RESUMEN
Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .
Asunto(s)
Benchmarking , Proteómica , Benchmarking/métodos , Proteómica/métodos , Reproducibilidad de los Resultados , Proteínas/análisis , Espectrometría de Masas en Tándem/métodos , Proteoma/análisisRESUMEN
Great advances have been made in sensitivity and acquisition speed on the Orbitrap mass analyzer, enabling increasingly deep proteome coverage. However, these advances have been mainly limited to the MS2 level, whereas ion beam sampling for the MS1 scans remains extremely inefficient. Here we report a data-acquisition method, termed BoxCar, in which filling multiple narrow mass-to-charge segments increases the mean ion injection time more than tenfold as compared to that of a standard full scan. In 1-h analyses, the method provided MS1-level evidence for more than 90% of the proteome of a human cancer cell line that had previously been identified in 24 fractions, and it quantified more than 6,200 proteins in ten of ten replicates. In mouse brain tissue, we detected more than 10,000 proteins in only 100 min, and sensitivity extended into the low-attomolar range.
Asunto(s)
Bases de Datos de Proteínas , Péptidos/metabolismo , Proteómica/métodos , Animales , Cerebelo/metabolismo , Cromatografía Liquida , Escherichia coli , Células HeLa , Humanos , Ratones , Péptidos/química , Proteoma , Espectrometría de Masas en TándemRESUMEN
We developed EASI-tag (easily abstractable sulfoxide-based isobaric-tag), a new type of amine-derivatizing and sulfoxide-containing isobaric labeling reagents for highly accurate quantitative proteomics analysis using mass spectrometry. We observed that EASI-tag labels dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12C precursors and quantification at the MS2 level allowed accurate determination of quantitative differences between up to six multiplexed samples.
Asunto(s)
Espectrometría de Masas , Fraccionamiento Químico , Cromatografía Liquida/métodos , Biología Computacional , Células HeLa , Humanos , Iones , Proteoma/análisis , Proteómica/métodos , Coloración y EtiquetadoRESUMEN
Characterization of tumors at the molecular level has improved our knowledge of cancer causation and progression. Proteomic analysis of their signaling pathways promises to enhance our understanding of cancer aberrations at the functional level, but this requires accurate and robust tools. Here, we develop a state of the art quantitative mass spectrometric pipeline to characterize formalin-fixed paraffin-embedded tissues of patients with closely related subtypes of diffuse large B-cell lymphoma. We combined a super-SILAC approach with label-free quantification (hybrid LFQ) to address situations where the protein is absent in the super-SILAC standard but present in the patient samples. Shotgun proteomic analysis on a quadrupole Orbitrap quantified almost 9,000 tumor proteins in 20 patients. The quantitative accuracy of our approach allowed the segregation of diffuse large B-cell lymphoma patients according to their cell of origin using both their global protein expression patterns and the 55-protein signature obtained previously from patient-derived cell lines (Deeb, S. J., D'Souza, R. C., Cox, J., Schmidt-Supprian, M., and Mann, M. (2012) Mol. Cell. Proteomics 11, 77-89). Expression levels of individual segregation-driving proteins as well as categories such as extracellular matrix proteins behaved consistently with known trends between the subtypes. We used machine learning (support vector machines) to extract candidate proteins with the highest segregating power. A panel of four proteins (PALD1, MME, TNFAIP8, and TBC1D4) is predicted to classify patients with low error rates. Highly ranked proteins from the support vector analysis revealed differential expression of core signaling molecules between the subtypes, elucidating aspects of their pathobiology.
Asunto(s)
Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Aprendizaje Automático , Proteínas de Neoplasias/genética , Proteoma/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Formaldehído , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Marcaje Isotópico/métodos , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Proteínas de Neoplasias/metabolismo , Neprilisina/genética , Neprilisina/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Análisis de Componente Principal , Proteoma/metabolismo , Proteómica/métodos , Transducción de Señal , Adhesión del Tejido , Fijación del TejidoRESUMEN
Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum-the highest proteome coverage reported with a QTOF instrument so far.
