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 Fas-induced apoptosis analyzed by high throughput proteome analysis

The fate of cytosolic proteins was studied during Fas-induced cell death of Jurkat T-lymphocytes by proteome analysis. Among 1000 spots resolved in two-dimensional gels, comparison of control versus apoptotic cells revealed that the signal intensity of 19 spots decreased or even disappeared, whereas 38 novel spots emerged. These proteins were further analyzed with respect to de novo protein synthesis, phosphorylation status, and intracellular localization by metabolic labeling and analysis of subcellular protein fractions in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that e.g. hsp27, hsp70B, calmodulin, and H-ras synthesis was induced upon Fas signaling. 34 proteins were affected by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-1alpha became detectable in the nucleus. In addition, degradation of 12 proteins was observed; among them myosin heavy chain was identified as a novel caspase target. Fas-induced proteome alterations were compared with those of other cell death inducers, indicating specific physiological characteristics of different cell death mechanisms, consequent to as well as independent of caspase activation. Characteristic proteome alterations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo.     

Annotated proteome of a human T-cell lymphoma.

As the reliable identification of proteins by tandem mass spectrometry becomes increasingly common, the full characterization of large data sets of proteins remains a difficult challenge. Our goal was to survey the proteome of a human T-cell lymphoma-derived cell line in a single set of experiments and present an automated method for the annotation of lists of proteins. A downstream application of these data includes the identification of novel pathogenetic and candidate diagnostic markers of T-cell lymphoma. Total protein isolated from cytoplasmic, membrane, and nuclear fractions of the SUDHL-1 T-cell lymphoma cell line was resolved by SDS-PAGE, and the entire gel lanes digested and analyzed by tandem mass spectrometry. Acquired data files were searched against the UniProt protein database using the SEQUEST algorithm. Search results for each subcellular fraction were analyzed using INTERACT and ProteinProphet. All protein identifications with an error rate of less than 10% were directly exported into excel and analyzed using GOMiner (NIH/NCI). The Gene ontology molecular function and cell location data were summarized for the identified proteins and results exported as user-interactive directed acyclic graphs. A total of 1105 unique proteins were identified and fully annotated, including numerous proteins that had not been previously characterized in lymphoma, in functional categories such as cell adhesion, migration, signaling, and stress response. This study demonstrates the utility of currently available bioinformatics tools for the robust identification and annotation of large numbers of proteins in a batchwise fashion.     

种子蛋白质组的研究进展

蛋白质组学是通过对全套蛋白质动态的研究,来阐明生物体、组织、细胞和亚细胞全部蛋白质的表达模式及功能模式。大量可用的核苷酸序列信息和灵敏高速的质谱鉴定技术,使得蛋白质组学方法为分析模式植物和农作物的复杂功能开辟了新的途径。目前,种子蛋白质组研究主要集中在两个方面：一方面是鉴定尽可能多的蛋白,以创建种子特定生命时期的蛋白质组参照图谱;另一方面主要集中在差异蛋白质组,通过比较分析不同蛋白质组,以探明关键功能蛋白。该文综述了近年来种子蛋白质组的研究进展,内容包括种子发育过程中蛋白质组的变化,与种子休眠/萌发相关的蛋白质组、翻译后修饰蛋白质组、细胞与亚细胞差异蛋白质组以及环境因子对种子蛋白质组的影响;并对种子蛋白质组研究的热点问题进行了展望。     

种子蛋白质组的研究进展

蛋白质组学是通过对全套蛋白质动态的研究, 来阐明生物体、组织、细胞和亚细胞全部蛋白质的表达模式及功能模式。大量可用的核苷酸序列信息和灵敏高速的质谱鉴定技术, 使得蛋白质组学方法为分析模式植物和农作物的复杂功能开辟了新的途径。目前, 种子蛋白质组研究主要集中在两个方面: 一方面是鉴定尽可能多的蛋白, 以创建种子特定生命时期的蛋白质组参照图谱; 另一方面主要集中在差异蛋白质组, 通过比较分析不同蛋白质组, 以探明关键功能蛋白。该文综述了近年来种子蛋白质组的研究进展, 内容包括种子发育过程中蛋白质组的变化, 与种子休眠/萌发相关的蛋白质组、翻译后修饰蛋白质组、细胞与亚细胞差异蛋白质组以及环境因子对种子蛋白质组的影响; 并对种子蛋白质组研究的热点问题进行了展望。     

Proteomic characterization of plasma membrane-proximal T cell activation responses

Early downstream responses of T lymphocytes following T cell antigen receptor (TCR) activation are mediated by protein complexes that assemble in domains of the plasma membrane. Using stable isotope labeling with amino acids in cell culture and mass spectrometry, we quantitatively related the proteome of αCD3 immunoisolated native TCR signaling plasma membrane domains to that of control plasma membrane fragments not engaged in TCR signaling. Proteins were sorted according to their relative enrichment in isolated TCR signaling plasma membrane domains, identifying a complex protein network that is anchored in the vicinity of activated TCR. These networks harbor widespread mediators of plasma membrane-proximal T cell activities, including propagation, balancing, and attenuation of TCR signaling, immune synapse formation, as well as cytoskeletal arrangements relative to TCR activation clusters. These results highlight the unique potential of systematic characterizations of plasma membrane-proximal T cell activation proteome in the context of its native lipid bilayer platform.     

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.     

