An analysis of the Caenorhabditis elegans lipid raft proteome using geLC-MS/MS

Lipid rafts are microdomains of the phospholipid bilayer, proposed to form semi-stable "islands" that act as a platform for several important cellular processes; major classes of raft-resident proteins include signalling proteins and glycosylphosphatidylinositol (GPI)-anchored proteins. Proteomic studies into lipid rafts have been mainly carried out in mammalian cell lines and single cell organisms. The nematode Caenorhabditis elegans, the model organism with a well-defined developmental profile, is ideally suited for the study of this subcellular locale in a complex developmental context. A study of the lipid raft proteome of C. elegans is presented here. A total of 44 proteins were identified from the lipid raft fraction using geLC-MS/MS, of which 40 have been determined to be likely raft proteins after analysis of predicted functions. Prediction of GPI-anchoring of the proteins found 21 to be potentially modified in this way, two of which were experimentally confirmed to be GPI-anchored. This work is the first reported study of the lipid raft proteome in C. elegans. The results show that raft proteins, including numerous GPI-anchored proteins, may have a variety of potentially important roles within the nematode, and will hopefully lead to C. elegans becoming a useful model for the study of lipid rafts.     

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.     

Optimization of protein solubilization for the analysis of the CD14 human monocyte membrane proteome using LC-MS/MS

Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), iii) a combination of both agents (methanol + PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was ~ 98% positive for CD14 expression as determined by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46%) were mapped integral membrane proteins. However, employing PPS 216 proteins were identified of which 75 (35%) were mapped as integral membrane proteins. These results indicate that methanol alone or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.     

Optimization of protein solubilization for the analysis of the CD14 human monocyte membrane proteome using LC-MS/MS

Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), iii) a combination of both agents (methanol + PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was ~ 98% positive for CD14 expression as determined by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46%) were mapped integral membrane proteins. However, employing PPS 216 proteins were identified of which 75 (35%) were mapped as integral membrane proteins. These results indicate that methanol alone or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.     

Robust method for proteome analysis by MS/MS using an entire translated genome: demonstration on the ciliome of Tetrahymena thermophila

To improve the utility of increasingly large numbers of available unannotated and initially poorly annotated genomic sequences for proteome analysis, we demonstrate that effective protein identification can be made on a large and unannotated genome. The strategy developed is to translate the unannotated genome sequence into amino acid sequence encoding putative proteins in all six reading frames, to identify peptides by tandem mass spectrometry (MS/MS), to localize them on the genome sequence, and to preliminarily annotate the protein via a similarity search by BLAST. These tasks have been optimized and automated. Optimization to obtain multiple peptide matches in effect extends the searchable region and results in more robust protein identification. The viability of this strategy is demonstrated with the identification of 223 cilia proteins in the unicellular eukaryotic model organism Tetrahymena thermophila, whose initial genomic sequence draft was released in November 2003. To the best of our knowledge, this is the first demonstration of large-scale protein identification based on such a large, unannotated genome. Of the 223 cilia proteins, 84 have no similarity to proteins in NCBI's nonredundant (nr) database. This methodology allows identifying the locations of the genes encoding these novel proteins, which is a necessary first step to downstream functional genomic experimentation.     

An improved method of sample preparation on AnchorChip targets for MALDI-MS and MS/MS and its application in the liver proteome project

An improved method for sample preparation for MALDI-MS and MS/MS using AnchorChip targets is presented. The method, termed the SMW method (sample, matrix wash), results in better sensitivity for peptide mass fingerprinting as well as for sequencing by MS/MS than previously published methods. The method allows up-concentration and desalting directly on the mass spectrometric target and should be amenable for automation. A draw back caused by extensive oxidation of methionine and tryptophan in the SMW method can be alleviated by the addition of n-octyl glucopyranoside and DTT to the sample solution. The method was validated for protein identification from a 2-DE based liver proteome study. The SMW method resulted in identification of many more proteins and in most cases with a better score than the previously published methods.     

Molecular weight assessment of proteins in total proteome profiles using 1D-PAGE and LC/MS/MS

Background  

The observed molecular weight of a protein on a 1D polyacrylamide gel can provide meaningful insight into its biological function. Differences between a protein's observed molecular weight and that predicted by its full length amino acid sequence can be the result of different types of post-translational events, such as alternative splicing (AS), endoproteolytic processing (EPP), and post-translational modifications (PTMs). The characterization of these events is one of the important goals of total proteome profiling (TPP). LC/MS/MS has emerged as one of the primary tools for TPP, but since this method identifies tryptic fragments of proteins, it has not generally been used for large-scale determination of the molecular weight of intact proteins in complex mixtures.     