Asunto(s)
Proteómica/instrumentación , Proteómica/métodos , Animales , Línea Celular , Cromatografía Liquida , Diploidia , Haploidia , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Iones , Espectrometría de Masas , Ratones , Peso Molecular , Péptidos/metabolismo , Proteoma/metabolismo , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/metabolismo , Factores de TiempoRESUMEN
Global analysis of lymphoma genome integrity and transcriptomes tremendously advanced our understanding of their biology. Technological advances in mass spectrometry-based proteomics promise to complete the picture by allowing the global quantification of proteins and their post-translational modifications. Here we use N-glyco FASP, a recently developed mass spectrometric approach using lectin-enrichment, in conjunction with a super-SILAC approach to quantify N-linked glycoproteins in lymphoma cells. From patient-derived diffuse large B-cell lymphoma cell lines, we mapped 2383 glycosites on 1321 protein groups, which were highly enriched for cell membrane proteins. This N-glyco subproteome alone allowed the segregation of the ABC from the GCB subtypes of diffuse large B-cell lymphoma, which before gene expression studies had been considered one disease entity. Encouragingly, many of the glycopeptides driving the segregation belong to proteins previously characterized as segregators in a deep proteome study of these subtypes (S. J. Deeb et al. MCP 2012 PMID 22442255). This conforms to the high correlation that we observed between the expression level of the glycosites and their corresponding proteins. Detailed examination of glycosites and glycoprotein expression levels uncovered, among other interesting findings, enrichment of transcription factor binding motifs, including known NF-kappa-B related ones. Thus, enrichment of a class of post-translationally modified peptides can classify cancer types as well as reveal cancer specific mechanistic changes.
Asunto(s)
Glicoproteínas/biosíntesis , Linfoma de Células B Grandes Difuso/genética , Proteínas de Neoplasias/biosíntesis , Proteómica , Regulación Neoplásica de la Expresión Génica , Glicosilación , Humanos , Linfoma de Células B Grandes Difuso/patologíaRESUMEN
Modern software platforms enable the analysis of shotgun proteomics data in an automated fashion resulting in high quality identification and quantification results. Additional understanding of the underlying data can be gained with the help of advanced visualization tools that allow for easy navigation through large LC-MS/MS datasets potentially consisting of terabytes of raw data. The updated MaxQuant version has a map navigation component that steers the users through mass and retention time-dependent mass spectrometric signals. It can be used to monitor a peptide feature used in label-free quantification over many LC-MS runs and visualize it with advanced 3D graphic models. An expert annotation system aids the interpretation of the MS/MS spectra used for the identification of these peptide features.
Asunto(s)
Gráficos por Computador , Proteómica/métodos , Programas Informáticos , Secuencia de Aminoácidos , Cromatografía Liquida , Espectrometría de Masas en TándemRESUMEN
Skeletal muscle has emerged as an important secretory organ that produces so-called myokines, regulating energy metabolism via autocrine, paracrine, and endocrine actions; however, the nature and extent of the muscle secretome has not been fully elucidated. Mass spectrometry (MS)-based proteomics, in principle, allows an unbiased and comprehensive analysis of cellular secretomes; however, the distinction of bona fide secreted proteins from proteins released upon lysis of a small fraction of dying cells remains challenging. Here we applied highly sensitive MS and streamlined bioinformatics to analyze the secretome of lipid-induced insulin-resistant skeletal muscle cells. Our workflow identified 1073 putative secreted proteins including 32 growth factors, 25 cytokines, and 29 metalloproteinases. In addition to previously reported proteins, we report hundreds of novel ones. Intriguingly, â¼40% of the secreted proteins were regulated under insulin-resistant conditions, including a protein family with signal peptide and EGF-like domain structure that had not yet been associated with insulin resistance. Finally, we report that secretion of IGF and IGF-binding proteins was down-regulated under insulin-resistant conditions. Our study demonstrates an efficient combined experimental and bioinformatics workflow to identify putative secreted proteins from insulin-resistant skeletal muscle cells, which could easily be adapted to other cellular models.
