Profiling human brain proteome by multi-dimensional separations coupled with MS

In our initial attempt to analyze the human brain proteome, we applied multi-dimensional protein separation and identification techniques using a combination of sample fractionation, 1-D SDS-PAGE, and MS analysis. The complexity of human brain proteome requires multiple fractionation strategies to extend the range and total number of proteins identified. According to the method of Klose (Methods Mol. Biol. 1999, 112, 67), proteins of the temporal lobe of human brain were fractionated into (i) cytoplasmic and nucleoplasmic, (ii) membrane and other structural, and (iii) DNA-binding proteins. Each fraction was then separated by SDS-PAGE, and the resulting gel line was cut into approximately 50 bands. After trypsin digestion, the resulting peptides from each band were analyzed by RP-LC/ESI-MS/MS using an LTQ spectrometer. The SEQUEST search program, which searched against the IPI database, was used for peptide sequence identification, and peptide sequences were validated by reversed sequence database search and filtered by the Protein Hit Score. Ultimately, 1533 proteins could be detected from the human brain. We classified the identified proteins according to their distribution on cellular components. Among these proteins, 24% were membrane proteins. Our results show that the multiple separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.     

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high‐salt protein fraction for quantitative proteome analysis

Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high‐molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high‐salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2‐D DIGE, MS and Western blotting.     

Characterization of the mouse brain proteome using global proteomic analysis complemented with cysteinyl-peptide enrichment

We report a global proteomic approach for analyzing brain tissue and for the first time a comprehensive characterization of the whole mouse brain proteome. Preparation of the whole brain sample incorporated a highly efficient cysteinyl-peptide enrichment (CPE) technique to complement a global enzymatic digestion method. Both the global and the cysteinyl-enriched peptide samples were analyzed by SCX fractionation coupled with reversed phase LC-MS/MS analysis. A total of 48,328 different peptides were confidently identified (>98% confidence level), covering 7792 nonredundant proteins ( approximately 34% of the predicted mouse proteome). A total of 1564 and 1859 proteins were identified exclusively from the cysteinyl-peptide and the global peptide samples, respectively, corresponding to 25% and 31% improvements in proteome coverage compared to analysis of only the global peptide or cysteinyl-peptide samples. The identified proteins provide a broad representation of the mouse proteome with little bias evident due to protein pI, molecular weight, and/or cellular localization. Approximately 26% of the identified proteins with gene ontology (GO) annotations were membrane proteins, with 1447 proteins predicted to have transmembrane domains, and many of the membrane proteins were found to be involved in transport and cell signaling. The MS/MS spectrum count information for the identified proteins was used to provide a measure of relative protein abundances. The mouse brain peptide/protein database generated from this study represents the most comprehensive proteome coverage for the mammalian brain to date, and the basis for future quantitative brain proteomic studies using mouse models. The proteomic approach presented here may have broad applications for rapid proteomic analyses of various mouse models of human brain diseases.     

Comparison of 2-D LC and 3-D LC with post- and pre-tryptic-digestion SEC fractionation for proteome analysis of normal human liver tissue

The current "shotgun" proteomic analysis, strong cation exchange-RPLC-MS/MS system, is a widely used method for proteome research. Currently, it is not suitable for complicated protein sample analysis, like mammal tissues or cells. To increase the protein identification confidence and number, an additional separation dimension for sample fractionation is necessary to be coupled prior to current multi-dimensional protein identification technology (MudPIT). In this work, SEC was elaborately selected and applied for sample prefractionation in consideration of its non-bias against sample and variety of choice of mobile phases. The analysis of the global lysate of normal human liver tissue sample provided by the China Human Liver Proteome Project, were performed to compare the proteome coverage, sequence coverage (peptide per protein identification) and protein identification efficiency in MudPIT, 3-D LC-MS/MS identification strategy with preproteolytic and postproteolytic fractionation. It was demonstrated that 3-D LC-MS/MS utilizing protein level fractionation was the most effective method. A MASCOT search using the MS/MS results acquired by QSTAR(XL) identified 1622 proteins from 3-D LC-MS/MS identification approaches. A primary analysis on molecular weight, pI and grand average hydrophobicity value distribution of the identified proteins in different approaches was made to further evaluate the 3-D LC-MS/MS analysis strategy.     

