RESUMO
Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP's. Mammalian MBP's, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the post-translational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial. The C1 component lacks any phosphorylated sites, a finding in agreement with the analysis of other MBP species. It also had a single methylation at R105 as did the components C2 and C3. The C2 component contains ten phosphorylated sites (S7, S18, S33, S64, S73, T96, S113, S141, S164, and S168), and modified arginine to citrulline residues at R24, and R165. Component C3 contains eight phosphorylated sites (S7, S33, S64, T96, S113, S141, S164, and S168), and citrulline residues at Arginine 41, R24 and R165. Partial deamidation of glutamine residues Q71, Q101 and Q146 were present in addition to asparagine N90 that was found in all three charge components. The glutamine at residue 3 is partially deamidated in isomers C1 and C2, whereas glutamine 74 and asparagine 83 were found not to be deamidated. Comparison of the PTM's of MBP's isolated from several vertebrate species reveals marked differences in their phosphate content. Chicken MBP does not share any phosphorylated sites with dogfish MBP; However, it does contain phosphorylated serine and threonine residues in common with mammalian MBP.
Assuntos
Proteína Básica da Mielina/metabolismo , Processamento de Proteína Pós-Traducional , Amidas/metabolismo , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Galinhas , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Iminas/metabolismo , Metilação , Dados de Sequência Molecular , Peso Molecular , Proteína Básica da Mielina/química , Proteína Básica da Mielina/isolamento & purificação , Fosforilação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cromatografia Líquida/métodos , Desulfovibrio vulgaris/metabolismo , Espectrometria de Massas/métodos , Proteoma/química , Proteoma/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Mapeamento Cromossômico/métodos , Desulfovibrio vulgaris/genética , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
We describe an improved artificial neural network (ANN)-based method for predicting peptide retention times in reversed-phase liquid chromatography. In addition to the peptide amino acid composition, this study investigated several other peptide descriptors to improve the predictive capability, such as peptide length, sequence, hydrophobicity and hydrophobic moment, and nearest-neighbor amino acid, as well as peptide predicted structural configurations (i.e., helix, sheet, coil). An ANN architecture that consisted of 1052 input nodes, 24 hidden nodes, and 1 output node was used to fully consider the amino acid residue sequence in each peptide. The network was trained using approximately 345,000 nonredundant peptides identified from a total of 12,059 LC-MS/MS analyses of more than 20 different organisms, and the predictive capability of the model was tested using 1303 confidently identified peptides that were not included in the training set. The model demonstrated an average elution time precision of approximately 1.5% and was able to distinguish among isomeric peptides based upon the inclusion of peptide sequence information. The prediction power represents a significant improvement over our earlier report (Petritis, K.; Kangas, L. J.; Ferguson, P. L.; Anderson, G. A.; Pasa-Tolic, L.; Lipton, M. S.; Auberry, K. J.; Strittmatter, E. F.; Shen, Y.; Zhao, R.; Smith, R. D. Anal. Chem. 2003, 75, 1039-1048) and other previously reported models.
Assuntos
Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Peptídeos/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isomerismo , Masculino , Camundongos , Redes Neurais de Computação , Sensibilidade e Especificidade , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de TempoRESUMO
Direct LC-MS/MS was used to examine the proteins extracted from exponential or stationary phase Desulfovibrio vulgaris cells that had been grown on a minimal medium containing either lactate or formate as the primary carbon source. Across all four growth conditions, 976 gene products were identified with high confidence, which is equal to approximately 28% of all predicted proteins in the D. vulgaris genome. Bioinformatic analysis showed that the proteins identified were distributed among almost all functional classes, with the energy metabolism category containing the greatest number of identified proteins. At least 154 ORFs originally annotated as hypothetical proteins were found to encode the expressed proteins, which provided verification for the authenticity of these hypothetical proteins. Proteomic analysis showed that proteins potentially involved in ATP biosynthesis using the proton gradient across membrane, such as ATPase, alcohol dehydrogenases, heterodisulfide reductases, and [NiFe] hydrogenase (HynAB-1) of the hydrogen cycling were highly expressed in all four growth conditions, suggesting they may be the primary pathways for ATP synthesis in D. vulgaris. Most of the enzymes involved in substrate-level phosphorylation were also detected in all tested conditions. However, no enzyme involved in CO cycling or formate cycling was detected, suggesting that they are not the primary ATP-biosynthesis pathways under the tested conditions. This study provides the first proteomic overview of the cellular metabolism of D. vulgaris. The complete list of proteins identified in this study and their abundances (peptide hits) is provided in Supplementary Table 1.