Asunto(s)
Citocinas/aislamiento & purificación , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/aislamiento & purificación , Péptidos y Proteínas de Señalización Intercelular/aislamiento & purificación , Metaloproteasas/aislamiento & purificación , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Línea Celular , Biología Computacional/métodos , Citocinas/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , Glucosa/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Insulina/farmacología , Resistencia a la Insulina , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metaloproteasas/genética , Metaloproteasas/metabolismo , Ratones , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Mioblastos/citología , Mioblastos/efectos de los fármacos , Ácido Palmítico/farmacología , Señales de Clasificación de Proteína/genética , Estructura Terciaria de Proteína , Albúmina Sérica Bovina/químicaRESUMEN
Identifying the building blocks of mammalian tissues is a precondition for understanding their function. In particular, global and quantitative analysis of the proteome of mammalian tissues would point to tissue-specific mechanisms and place the function of each protein in a whole-organism perspective. We performed proteomic analyses of 28 mouse tissues using high-resolution mass spectrometry and used a mix of mouse tissues labeled via stable isotope labeling with amino acids in cell culture as a "spike-in" internal standard for accurate protein quantification across these tissues. We identified a total of 7,349 proteins and quantified 6,974 of them. Bioinformatic data analysis showed that physiologically related tissues clustered together and that highly expressed proteins represented the characteristic tissue functions. Tissue specialization was reflected prominently in the proteomic profiles and is apparent already in their hundred most abundant proteins. The proportion of strictly tissue-specific proteins appeared to be small. However, even proteins with household functions, such as those in ribosomes and spliceosomes, can have dramatic expression differences among tissues. We describe a computational framework with which to correlate proteome profiles with physiological functions of the tissue. Our data will be useful to the broad scientific community as an initial atlas of protein expression of a mammalian species.
Asunto(s)
Aminoácidos/química , Mapeo Peptídico , Proteoma/química , Aminoácidos/metabolismo , Animales , Expresión Génica , Perfilación de la Expresión Génica , Marcaje Isotópico , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Proteoma/genética , Proteoma/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
MS-based proteomics generates rapidly increasing amounts of precise and quantitative information. Analysis of individual proteomic experiments has made great strides, but the crucial ability to compare and store information across different proteome measurements still presents many challenges. For example, it has been difficult to avoid contamination of databases with low quality peptide identifications, to control for the inflation in false positive identifications when combining data sets, and to integrate quantitative data. Although, for example, the contamination with low quality identifications has been addressed by joint analysis of deposited raw data in some public repositories, we reasoned that there should be a role for a database specifically designed for high resolution and quantitative data. Here we describe a novel database termed MaxQB that stores and displays collections of large proteomics projects and allows joint analysis and comparison. We demonstrate the analysis tools of MaxQB using proteome data of 11 different human cell lines and 28 mouse tissues. The database-wide false discovery rate is controlled by adjusting the project specific cutoff scores for the combined data sets. The 11 cell line proteomes together identify proteins expressed from more than half of all human genes. For each protein of interest, expression levels estimated by label-free quantification can be visualized across the cell lines. Similarly, the expression rank order and estimated amount of each protein within each proteome are plotted. We used MaxQB to calculate the signal reproducibility of the detected peptides for the same proteins across different proteomes. Spearman rank correlation between peptide intensity and detection probability of identified proteins was greater than 0.8 for 64% of the proteome, whereas a minority of proteins have negative correlation. This information can be used to pinpoint false protein identifications, independently of peptide database scores. The information contained in MaxQB, including high resolution fragment spectra, is accessible to the community via a user-friendly web interface at http://www.biochem.mpg.de/maxqb.
