Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system

Background  

Mass spectrometry has become a powerful tool for the analysis of large numbers of proteins in complex samples, enabling much of proteomics. Due to various analytical challenges, so far no proteome has been sequenced completely. O'Shea, Weissman and co-workers have recently determined the copy number of yeast proteins, making this proteome an excellent model system to study factors affecting coverage.     

Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine

Quantitative comparison of protein expression levels in 2D gels is complicated by the variables associated with protein separation and mass spectrometric responses. Metabolic labeling allows cells from different experiments to be mixed prior to analysis. This approach has been reported for prokaryotic cells. Here, we demonstrate that metabolic labeling can also be successfully applied to the eukaryote Saccharormyces cerevisiae. Yeast leucine auxotrophs grown on synthetic complete media containing natural abundance Leu or D10-Leu were mixed prior to 2D gel separation and MALDI analysis of the digested proteins. D10-Leu labeling provided an effective internal calibrant for peptide MS analysis, and the number of Leu residues yielded an additional parameter for peptide identification at low mass resolution (1000). Metabolic incorporation of D10-Leu into yeast proteins was found to be quantitative since the intensities of the peptide peaks corresponded to those expected on the basis of the percent label in the media. Thus, D10-Leu labeling should provide reliable data for comparing proteomes both quantitatively and qualitatively from wild-type and nonessential-gene-null-mutant strains of S. cerevisiae. Given the central role played by yeast in our understanding of eukaryotic gene and protein expression, it is anticipated that the quantitative expressional proteomic method outlined here will have widespread applications.     

Toward a definition of the complete proteome of plant peroxisomes: Where experimental proteomics must be complemented by bioinformatics

In the past few years, proteome analysis of Arabidopsis peroxisomes has been established by the complementary efforts of four research groups and has emerged as the major unbiased approach to identify new peroxisomal proteins on a large scale. Collectively, more than 100 new candidate proteins from plant peroxisomes have been identified, including long-awaited low-abundance proteins. More than 50 proteins have been validated as peroxisome targeted, nearly doubling the number of established plant peroxisomal proteins. Sequence homologies of the new proteins predict unexpected enzyme activities, novel metabolic pathways and unknown non-metabolic peroxisome functions. Despite this remarkable success, proteome analyses of plant peroxisomes remain highly material intensive and require major preparative efforts. Characterization of the membrane proteome or post-translational protein modifications poses major technical challenges. New strategies, including quantitative mass spectrometry methods, need to be applied to allow further identifications of plant peroxisomal proteins, such as of stress-inducible proteins. In the long process of defining the complete proteome of plant peroxisomes, the prediction of peroxisome-targeted proteins from plant genome sequences emerges as an essential complementary approach to identify additional peroxisomal proteins that are, for instance, specific to peroxisome variants from minor tissues and organs or to abiotically stressed model and crop plants.     

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.     

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.     

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.     

2D-DIGE proteome analysis on the platelet proteins of patients with major depression

Introduction

Platelet activation is related to the psychopathology of major depression. We attempted to search and identify protein biomarkers from the platelets of patients with major depression. High resolution two-dimensional Differential Gel Electrophoresis (2D-DIGE), the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), Western blot, and bioinformatic tools were applied to examine the platelet proteins of 10 patients with major depression and 10 healthy controls.

Results

The levels of 8 proteins were significantly different between the patients with major depression in the acute phase and healthy controls. The levels of protein disulfide-isomerase A3 (PDIA3) and F-actin-capping protein subunit beta (CAPZB) were higher in patients with major depression than in healthy controls. The levels of fibrinogen beta chain (FIBB), fibrinogen gamma chain (FIBG), retinoic acid receptor beta (RARB), glutathione peroxidase 1 (GPX1), SH3 domain-containing protein 19 (SH319), and T-complex protein 1 subunit beta (TCPB) were lower in patients with major depression than in healthy controls.

Conclusions

Platelet provided valuable information about the pathways and processes of inflammation/immunity, oxidative stress, and neurogenesis, related to major depression.     

Improve the coverage for the analysis of phosphoproteome of HeLa cells by a tandem digestion approach

Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.     

Evaluation of D10-Leu metabolic labeling coupled with MALDI-MS analysis in studying the response of the yeast proteome to H2O2 challenge

An efficient D10-Leu metabolic-labeling method combined with isotope-ratio quantitation by MALDI-TOF MS was used to probe the response of the yeast proteome to H2O2. Control cultures correct for effects not associated with H2O2 challenge. A stress-response index to H2O2 (SRIH2O2) is defined, and values are reported for seven proteins at 45-225 min following exposure to 0.4 mM H2O2. The time course of protein accumulation in unstressed cells following the H10- to D10-SCD switch suggests that proteome responses at <45 min could be monitored by addition of excess D10-Leu to H10-cultures.     

A relationship between gene expression and protein interactions on the proteome scale: analysis of the bacteriophage T7 and the yeast Saccharomyces cerevisiae

The relationship between the similarity of expression patterns for a pair of genes and interaction of the proteins they encode is demonstrated both for the simple genome of the bacteriophage T7 and the considerably more complex genome of the yeast Saccharomyces cerevisiae. Statistical analysis of large-scale gene expression and protein interaction data shows that protein pairs encoded by co-expressed genes interact with each other more frequently than with random proteins. Furthermore, the mean similarity of expression profiles is significantly higher for respective interacting protein pairs than for random ones. Such coupled analysis of gene expression and protein interaction data may allow evaluation of the results of large-scale gene expression and protein interaction screens as demonstrated for several publicly available datasets. The role of this link between expression and interaction in the evolution from monomeric to oligomeric protein structures is also discussed.     

Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome

Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.     

High-throughput analysis of rat liver plasma membrane proteome by a nonelectrophoretic in-gel tryptic digestion coupled with mass spectrometry identification

In-gel digestion is commonly used after proteins are resolved by polyacrylamide gel electrophoresis (SDS-PAGE, 2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the direct analysis of complex proteins. Here, we describe a strategy combining isolation of purified plasma membrane, efficient digestion of plasma membrane proteins in polyacrylamide gel, and high-sensitivity analysis by advanced mass spectrometry to create a new rapid and high-throughput method. The plasma membrane protein mixture is directly incorporated into a polyacrylamide gel matrix, After formation of the gel, proteins in the gel section are digested with trypsin, and the resulting peptides are subjected to reversed-phase, high-performance liquid chromatography followed by electrospray ion-trap tandem mass analysis. Using this optimized strategy, we have identified 883 rat liver membrane proteins, of which 490 had a gene ontology (GO) annotation indicating a cellular component, and 294 (60%) of the latter were known integral membrane proteins or membrane proteins. In total, 333 proteins are predicted by the TMHMM 2.0 algorithm to have transmembrane domains (TMDs) and 52% (175 of 333) proteins to contain 2-16 TMDs. The identified membrane proteins provide a broad representation of the rat plasma membrane proteome with little bias evident due to protein p I and molecular weight (MW). Also, membrane proteins with a high GRAVY score (grand average hydrophobicity score) were identified, and basic and acidic membrane proteins were evenly represented. This study not only offered an efficient and powerful method in shotgun proteomics for the identification of proteins of complex plasma membrane samples but also allowed in-depth study of liver membrane proteomes, such as of rat models of liver-related disease. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of rat liver plasma membrane in general.     

Rapid selection of peptide containing fractions in off‐line 2‐D HPLC in shotgun proteome analysis by screening with MALDI TOF MS

A simple and fast screening method for the selection of fractions of first dimension separation to be analyzed in second dimension‐MS/MS experiments in offline multidimensional liquid chromatographic separation schemes for shotgun proteome analysis was developed. The method is based on the measurement of total peptide content of the first dimension fractions by MALDI MS and was established using a tryptic digest of a bacterial proteome. The results of the screening process were in good agreement with those obtained in a detailed proteome analysis performed by RP×ion‐pair RP‐MALDI TOF/TOF MS analysis. The method supports a straightforward planning of experiments, also enabling a reduction of overall measurement time in shotgun proteome analysis.     

Large-scale evaluation of dynamically important residues in proteins predicted by the perturbation analysis of a coarse-grained elastic model

Backgrounds  

It is increasingly recognized that protein functions often require intricate conformational dynamics, which involves a network of key amino acid residues that couple spatially separated functional sites. Tremendous efforts have been made to identify these key residues by experimental and computational means.     

Purification of cytokinins on a polyvinylpyrrolidone column followed by analysis on a reversedphase C18ODS HPLC system

It has been found that cytokinins, as a class, can be separated from co-occurring phenolics by column chromatography using polyvinylpyrrolidone (PVP) with methanol as eluant. Subsequent fractionation of the cytokinins can then be achieved by HPLC on a C₁₈ODS reversed phase system using methanol: water (60: 40) as the mobile phase. The system shows considerable promise as an extremely mild separation process and has been used to separate two unknown cytokinins from seedlings ofHordeum vulgare cv. Steptoe.     

Identification of the thrombin light chain a as the single best mass for differentiation of gastric cancer patients from individuals with dyspepsia by proteome analysis

Gastric cancer mortality is second only to lung cancer, and its prognosis is dismal. Using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry, we previously identified a single best mass, which could separate gastric cancer from patients without cancer, with a sensitivity of 89.9% and a specificity of 90%. Using protein liquid chromatography systems with various chromatography media and MS/MS analysis, we were able to identify thrombin light chain A, a proteolytic fragment of prothrombin, as the single best mass for early detection of gastric cancer patients. These findings indicate that disturbances in the coagulation-system are early events in gastric cancer biology and that a decrease or loss of thrombin light chain A, which we termed negative serum protein profiling, may contribute to the diagnosis of cancer patients.     

Update on the detection of beta-exotoxin in Bacillus thuringiensis strains by HPLC analysis

AIMS: The current work aimed to study the presence of beta-exotoxin by high-performance liquid chromatography (HPLC) in supernatant fluids from final whole cultures of the 69 type strains and 13 subtypes of Bacillus thuringiensis strains, as well as from some insecticidal strains. METHODS AND RESULTS: Results from HPLC and bioassays with Ephestia kuhniella (Lepidoptera Pyralidae) were compared. Type I beta-exotoxin was only detected in type strains representing serotypes H1, H9 and H10a,10b. Discrepancies between HPLC and bioassays were found in H8a,8b and some insecticidal strains, which suggests the occurrence of another soluble toxin different from type I beta-exotoxin, possibly type II beta-exotoxin. CONCLUSION: This study shows the need to use bioassays to determine the presence of beta-exotoxin activity. However, HPLC is a fast and sensitive technique if only type I beta-exotoxin is to be determined. The occurrence of beta-exotoxin in a type strain does not imply production of this metabolite by other strains belonging to the same serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: These results complete the characterization of type strains belonging to the International Entomopathogenic Bacillus Collection (Institut Pasteur, Paris, France).