Assuntos
Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Desulfovibrio vulgaris/metabolismo , Espectrometria de Massas/métodos , Proteômica/métodos , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Proteínas de Bactérias/genética , Desulfovibrio vulgaris/genética , Desulfovibrio vulgaris/crescimento & desenvolvimento , Eletroforese em Gel Bidimensional , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Formiatos/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidrogenase/genética , Hidrogenase/metabolismo , Ácido Láctico/metabolismo , Modelos Biológicos , Fases de Leitura Aberta/genética , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Sulfatos/metabolismoRESUMO
A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes.
Assuntos
Fibroblastos/efeitos da radiação , Fosfoproteínas/metabolismo , Fosfoproteínas/efeitos da radiação , Proteômica/métodos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Motivos de Aminoácidos , Sequência de Aminoácidos , Células Cultivadas , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/efeitos da radiação , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Proteína Oncogênica v-akt/metabolismo , Proteína Oncogênica v-akt/efeitos da radiação , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosforilação , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Proteome comparison of cell lines derived from cancer and normal breast epithelium provide opportunities to identify differentially expressed proteins and pathways associated with specific phenotypes. We employed 16O/18O peptide labeling, FT-ICR MS, and an accurate mass and time (AMT) tag strategy to simultaneously compare the relative abundance of hundreds of proteins in non-cancer and cancer cell lines derived from breast tissue. A cell line reference panel allowed relative protein abundance comparisons among multiple cell lines and across multiple experiments. A peptide database generated from multidimensional LC separations and MS/MS analysis was used for subsequent AMT tag-based peptide identifications. This peptide database represented a total of 2299 proteins, including 514 that were quantified in five cell lines using the AMT tag and 16O/18O strategies. Eighty-six proteins showed at least a threefold protein abundance change between cancer and non-cancer cell lines. Hierarchical clustering of protein abundance ratios revealed that several groups of proteins were differentially expressed between the cancer cell lines.
Assuntos
Neoplasias da Mama/química , Proteoma , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Espectrometria de Massas , Isótopos de Oxigênio , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Normal and cancer cell line proteomes were profiled using high throughput mass spectrometry techniques. Application of protein-level and peptide-level sample fractionation combined with LC-MS/MS analysis enabled identification of 2235 unmodified proteins representing a broad range of functional and compartmental classes. An iterative multistep search strategy was used to identify post-translational modifications, revealing several proteins that are preferentially modified in cancer cells. Information regarding both unmodified and modified protein forms was combined with publicly available gene expression and protein-protein interaction data. The resulting integrated dataset revealed several functionally related proteins that are differentially regulated between normal and cancer cell lines.
