Capillary zone electrophoresis for analysis of complex proteomes using an electrokinetically pumped sheath flow nanospray interface (original) (raw)
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Mass Spectrometry Reviews, 2009
Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry( i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed.
Analytical Chemistry, 2005
The throughput of proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations as well as the subsequent mass spectrometric analysis; at present, analysis times can range anywhere from hours (high throughput) to days or longer (low throughput). We have explored the basis for proteomics analyses conducted on the order of minutes using high-speed capillary RPLC combined through online electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.8-µm porous C18 particle-packed 50-µm-i.d. capillaries were used to speed the RPLC separations while still providing high-quality separations. Both time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS were applied for identifying peptides using the accurate mass and time (AMT) tag approach. Peptide RPLC relative retention (elution) times that were generated by solvent gradients that differed by at least 25-fold were found to provide relative elution times that agreed to within 5%, which provides the basis for using peptide AMT tags for higher throughput proteomics measurements. For fast MS acquisition speeds (e.g., 0.2 s for TOF and either ∼0.3 or ∼0.6 s for FTICR), peptide mass measurement accuracies of better than (15 ppm were obtained with the highspeed RPLC separations. The ability to identify peptides and the overall proteome coverage was determined by factors that include the separation peak capacity, the sensitivity of the MS (with fast scanning), and the accuracy of both the mass measurements and the relative RPLC peptide elution times. The experimental RPLC relative elution time accuracies of 5% (using high-speed capillary RPLC) and mass measurement accuracies of better than (15 ppm allowed for the confident identification of >2800 peptides and >760 proteins from >13 000 dif-ferent putative peptides detected from a Shewanella oneidensis tryptic digest. Initial results for both RPLC-ESI-TOF and RPLC-ESI-FTICR MS were similar, with ∼2000 different peptides from ∼600 different proteins identified within 2-3 min. For <120-s proteomic analysis, TOF MS analyses were more effective, while FTICR MS was more effective for the >150-s analysis due to the improved mass accuracies attained using longer spectrum acquisition times.
Analytical Chemistry, 2005
The throughput of proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations as well as the subsequent mass spectrometric analysis; at present, analysis times can range anywhere from hours (high throughput) to days or longer (low throughput). We have explored the basis for proteomics analyses conducted on the order of minutes using high-speed capillary RPLC combined through online electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.8-µm porous C18 particle-packed 50-µm-i.d. capillaries were used to speed the RPLC separations while still providing high-quality separations. Both time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS were applied for identifying peptides using the accurate mass and time (AMT) tag approach. Peptide RPLC relative retention (elution) times that were generated by solvent gradients that differed by at least 25-fold were found to provide relative elution times that agreed to within 5%, which provides the basis for using peptide AMT tags for higher throughput proteomics measurements. For fast MS acquisition speeds (e.g., 0.2 s for TOF and either ∼0.3 or ∼0.6 s for FTICR), peptide mass measurement accuracies of better than (15 ppm were obtained with the highspeed RPLC separations. The ability to identify peptides and the overall proteome coverage was determined by factors that include the separation peak capacity, the sensitivity of the MS (with fast scanning), and the accuracy of both the mass measurements and the relative RPLC peptide elution times. The experimental RPLC relative elution time accuracies of 5% (using high-speed capillary RPLC) and mass measurement accuracies of better than (15 ppm allowed for the confident identification of >2800 peptides and >760 proteins from >13 000 dif-ferent putative peptides detected from a Shewanella oneidensis tryptic digest. Initial results for both RPLC-ESI-TOF and RPLC-ESI-FTICR MS were similar, with ∼2000 different peptides from ∼600 different proteins identified within 2-3 min. For <120-s proteomic analysis, TOF MS analyses were more effective, while FTICR MS was more effective for the >150-s analysis due to the improved mass accuracies attained using longer spectrum acquisition times.
Analytical …, 2009
An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by a microcolumn packed with mixed weak anion and weak cation exchange (WAX/WCX) particles under a series of salt steps, online digested by a trypsin immobilized microenzymatic reactor (IMER), trapped and desalted by two parallel C8 precolumns, separated by microreversedphase liquid chromatography (µRPLC) under a linear gradient of organic modifier concentration, and finally identified by electrospray ionization-MS/MS (ESI-MS/ MS). To evaluate the performance of such a platform, a mixture of myoglobin, cytochrome c, bovine serum albumin (BSA), and r-casein, with mass ranging from 25 ng to 2 µg, was analyzed. Compared to the methods by offline protein fractionation and shotgun based strategy, the analysis time, including sample preparation, digestion, desalting, separation, and detection, was shortened from ca. 30 to 5 h, and cytochrome c with abundance of 25 ng could be identified with improved sequence coverage. Furthermore, such an integrated platform was successfully applied into the analysis of proteins extracted from human lung cancer cells. Compared with the results obtained by the shotgun approach, the identified protein number was increased by 30%. All these results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.