An alternative strategy for the membrane proteome analysis of the green sulfur bacterium Chlorobium tepidum using blue native PAGE and 2-D PAGE on purified membranes

To avoid the specific problems concerning intrinsic membrane proteins in proteome analysis, an alternative strategy is described that is complementary to previous investigations using 2-D polyacrylamide gel electrophoresis (PAGE) techniques. The strategy involves (a) obtaining purified preparations of the membranes from Chlorobium tepidum by washing with 2 M NaBr, which removed membrane-associated soluble proteins and membrane-associated organelles; (b) separation of membrane protein complexes using 1-D Blue-native polyacrylamide gel electrophoresis (BN-PAGE) after solubilization with n-dodecyl-beta-d-maltoside (DDM); (c) combination of the BN with Tricine-SDS-PAGE; (d) high-throughput mass spectrometric analysis after gel band excision, in-gel digestion, and MALDI target spotting; and (e) protein identification from mixtures of tryptic peptides by peptide mass fingerprinting. Using this approach, we identified 143 different proteins, 70 of which have not been previously reported using 2-D PAGE techniques. Membrane proteins with up to 14 transmembrane helices were found, and this procedure proved to be efficient with proteins within a wide pI range (4.4-11.6). About 54% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, and protein translocation, while for the others, a function is not yet known, indicating the potential of the method for the elucidation of the membrane proteomes in general.     

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.     

Mapping the membrane proteome of Corynebacterium glutamicum

In order to avoid the specific problems with intrinsic membrane proteins in proteome analysis, a new procedure was developed which is superior to the classical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) method in terms of intrinsic membrane proteins. For analysis of the membrane proteome from Corynebacterium glutamicum, we replaced the first separation dimension, i.e., the isoelectric focusing step, by anion-exchange chromatography, followed by sodium dodecyl sulfate (SDS)-PAGE in the second separation dimension. Enrichment of the membrane intrinsic subproteome was achieved by washing with 2.5 M NaBr which removed more than 35% of the membrane-associated soluble proteins. For the extraction and solubilization of membrane proteins, the detergent amidosulfobetaine 14 (ASB-14) was most efficient in a detailed screening procedure and proved also suitable for chromatography. 356 gel bands were spotted, and out of 170 different identified proteins, 50 were membrane-integral. Membrane proteins with one up to 13 transmembrane helices were found. Careful analysis revealed that this new procedure covers proteins from a wide pI range (3.7-10.6) and a wide mass range of 10-120 kDa. About 50% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, protein translocation, and proteolysis while for the others a function is not yet known, indicating the potential of the developed method for elucidation of membrane proteomes in general.     

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.     

Identification and characterization of the Sulfolobus solfataricus P2 proteome

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC-ESI-MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.     

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).     

Analysis of the mouse liver proteome using advanced mass spectrometry

We report a large-scale analysis of mouse liver tissue comprising a novel fractionation approach and high-accuracy mass spectrometry techniques. Two fractions enriched for soluble and membrane proteins from 20 mg of frozen tissue were separated by one-dimensional electrophoresis followed by LC-MS/MS on the hybrid linear ion trap (LTQ)-Orbitrap mass spectrometer. Confident identification of 2210 proteins relied on at least two peptides. We combined this proteome with our previously reported organellar map (Foster et al. Cell 2006, 125, 187-199) to generate a very high confidence mouse liver proteome of 3244 proteins. The identified proteins represent the liver proteome with no discernible bias due to protein physicochemical properties, subcellular distribution, or biological function. Forty-seven percent of identified proteins were annotated as membrane-bound, and for 35.3%, transmembrane domains were predicted. For potential application in toxicology or clinical studies, we demonstrate that it is possible to consistently identify more than 1000 proteins in a single run.     