Assuntos
Neoplasias da Mama/patologia , Genômica/métodos , Proteômica/métodos , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cromatografia Líquida , Análise por Conglomerados , Bases de Dados de Proteínas , Regulação Neoplásica da Expressão Gênica , Humanos , Espectrometria de Massas , Metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Peptídeos/química , Análise Serial de Proteínas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas/químicaRESUMO
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 on-line electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.8-microm porous C18 particle-packed 50-microm-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 approximately 0.3 or approximately 0.6 s for FTICR), peptide mass measurement accuracies of better than +/-15 ppm were obtained with the high-speed 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 different putative peptides detected from a Shewanellaoneidensis tryptic digest. Initial results for both RPLC-ESI-TOF and RPLC-ESI-FTICR MS were similar, with approximately 2000 different peptides from approximately 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.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Proteoma/análise , Proteoma/química , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteoma/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de Tempo , Tripsina/metabolismoRESUMO
Ion mobility spectrometry (IMS) has been explored for decades, and its versatility in separation and identification of gas-phase ions is well established. Recently, field asymmetric waveform IMS (FAIMS) has been gaining acceptance in similar applications. Coupled to mass spectrometry (MS), both IMS and FAIMS have shown the potential for broad utility in proteomics and other biological analyses. A major attraction of these separations is extremely high speed, exceeding that of condensed-phase alternatives by orders of magnitude. However, modest separation peak capacities have limited the utility of FAIMS and IMS for analyses of complex mixtures. We report 2-D gas-phase separations that join FAIMS to IMS, in conjunction with high-resolution and accuracy time-of-flight (TOF) MS. Implementation of FAIMS/IMS and IMS/MS interfaces using electrodynamic ion funnels greatly improves sensitivity. Evaluation of FAIMS/IMS/TOF performance for a protein mixture tryptic digest reveals high orthogonality between FAIMS and IMS dimensions and, hence, the benefit of FAIMS filtering prior to IMS/MS. The effective peak capacities in analyses of tryptic peptides are approximately 500 for FAIMS/IMS separations and approximately 10(6) for 3-D FAIMS/IMS/MS, providing a potential platform for ultrahigh-throughput analyses of complex mixtures.
Assuntos
Espectrometria de Massas em Tandem/métodos , GasesRESUMO
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.
Assuntos
Plasma/química , Proteoma , Animais , Cromatografia de Afinidade , Cromatografia Líquida/métodos , Eletroforese em Gel Bidimensional , Humanos , Imunoglobulina G/isolamento & purificação , Espectrometria de Massas/métodos , Mapeamento de Peptídeos , Peptídeos/isolamento & purificação , Proteômica/métodos , Albumina Sérica/isolamento & purificaçãoRESUMO
We describe methods for mass spectrometric identification of heme-containing peptides from c-type cytochromes that contain the CXXCH (X=any amino acid) sequence motif. The heme fragment ion yielded the most abundant MS/MS peak for standard heme-containing peptides with one amino acid difference for both 2+ and 3+ peptide charge states; both sequence and charge affect the extent of heme loss. Application to Shewanella oneidenis demonstrated the utility of this approach for identifying c-type heme-containing peptides from complex proteome samples.
Assuntos
Proteínas de Bactérias/análise , Grupo dos Citocromos c/análise , Shewanella/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Heme , Espectrometria de Massas , Fragmentos de Peptídeos/isolamento & purificaçãoRESUMO
The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation to develop a fundamental understanding of the bystander response. Chinese hamster ovary cells were chosen because they have been widely used for radiation studies and are reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and Fourier transform-ion cyclotron resonance (FT-ICR)-mass spectrometry (MS) analyses. Since the hamster genome has not been sequenced, MS data was searched against the mouse and human protein databases. Nearly 150 proteins identified by tandem mass spectrometry were confirmed by FT-ICR. When both types of MS data were evaluated, using a new confidence scoring tool based on discriminant analyses, about 500 proteins were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface and, hence were likely shed. However, estimates of quantitative changes, based on two independent MS approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using MS in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.
Assuntos
Células CHO/metabolismo , Bases de Dados de Proteínas , Espectrometria de Massas/estatística & dados numéricos , Proteínas/metabolismo , Animais , Intervalos de Confiança , Cricetinae , Cricetulus , Humanos , Espectrometria de Massas/métodos , CamundongosRESUMO
Large-scale protein identifications from highly complex protein mixtures have recently been achieved using multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) and subsequent database searching with algorithms such as SEQUEST. Here, we describe a probability-based evaluation of false positive rates associated with peptide identifications from three different human proteome samples. Peptides from human plasma, human mammary epithelial cell (HMEC) lysate, and human hepatocyte (Huh)-7.5 cell lysate were separated by strong cation exchange (SCX) chromatography coupled offline with reversed-phase capillary LC-MS/MS analyses. The MS/MS spectra were first analyzed by SEQUEST, searching independently against both normal and sequence-reversed human protein databases, and the false positive rates of peptide identifications for the three proteome samples were then analyzed and compared. The observed false positive rates of peptide identifications for human plasma were significantly higher than those for the human cell lines when identical filtering criteria were used, suggesting that the false positive rates are significantly dependent on sample characteristics, particularly the number of proteins found within the detectable dynamic range. Two new sets of filtering criteria are proposed for human plasma and human cell lines, respectively, to provide an overall confidence of >95% for peptide identifications. The new criteria were compared, using a normalized elution time (NET) criterion (Petritis et al. Anal. Chem. 2003, 75, 1039-1048), with previously published criteria (Washburn et al. Nat. Biotechnol. 2001, 19, 242-247). The results demonstrate that the present criteria provide significantly higher levels of confidence for peptide identifications from mammalian proteomes without greatly decreasing the number of identifications.