Free‐flow electrophoresis in proteome sample preparation
PROTEOMICS, 2013
An aim of proteome research is to identify the entire complement of proteins expressed in defined cell types of humans, animals, plants, and microorganisms. The approach requires searching for low abundant or even rarely expressed proteins in many cell types, as well as the determination of the protein expression levels in subcellular compartments and organelles. In recent years, rather powerful MS technologies have been developed. At this stage of MS device development, it is of highest interest to purify intact cell types or isolate subcellular compartments, where the proteins of interest are originating from, which determine the final composition of a peptide mixture. Free‐flow electrophoresis proved to be useful to prepare meaningful peptide mixtures because of its improved capabilities in particle electrophoresis and the enhanced resolution in protein separation. Sample preparation by free‐flow electrophoresis mediated particle separation was preferentially performed for purifi...
Ion-interaction–capillary zone electrophoresis of cationic proteomic peptide standards
Journal of Chromatography A, 2006
We have employed a novel capillary electrophoresis (CE) approach recently developed in our laboratory, termed ion-interaction-capillary zone electrophoresis (II-CZE), to the resolution of a mixture of 27 synthetic cationic proteomic peptide standards. These peptides were comprised of three groups of nine peptides (with net charges of +1, +2 and +3 for all nine peptides within a group), the hydrophobicity of the nine peptides within a group varying only subtly between adjacent peptides. This bidimensional CE approach achieved excellent resolution of the peptides with high peak capacity by combining the powerful CZE mechanism located in the background electrolyte (BGE) with an hydrophobicity-based mechanism also located in the BGE, the latter consisting of high concentrations (up to 0.4 M) of aqueous perfluorinated acids (trifluoroacetic acid, pentafluoropropionic acid and heptafluorobutyric acid). Thus, concomitant with a CZE separation of the three differently charged groups of peptides, there is an hydrophobicallymediated separation of the peptides within these groups effected through interaction of the hydrophobic anions of the perfluorinated acids with hydrophobic amino acid side-chains in the peptides. This methodology is dramatically different from other CE methods that have used complexing agents such as micelles or cyclodextrins in MEKC. Overall, the results presented here demonstrate the value of CE as a peptide separative tool in its own right, including its use for proteomic applications, and not merely as a complementary technique to reversed-phase highperformance liquid chromatography (RP-HPLC).
Journal of Proteome Research, 2006
Multidimensional LC-MS based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed-phase liquid chromatography in the second. Recently, it has been shown that isoelectric focusing (IEF) is an interesting alternative approach to ion exchange separation of peptides in the first dimension. Here we present an improved protocol for peptide separation by continuous free-flow electrophoresis (FFE) as the first dimension in a two-dimensional peptide separation work flow. By the use of a flat pI gradient and a mannitol and urea based separation media we were able to perform high-throughput proteome analysis with improved interfacing between FFE and RPLC-MS/MS. The developed protocol was applied to a cytosolic fraction from Schneider S2 cells from Drosophila melanogaster, resulting in the identification of more than 10 000 unique peptides with high probability. To improve the accuracy of the peptide identification following FFE-IEF we incorporated the pI information as an additional parameter into a statistical model for discrimination between correct and incorrect peptide assignments to MS/MS spectra.
Increased Selectivity, Analytical Precision, and Throughput in Targeted Proteomics
Molecular & Cellular Proteomics, 2011
Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring (SRM) mode are characterized by a high degree of selectivity and low limit of detection; however the concurrent analysis of multiple analytes occurs at the expense of sensitivity due to reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented, in which SRM is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions are acquired in a data dependent event, triggered when all the primary transitions exceed a preset threshold.
Advances in proteomic workflows for systems biology
Current Opinion in Biotechnology, 2007
Mass spectrometry, specifically the analysis of complex peptide mixtures by liquid chromatography and tandem mass spectrometry (shotgun proteomics) has been at the center of proteomics research for the last decade. To overcome some of the fundamental limitations of the approach, including its limited sensitivity and high degree of redundancy, new proteomics workflows are being developed. Among these, targeting methods in which specific peptides are selectively isolated, identified and quantified are particularly promising. Here we summarize recent incremental advances in shotgun proteomics methods and outline emerging targeted workflows. The development of the target driven approaches with their ability to detect and quantify identical, non-redundant sets of proteins in multiple repeat analyses will be critically important for the application of proteomics to biomarker discovery and validation, and to systems biology research. Incremental improvements of non targeted mass spectrometry based proteomics For the last few years shotgun tandem mass spectrometry has been the most popular and widely used method in proteomics. In this method, complex protein mixtures are digested to peptides, usually using trypsin as the protease, and the resulting peptides are fractionated by one, two or three dimensional separation and analyzed by tandem mass spectrometry [1,2]. Optionally, stable isotope signatures are introduced into proteins or peptides to allow quantitatively accurate comparisons of samples [3-6]. Over the last years incremental improvements have increased the reproducibility of peptide separation, the speed and accuracy of data collection and the confidence of inferring the sequence of peptides and proteins from the fragment ion spectra. Figure 1 illustrates the general workflow and indicates significant recent technical advances that are further described in the following sections. Advances in sample preparation To improve on the resolution achievable by the classical two-dimensional (cation exchange/ reversed-phase) chromatography peptide separation, iso-electric focusing techniques in gels and in solution have been described [7-10]. Since the pI of peptides can be accurately calculated from the amino acid sequence of a peptide, the pI information obtained by such experiments has also proven beneficial for the correct assignment of fragment ion spectra to peptide sequences (see below). It can be expected that with the development of instruments supporting robust [7,10] and preferentially multiplexed peptide IEF separations [8] these methods will gain in importance in proteomics research. The development of highly reproducible capillary