Asunto(s)
Bases de Datos de Proteínas , Fragmentos de Péptidos/metabolismo , Proteoma/análisis , Proteómica , Motor de Búsqueda , Animales , Células Cultivadas , Cromatografía Liquida , Humanos , Espectrometría de Masas , Ratones , Reproducibilidad de los ResultadosRESUMEN
Deep proteomic analysis of mammalian cell lines would yield an inventory of the building blocks of the most commonly used systems in biological research. Mass spectrometry-based proteomics can identify and quantify proteins in a global and unbiased manner and can highlight the cellular processes that are altered between such systems. We analyzed 11 human cell lines using an LTQ-Orbitrap family mass spectrometer with a "high field" Orbitrap mass analyzer with improved resolution and sequencing speed. We identified a total of 11,731 proteins, and on average 10,361 ± 120 proteins in each cell line. This very high proteome coverage enabled analysis of a broad range of processes and functions. Despite the distinct origins of the cell lines, our quantitative results showed surprisingly high similarity in terms of expressed proteins. Nevertheless, this global similarity of the proteomes did not imply equal expression levels of individual proteins across the 11 cell lines, as we found significant differences in expression levels for an estimated two-third of them. The variability in cellular expression levels was similar for low and high abundance proteins, and even many of the most highly expressed proteins with household roles showed significant differences between cells. Metabolic pathways, which have high redundancy, exhibited variable expression, whereas basic cellular functions such as the basal transcription machinery varied much less. We harness knowledge of these cell line proteomes for the construction of a broad coverage "super-SILAC" quantification standard. Together with the accompanying paper (Schaab, C. MCP 2012, PMID: 22301388) (17) these data can be used to obtain reference expression profiles for proteins of interest both within and across cell line proteomes.
Asunto(s)
Marcaje Isotópico , Fragmentos de Péptidos/metabolismo , Proteoma/análisis , Proteómica , Células Cultivadas , Cromatografía Liquida , Biología Computacional , Humanos , Proteoma/metabolismo , Espectrometría de Masas en TándemRESUMEN
Yeast remains an important model for systems biology and for evaluating proteomics strategies. In-depth shotgun proteomics studies have reached nearly comprehensive coverage, and rapid, targeted approaches have been developed for this organism. Recently, we demonstrated that single LC-MS/MS analysis using long columns and gradients coupled to a linear ion trap Orbitrap instrument had an unexpectedly large dynamic range of protein identification (Thakur, S. S., Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., and Mann, M. (2011) Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol. Cell Proteomics 10, 10.1074/mcp.M110.003699). Here we couple an ultra high pressure liquid chromatography system to a novel bench top Orbitrap mass spectrometer (Q Exactive) with the goal of nearly complete, rapid, and robust analysis of the yeast proteome. Single runs of filter-aided sample preparation (FASP)-prepared and LysC-digested yeast cell lysates identified an average of 3923 proteins. Combined analysis of six single runs improved these values to more than 4000 identified proteins/run, close to the total number of proteins expressed under standard conditions, with median sequence coverage of 23%. Because of the absence of fractionation steps, only minuscule amounts of sample are required. Thus the yeast model proteome can now largely be covered within a few hours of measurement time and at high sensitivity. Median coverage of proteins in Kyoto Encyclopedia of Genes and Genomes pathways with at least 10 members was 88%, and pathways not covered were not expected to be active under the conditions used. To study perturbations of the yeast proteome, we developed an external, heavy lysine-labeled SILAC yeast standard representing different proteome states. This spike-in standard was employed to measure the heat shock response of the yeast proteome. Bioinformatic analysis of the heat shock response revealed that translation-related functions were down-regulated prominently, including nucleolar processes. Conversely, stress-related pathways were up-regulated. The proteomic technology described here is straightforward, rapid, and robust, potentially enabling widespread use in the yeast and other biological research communities.
Asunto(s)
Marcaje Isotópico , Proteoma/análisis , Proteoma/metabolismo , Proteómica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Western Blotting , Cromatografía Liquida , Biología Computacional , Electroforesis en Gel Bidimensional , Espectrometría de Masas , Fragmentos de Péptidos/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.