Assuntos
Espectrometria de Massas/métodos , Peptídeos/análise , Probabilidade , Proteínas/análise , Proteômica/métodos , Proteínas Sanguíneas/análise , Células Epiteliais/química , Hepatócitos/química , Humanos , Proteoma/análiseRESUMO
We describe the application of a peptide retention time reversed phase liquid chromatography (RPLC) prediction model previously reported (Petritis et al. Anal. Chem. 2003, 75, 1039) for improved peptide identification. The model uses peptide sequence information to generate a theoretical (predicted) elution time that can be compared with the observed elution time. Using data from a set of known proteins, the retention time parameter was incorporated into a discriminant function for use with tandem mass spectrometry (MS/MS) data analyzed with the peptide/protein identification program SEQUEST. For singly charged ions, the number of confident identifications increased by 12% when the elution time metric is included compared to when mass spectral data is the sole source of information in the context of a Drosophila melanogaster database. A 3-4% improvement was obtained for doubly and triply charged ions for the same biological system. Application to the larger Rattus norvegicus (rat) and human proteome databases resulted in an 8-9% overall increase in the number of confident identifications, when both the discriminant function and elution time are used. The effect of adding "runner-up" hits (peptide matches that are not the highest scoring for a spectra) from SEQUEST is also explored, and we find that the number of confident identifications is further increased by 1% when these hits are also considered. Finally, application of the discriminant functions derived in this work with approximately 2.2 million spectra from over three hundred LC-MS/MS analyses of peptides from human plasma protein resulted in a 16% increase in confident peptide identifications (9022 vs 7779) using elution time information. Further improvements from the use of elution time information can be expected as both the experimental control of elution time reproducibility and the predictive capability are improved.
Assuntos
Cromatografia Líquida , Espectrometria de Massas , Peptídeos/análise , Proteoma/química , Software , Animais , Proteínas de Drosophila/química , Peptídeos/química , Peptídeos/isolamento & purificação , RatosRESUMO
A new quantitative cysteinyl-peptide enrichment technology (QCET) was developed to achieve higher efficiency, greater dynamic range, and higher throughput in quantitative proteomics that use stable-isotope labeling techniques combined with high-resolution liquid chromatography (LC)-mass spectrometry (MS). This approach involves (18)O labeling of tryptic peptides, high-efficiency enrichment of cysteine-containing peptides, and confident protein identification and quantification using the accurate mass and time tag strategy. Proteome profiling of naïve and in vitro-differentiated human mammary epithelial cells using QCET resulted in the identification and quantification of 603 proteins in a single LC-Fourier transform ion cyclotron resonance MS analysis. Advantages of this technology include the following: (1) a simple, highly efficient method for enriching cysteinyl-peptides; (2) a high-throughput strategy suitable for extensive proteome analysis; and (3) improved labeling efficiency for better quantitative measurements. This technology enhances both the functional analysis of biological systems and the detection of potential clinical biomarkers.