Nicotine‐induced protein expression profiling reveals mutually altered proteins across four human cell lines

Mass spectrometry‐based proteomic strategies can profile the expression level of proteins in response to external stimuli. Nicotine affects diverse cellular pathways, however, the nicotine‐induced alterations on the global proteome across human cell lines have not been fully elucidated. We measured perturbations in protein levels resulting from nicotine treatment in four cell lines—HEK, HeLa, PaSC, and SH‐SY5Y—in a single experiment using tandem mass tags (TMT10‐plex) and high‐resolution mass spectrometry. We quantified 8590 proteins across all cell lines. Of these, nicotine increased the abundance of 31 proteins 1.5‐fold or greater in all cell lines. Likewise, considering proteins with altered levels in at least three of the four cell lines, 64 were up‐regulated, while one was down‐regulated. Gene ontology analysis revealed that ～40% of these proteins were membrane bound, and functioned in transmembrane signaling and receptor activity. We highlighted proteins, including APP, APLP2, LAPTM4B, and NCOA4, which were dysregulated by nicotine in all cell lines investigated and may have implications in downstream signaling pathways, particularly autophagy. Using the outlined methodology, studies in additional (including primary) cell lines will provide further evidence that alterations in the levels of these proteins are indeed a general response to nicotine and thereby merit further investigation.     

In-depth analysis of the adipocyte proteome by mass spectrometry and bioinformatics

Adipocytes are central players in energy metabolism and the obesity epidemic, yet their protein composition remains largely unexplored. We investigated the adipocyte proteome by combining high accuracy, high sensitivity protein identification technology with subcellular fractionation of nuclei, mitochondria, membrane, and cytosol of 3T3-L1 adipocytes. We identified 3,287 proteins while essentially eliminating false positives, making this one of the largest high confidence proteomes reported to date. Comprehensive bioinformatics analysis revealed that the adipocyte proteome, despite its specialized role, is very complex. Comparison with microarray data showed that the mRNA abundance of detected versus non-detected proteins differed by less than 2-fold and that proteomics covered as large a proportion of the insulin signaling pathway. We used the Endeavour gene prioritization algorithm to associate a number of factors with vesicle transport in response to insulin stimulation, a key function of adipocytes. Our data and analysis can serve as a model for cellular proteomics. The adipocyte proteome is available as supplemental material and from the Max-Planck Unified Proteome database.     

Integrative analysis of the mitochondrial proteome in yeast

In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.     

Zooming in: fractionation strategies in proteomics

The recent development of mass spectrometry, i.e., high sensitivity, automation of protein identification and some post-translational modifications (PTMs) significantly increased the number of large-scale proteomics projects. However, there are still considerable limitations as none of the currently available proteomics techniques allows the analysis of an entire proteome in a single step procedure. On the other hand, there are several successful studies analyzing well defined groups of proteins, e.g., proteins of purified organelles, membrane microdomains or isolated proteins with certain PTMs. Coupling of advanced separation methodologies (different prefractionation strategies, such as subcellular fractionation, affinity purification, fractionation of proteins and peptides according to their physicochemical properties) to highly sensitive mass spectrometers provides powerful means to detect and analyze dynamic changes of low abundant regulatory proteins in eukaryotic cells on the subcellular level. This review summarizes and discusses recent strategies in proteomics approaches where different fractionation strategies were successfully applied.     

A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue

Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.     

A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers

The non-mineral component of bone matrix consists of 90% collagenous, 10% non-collagenous proteins. These proteins regulate mineralization, growth, cell signaling and differentiation, and provide bone with its tensile strength. Expression of bone matrix proteins have historically been studied individually or in small numbers owing to limitations in analytical technologies. Current mass-spectrometric and separations technologies allow a global view of protein expression patterns in complex samples. To our knowledge, no proteome profile of bone matrix has yet been reported. Therefore, we have used mass spectrometry as a tool to generate a profile of proteins present in the extracellular matrix of adult rat bone. Overall, 108 and 25 proteins were identified with high confidence in the metaphysis and diaphysis, respectively, using a bottom up proteomic technique. Twenty-one of these proteins were present in both the metaphysis and diaphysis including the bone specific proteins, osteocalcin, type I collagen, osteopontin, osteoregulin, and bone sialoprotein. Interestingly, type II collagen, a protein thought to be exclusively expressed in cartilage, was identified in both the metaphysis and diaphysis. This observation was validated by Western blot. Additionally, the presence of aggrecan, another protein expressed in cartilage was identified in the bone matrix extracts by Western blot. The proteome profile generated using this technology represents an initial survey of the acid soluble proteins of bone matrix which provides a reference for the analysis of deviations from the normal composition due to perturbations or disease states.     

Initial proteome analysis of model microorganism Haemophilus influenzae strain Rd KW20

The proteome of Haemophilus influenzae strain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS). This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome. In order to gain insight into the central metabolism of H. influenzae, cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS. Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification. Approximately 25% of all predicted proteins (open reading frames) of H. influenzae strain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra. Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies. The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies. At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins. Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria. The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism.     

Combining results from lectin affinity chromatography and glycocapture approaches substantially improves the coverage of the glycoproteome

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometry-based analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycoproteins. The plasma membrane localization of a subset of proteins identified was validated, and the dynamics of their expression in HeLa cells was evaluated during the cell cycle. Results obtained from the cell cycle studies demonstrate that plasma membrane protein expression can change up to 4-fold as cells transit the cell cycle and demonstrate the need to consider such changes when carrying out quantitative proteomics comparison of cell lines.