The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins

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.     

Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis

The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW. Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.     

Proteomic Profiling and Neurodegeneration in Alzheimer's Disease

Quantitative proteome analysis of Alzheimer's disease (AD) brains was performed using 2-D gels to identify disease specific changes in protein expression. The task of characterizing the proteome and its components is now practically achievable because of the development and integration of four important tools: protein, EST, and complete genome sequence databases, mass spectrometry, matching software for protein sequences and protein separation technology. Mass spectrometry (MS) instrumentation has undergone a tremendous change over the past decade, culminating in the development of highly sensitive, robust instruments that can reliably analyze biomolecules, particularly proteins and peptides; we identified 35 proteins from over 100 protein spots on a 2-D gel. Using this current technology, protein-expression profiling, which is actually a specialized form of mining, is an important principal application of proteomics. The information obtained has tremendous potential as a means of determining the pathogenesis, and detecting disease markers and potential targets for drug therapy in AD.     

Comprehensive royal jelly (RJ) proteomics using one- and two-dimensional proteomics platforms reveals novel RJ proteins and potential phospho/glycoproteins

Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html).     

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.     

LC‐MS/MS as an alternative for SDS‐PAGE in blue native analysis of protein complexes

Two‐dimensional blue native/SDS‐PAGE is widely applied to investigate native protein–protein interactions, particularly those within membrane multi‐protein complexes. MS has enabled the application of this approach at the proteome scale, typically by analysis of picked protein spots. Here, we investigated the potential of using LC‐MS/MS as an alternative for SDS‐PAGE in blue native (BN) analysis of protein complexes. By subjecting equal slices from BN gel lanes to label‐free semi‐quantitative LC‐MS/MS, we determined an abundance profile for each protein across the BN gel, and used these profiles to identify potentially interacting proteins by protein correlation profiling. We demonstrate the feasibility of this approach by considering the oxidative phosphorylation complexes I–V in the native human embryonic kidney 293 mitochondrial fraction, showing that the method is capable of detecting both the fully assembled complexes as well as assembly/turnover intermediates of complex I (NADH:ubiquinone oxidoreductase). Using protein correlation profiling with a profile for subunits NDUFS2, 3, 7 and 8 we identified multiple proteins possibly involved in the biogenesis of complex I, including the recently implicated chaperone C6ORF66 and a novel candidate, C3ORF60.     

Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis

Normal Chinese Liver Proteome Expression Profile is one of the major parts of Human Liver Proteome Project. Before starting the studies, it is necessary to examine the interindividual variation of normal liver proteome and evaluate the minimal size of samples for proteomic analysis. In this study, normal liver samples from ten individual volunteers were collected and the proteome profiles of these samples were analyzed using 2-D difference gel electrophoresis (DIGE) combined with MALDI-TOF/TOF MS. The individual liver tissue lysates were labeled with Cy3 and Cy5 while the pooled sample was labeled with Cy2 as an internal standard, which minimized gel-to-gel variation. After analysis by the DeCyder software, up to 2056 protein spots were detected on the master gel. The CV of standardized abundance was calculated for the protein spots that were matched across all ten gels. The CV values of these protein spots ranged from 6.4 to 108.5% and the median CV was approximately 19%, which demonstrated that the protein expression of normal liver among different individuals was relatively stable. The eight proteins with CV values over 50% were identified which would be a caveat when considering these proteins as potential disease-related markers. Moreover, the one-way ANOVA feature showed a correlation between sample size and individual variations. The results showed that when the sample size exceeded 7, the individual variations were not significant to the whole pool. Our results are an important basis for liver protein expression profiles and comparative proteomics of liver disease.