Assuntos
Cisteína/química , Peptídeos/análise , Peptídeos/química , Proteoma/análise , Proteômica/métodos , Células Cultivadas , Humanos , Proteoma/química , Proteômica/instrumentaçãoRESUMO
Recent multidimensional liquid chromatography MS/MS studies have contributed to the identification of large numbers of expressed proteins for numerous species. The present study couples size exclusion chromatography of intact proteins with the separation of tryptically digested peptides using a combination of strong cation exchange and high resolution, reversed phase capillary chromatography to identify proteins extracted from human mammary epithelial cells (HMECs). In addition to conventional conservative criteria for protein identifications, the confidence levels were additionally increased through the use of peptide normalized elution times (NET) for the liquid chromatographic separation step. The combined approach resulted in a total of 5838 unique peptides identified covering 1574 different proteins with an estimated 4% gene coverage of the human genome, as annotated by the National Center for Biotechnology Information (NCBI). This database provides a baseline for comparison against variations in other genetically and environmentally perturbed systems. Proteins identified were categorized based upon intracellular location and biological process with the identification of numerous receptors, regulatory proteins, and extracellular proteins, demonstrating the usefulness of this application in the global analysis of human cells for future comparative studies.
Assuntos
Células Epiteliais/química , Glândulas Mamárias Humanas/citologia , Proteínas/análise , Proteômica/métodos , Linhagem Celular , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de PeptídeosRESUMO
We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dual-channel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (<20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ion-energy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10(3), and >10(5) for peptide abundances in the overall separation.
Assuntos
Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Peptídeos/análise , Proteômica/métodos , Sequência de Aminoácidos , Automação , Cromatografia Líquida de Alta Pressão , Ciclotrons , Deinococcus/química , Deinococcus/citologia , Eletroforese Capilar , Análise de Fourier , Dados de Sequência Molecular , Peptídeos/química , Proteômica/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
When combined with capillary LC separations, electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) has demonstrated capabilities for advanced characterization of proteomes based upon analyses of proteolytic digests. Incorporation of external (to the ICR cell) multipole devices with FTICR for ion selection and ion accumulation has enhanced the dynamic range, sensitivity, and duty cycle of measurements. However, the highly variable ion production rate from an LC separation can result in "overfilling" of the external trap during the elution of major peaks and result in m/z discrimination and fragmentation of peptide ions. Excessive space charge trapped in the ICR cell also causes significant shifts in the detected ion cyclotron frequencies, reducing the achievable mass measurement accuracy (MMA) and making protein identification less effective. To eliminate m/z discrimination in the external ion trap, further increase duty cycle, and improve MMA, we have developed the capability for data-dependent adjustment of ion accumulation times in the course of an LC separation, referred to as automated gain control (AGC). This development has been implemented in combination with low kinetic energy gated ion trapping and internal calibration using a dual-channel electrodynamic ion funnel. The overall system was initially evaluated in the analysis of a tryptic digest of bovine serum albumin. In conjunction with internal calibration, the capillary LC-ESI-AGC-FTICR instrumentation provided a approximately 10-fold increase in the number of identified tryptic peptides compared to that obtained using a fixed ion accumulation time and external calibration methods.
RESUMO
We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are captured as an accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e., using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; approximately 3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, approximately 900 ORFs detected using LC-TOFMS, with approximately 500 ORFs covered by at least two AMT tags. These results indicate that AMT database searches with modest mass and elution time criteria can provide proteomic information for approximately one thousand proteins in a single run of <3 h. The advantage of this method over using MS/MS based techniques is the large number of identifications that occur in a single experiment as well as the basis for improved quantitation. For MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually. These results demonstrate the utility of the AMT tag approach using capillary LC-TOF MS instruments, and also show that AMT tags developed using other instrumentation can be effectively utilized.
Assuntos
Cromatografia Líquida/métodos , Deinococcus/química , Espectrometria de Massas/métodos , Peptídeos/análise , Peptídeos/química , Proteoma/análise , Proteoma/química , Sequência de Aminoácidos , Deinococcus/genética , Eletroforese em Gel Bidimensional , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Peptídeos/genética , Proteoma/genética , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Characterization of the surface exposed membrane subproteome of human mammary epithelial cells (strain 184 A1L5) implemented lysine specific in situ labeling of the proteins using sulfosuccinimidyl-6-(biotinamido)hexanoate, followed by enrichment of the biotinylated, tryptically digested peptides, and then liquid chromatography-tandem mass spectrometry analysis of the labeled peptides. Probing the membrane subproteome in this manner yielded unambiguous identification of proteins situated on the cell surface. The method reported can be adapted to include stable isotope labeling of proteins for quantitation of changes occurring on the cell surface in response to specific perturbations.