Asunto(s)
Cromatografía Liquida , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Proteómica/instrumentación , Espectrometría de Masas en Tándem/instrumentación , Células HeLa , Humanos , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
We describe a method to accurately quantify human tumor proteomes by combining a mixture of five stable-isotope labeling by amino acids in cell culture (SILAC)-labeled cell lines with human carcinoma tissue. This generated hundreds of thousands of isotopically labeled peptides in appropriate amounts to serve as internal standards for mass spectrometry-based analysis. By decoupling the labeling from the measurement, this super-SILAC method broadens the scope of SILAC-based proteomics.
Asunto(s)
Neoplasias de la Mama/química , Marcaje Isotópico/métodos , Proteínas de Neoplasias/análisis , Proteómica/métodos , Astrocitoma/química , Isótopos de Carbono , Línea Celular Tumoral , Femenino , Glioblastoma/química , Humanos , Isótopos de NitrógenoRESUMEN
In-depth MS-based proteomics has necessitated fractionation of either proteins or peptides or both, often requiring considerable analysis time. Here we employ long liquid chromatography runs with high resolution coupled to an instrument with fast sequencing speed to investigate how much of the proteome is directly accessible to liquid chromatography-tandem MS characterization without any prefractionation steps. Triplicate single-run analyses identified 2990 yeast proteins, 68% of the total measured in a comprehensive yeast proteome. Among them, we covered the enzymes of the glycolysis and gluconeogenesis pathway targeted in a recent multiple reaction monitoring study. In a mammalian cell line, we identified 5376 proteins in a triplicate run, including representatives of 173 out of 200 KEGG metabolic and signaling pathways. Remarkably, the majority of proteins could be detected in the samples at sub-femtomole amounts and many in the low attomole range, in agreement with absolute abundance estimation done in previous works (Picotti et al. Cell, 138, 795-806, 2009). Our results imply an unexpectedly large dynamic range of the MS signal and sensitivity for liquid chromatography-tandem MS alone. With further development, single-run analysis has the potential to radically simplify many proteomic studies while maintaining a systems-wide view of the proteome.
Asunto(s)
Proteoma/metabolismo , Espectrometría de Masas en Tándem , Técnicas de Cultivo de Célula , Fraccionamiento Celular , Cromatografía Líquida de Alta Presión , Células HEK293 , Humanos , Marcaje Isotópico , Límite de Detección , Redes y Vías Metabólicas , Proteoma/aislamiento & purificación , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This "Q Exactive" instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top 10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate- a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.
Asunto(s)
Espectrometría de Masas , Fragmentos de Péptidos/análisis , Proteínas/análisis , Proteómica/métodos , Algoritmos , Secuencia de Aminoácidos , Femenino , Células HeLa , Humanos , Iones , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Proteínas/químicaRESUMEN
Along the transformation process, cells accumulate DNA aberrations, including mutations, translocations, amplifications, and deletions. Despite numerous studies, the overall effects of amplifications and deletions on the end point of gene expression--the level of proteins--is generally unknown. Here we use large-scale and high-resolution proteomics combined with gene copy number analysis to investigate in a global manner to what extent these genomic changes have a proteomic output and therefore the ability to affect cellular transformation. We accurately measure expression levels of 6,735 proteins and directly compare them to the gene copy number. We find that the average effect of these alterations on the protein expression is only a few percent. Nevertheless, by using a novel algorithm, we find the combined impact that many of these regional chromosomal aberrations have at the protein level. We show that proteins encoded by amplified oncogenes are often overexpressed, while adjacent amplified genes, which presumably do not promote growth and survival, are attenuated. Furthermore, regulation of biological processes and molecular complexes is independent of general copy number changes. By connecting the primary genome alteration to their proteomic consequences, this approach helps to interpret the data from large-scale cancer genomics efforts.
