. 1-5 An organism's proteome is not fixed, but changes with the state of the development, the tissue, and the environmental conditions. 6,7 To delineate key proteins and unravel the complex molecular pathways and networks involved in cellular responses, a set of proteomes in response to various environmental "perturbation" can be analyzed and exploited. This requires that the proteome analysis methodology be sensitive, robust, quantitative, and high-throughput. Mass spectrometry (MS) is playing an increasingly important role in proteome analysis, 8-10 and current proteome analysis strategies primarily involve its combination with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separations of proteins. 11-13 However, limitations of 2-D PAGE that arise due to measurement dynamic range, protein solubility issues, and extremes of protein isoelectric points and molecular weights have impeded complete proteome characterization, 6,14 and thus true proteome-wide analysis will only be realized by implementation of more effective approaches that likely will include higher resolution and/or multidimensional separation strategies. In one approach for improving proteome coverage, Yates and co-workers demonstrated the use of global protein digests with two-dimensional capillary liquid chromatography (LC/LC) coupled