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1.
A resurgence of interest in the human plasma proteome has occurred in recent years because it holds great promise of revolution in disease diagnosis and therapeutic monitoring. As one of the most powerful separation techniques, multidimensional liquid chromatography has attracted extensive attention, but most published works have focused on the fractionation of tryptic peptides. In this study, proteins from human plasma were prefractionated by online sequential strong cation exchange chromatography and reversed-phase chromatography. The resulting 30 samples were individually digested by trypsin, and analyzed by capillary reversed-phase liquid chromatography coupled with linear ion trap mass spectrometry. After meeting stringent criteria, a total of 1292 distinct proteins were successfully identified in our work, among which, some proteins known to be present in serum in <10 ng/mL were detected. Compared with other works in published literatures, this analysis offered a more full-scale list of the plasma proteome. Considering our strategy allows high throughput of protein identification in serum, the prefractionation of proteins before MS analysis is a simple and effective method to facilitate human plasma proteome research.  相似文献   

2.
Carter C  Pan S  Zouhar J  Avila EL  Girke T  Raikhel NV 《The Plant cell》2004,16(12):3285-3303
Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplast-enriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N- and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.  相似文献   

3.
Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.  相似文献   

4.
Ueberle B  Frank R  Herrmann R 《Proteomics》2002,2(6):754-764
An existing proteome map of the bacterium Mycoplasma pneumoniae comprising proteins from 224 genes was extended to 305 genes. This corresponds to about 44% of the 688 proposed genome sequence derived open reading frames (ORFs). The newly assigned gene products were enriched, separated by one-dimensional or two-dimensional (2-D) gel electrophoresis and identified by mass spectrometry. The enrichment procedures included differential centrifugation, anion and cation exchange chromatography, affinity chromatography with heparin as a ligand and isolation of biotinylated proteins by binding to immobilized streptavidin. A comparative analysis of the identified proteins from 305 genes with the as yet unverified 383 ORFs concerning isoelectric point, molecular weight and number of transmembrane segments revealed that proteins with more than three predicted transmembrane segments and an isoelectric point above 10.5 are most likely not to be separated by 2-D gel electrophoresis. The mutual benefits of genomics and proteomics were shown by the identification of a todate unannotated 128 amino acid long protein.  相似文献   

5.
We describe methods for broad characterization of the human plasma proteome. The combination of stepwise immunoglobulin G (IgG) and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of > 94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (< 30 pg/mL to approximately 30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin, and the human plasma protein loss in the affinity chromatography/strong cation exchange/reversed-phase liquid chromatography-tandem mass spectrometry methodology was investigated in detail. The results of this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies of the identification of novel protein disease markers, as well as further studies of protein-protein interactions.  相似文献   

6.
In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6M guanidine hydrochloride, 20mM Tris-HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0-1.0M) gradient, the bound proteins were eluted out at 0.1-1.0M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver.  相似文献   

7.
Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.  相似文献   

8.
Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M.S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M.S. and non-M.S. patients using ELISA.  相似文献   

9.
Simplified proteomics approach to discover protein-ligand interactions   总被引:1,自引:0,他引:1  
Identifying targets of biologically active small molecules is an essential but still challenging task in drug research and chemical genetics. Energetics-based target identification is an approach that utilizes the change in the conformational stabilities of proteins upon ligand binding in order to identify target proteins. Different from traditional affinity-based capture approaches, energetics-based methods do not require any labeling or immobilization of the test molecule. Here, we report a surprisingly simple version of energetics-based target identification, which only requires ion exchange chromatography, SDS PAGE, and minimal use of mass spectrometry. The complexity of a proteome is reduced through fractionation by ion exchange chromatography. Urea-induced unfolding of proteins in each fraction is then monitored by the significant increase in proteolytic susceptibility upon unfolding in the presence and the absence of a ligand. Proteins showing a different degree of unfolding with the ligand are identified by SDS PAGE followed by mass spectrometry. Using this approach, we identified ATP-binding proteins in the Escherichia coli proteome. In addition to known ATP-binding proteins, we also identified a number of proteins that were not previously known to interact with ATP. To validate one such finding, we cloned and purified phosphoglyceromutase, which was not previously known to bind ATP, and confirmed that ATP indeed stabilizes this protein. The combination of fractionation and pulse proteolysis offers an opportunity to investigate protein-drug or protein-metabolite interactions on a proteomic scale with minimal instrumentation and without modification of a molecule of interest.  相似文献   

10.
Peptide mass fingerprinting   总被引:10,自引:0,他引:10  
Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.  相似文献   

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