Asunto(s)
Neoplasias de la Mama/genética , Variaciones en el Número de Copia de ADN/genética , Proteómica , Línea Celular , Aberraciones Cromosómicas , Bases de Datos Genéticas , Femenino , Amplificación de Genes/genética , Dosificación de Gen/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias/genética , Humanos , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Polimorfismo de Nucleótido Simple/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteoma/genética , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Quantitative proteomics now provides abundance ratios for thousands of proteins upon perturbations. These need to be functionally interpreted and correlated to other types of quantitative genome-wide data such as the corresponding transcriptome changes. We describe a new method, 2D annotation enrichment, which compares quantitative data from any two 'omics' types in the context of categorical annotation of the proteins or genes. Suitable genome-wide categories are membership of proteins in biochemical pathways, their annotation with gene ontology terms, sub-cellular localization, presence of protein domains or membership in protein complexes. 2D annotation enrichment detects annotation terms whose members show consistent behavior in one or both of the data dimensions. This consistent behavior can be a correlation between the two data types, such as simultaneous up- or down-regulation in both data dimensions, or a lack thereof, such as regulation in one dimension but no change in the other. For the statistical formulation of the test we introduce a two-dimensional generalization of the nonparametric two-sample test. The false discovery rate is stringently controlled by correcting for multiple hypothesis testing. We also describe one-dimensional annotation enrichment, which can be applied to single omics data. The 1D and 2D annotation enrichment algorithms are freely available as part of the Perseus software.
Asunto(s)
Genes , Proteínas/química , Proteómica/estadística & datos numéricos , Algoritmos , Interpretación Estadística de Datos , Programas InformáticosRESUMEN
While the number and identity of proteins expressed in a single human cell type is currently unknown, this fundamental question can be addressed by advanced mass spectrometry (MS)-based proteomics. Online liquid chromatography coupled to high-resolution MS and MS/MS yielded 166 420 peptides with unique amino-acid sequence from HeLa cells. These peptides identified 10 255 different human proteins encoded by 9207 human genes, providing a lower limit on the proteome in this cancer cell line. Deep transcriptome sequencing revealed transcripts for nearly all detected proteins. We calculate copy numbers for the expressed proteins and show that the abundances of > 90% of them are within a factor 60 of the median protein expression level. Comparisons of the proteome and the transcriptome, and analysis of protein complex databases and GO categories, suggest that we achieved deep coverage of the functional transcriptome and the proteome of a single cell type.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Proteoma , Proteómica/métodos , Transcriptoma , Secuencia de Bases , Línea Celular Tumoral , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Espectrometría de Masas/métodos , Modelos Biológicos , Proteoma/genética , Proteoma/metabolismoRESUMEN
The orbitrap mass analyzer combines high sensitivity, high resolution, and high mass accuracy in a compact format. In proteomics applications, it is used in a hybrid configuration with a linear ion trap (LTQ-Orbitrap) where the linear trap quadrupole (LTQ) accumulates, isolates, and fragments peptide ions. Alternatively, isolated ions can be fragmented by higher energy collisional dissociation. A recently introduced stand-alone orbitrap analyzer (Exactive) also features a higher energy collisional dissociation cell but cannot isolate ions. Here we report that this instrument can efficiently characterize protein mixtures by alternating MS and "all-ion fragmentation" (AIF) MS/MS scans in a manner similar to that previously described for quadrupole time-of-flight instruments. We applied the peak recognition algorithms of the MaxQuant software at both the precursor and product ion levels. Assignment of fragment ions to co-eluting precursor ions was facilitated by high resolution (100,000 at m/z 200) and high mass accuracy. For efficient fragmentation of different mass precursors, we implemented a stepped collision energy procedure with cumulative MS readout. AIF on the Exactive identified 45 of 48 proteins in an equimolar protein standard mixture and all of them when using a small database. The technique also identified proteins with more than 100-fold abundance differences in a high dynamic range standard. When applied to protein identification in gel slices, AIF unambiguously characterized an immunoprecipitated protein that was barely visible by Coomassie staining and quantified it relative to contaminating proteins. AIF on a benchtop orbitrap instrument is therefore an attractive technology for a wide range of proteomics analyses.