Validation of peptide MS/MS spectra using metabolic isotope labeling for spectral matching-based shotgun proteome analysis

We report an isotope labeling shotgun proteome analysis strategy to validate the spectrum-to-sequence assignments generated by using sequence-database searching for the construction of a more reliable MS/MS spectral library. This strategy is demonstrated in the analysis of the E. coli K12 proteome. In the workflow, E. coli cells were cultured in normal and (15)N-enriched media. The differentially labeled proteins from the cell extracts were subjected to trypsin digestion and two-dimensional liquid chromatography quadrupole time-of-flight tandem mass spectrometry (2D-LC QTOF MS/MS) analysis. The MS/MS spectra of the two samples were individually searched using Mascot against the E. coli proteome database to generate lists of peptide sequence matches. The two data sets were compared by overlaying the spectra of unlabeled and labeled matches of the same peptide sequence for validation. Two cutoff filters, one based on the number of common fragment ions and another one on the similarity of intensity patterns among the common ions, were developed and applied to the overlaid spectral pairs to reject the low quality or incorrectly assigned spectra. By examining 257,907 and 245,156 spectra acquired from the unlabeled and (15)N-labeled samples, respectively, an experimentally validated MS/MS spectral library of tryptic peptides was constructed for E. coli K12 that consisted of 9,302 unique spectra with unique sequence and charge state, representing 7,763 unique peptide sequences. This E. coli spectral library could be readily expanded, and the overall strategy should be applicable to other organisms. Even with this relatively small library, it was shown that more peptides could be identified with higher confidence using the spectral search method than by sequence-database searching.     

The human proteome project: current state and future direction

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.     

Improved 2D nano-LC/MS for proteomics applications: a comparative analysis using yeast proteome.

The most commonly used method for protein identification with two-dimensional (2D) online liquid chromatography-mass spectrometry (LC/MS) involves the elution of digest peptides from a strong cation exchange column by an injected salt step gradient of increasing salt concentration followed by reversed phase separation. However, in this approach ion exchange chromatography does not perform to its fullest extent, primarily because the injected volume of salt solution is not optimized to the SCX column. To improve the performance of strong cation exchange chromatography, we developed a new method for 2D online nano-LC/MS that replaces the injected salt step gradient with an optimized semicontinuous pumped salt gradient. The viability of this method is demonstrated in the results of a comparative analysis of a complex tryptic digest of the yeast proteome using the injected salt solution method and the semicontinuous pump salt method. The semicontinuous pump salt method compares favorably with the commonly used injection method and also with an offline 2D-LC method.     

Characterization of the human skeletal muscle proteome by one-dimensional gel electrophoresis and HPLC-ESI-MS/MS

Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.     

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

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

Method for therapeutic drug monitoring of azole antifungal drugs in human serum using LC/MS/MS

Fungal infections occur in immunocompromised patients. Azole antifungal agents are used for the prophylaxis and treatment of these infections. The interest in therapeutic drug monitoring azole agents has increased over the last few years. Inter- and intra-patient variability of pharmacokinetics, drug–drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of the drugs, belonging to the group of azoles. Therefore a simple, rapid and flexible method of analysis is required. This method is based on the precipitation of proteins in human serum with LC/MS/MS detection. Validation was performed according to the guidelines for bioanalytical method validation of the food and drug administration agency. The four most used azole drugs can be detected in human serum within the clinical relevant serum levels with good accuracy and reproducibility at the limit of quantification. Intra- and inter-day validation demonstrated good accuracy and reproducibility. A rapid, sensitive and flexible LC/MS/MS method has been developed and validated to measure voriconazole (VRZ), fluconazole (FLZ), itraconazole (ITZ) and posaconazole (PSZ) in human serum. This new method is suitable for clinical pharmacokinetic studies and routine monitoring in daily practice.     

Label-free quantitative analysis of one-dimensional PAGE LC/MS/MS proteome: application on angiotensin II-stimulated smooth muscle cells secretome

A widely used method for protein identification couples prefractionation of protein samples by one-dimensional (1D) PAGE with LC/MS/MS. We developed a new label-free quantitative algorithm by combining measurements of spectral counting, ion intensity, and peak area on 1D PAGE-based proteomics. This algorithm has several improvements over other label-free quantitative algorithms: (i) Errors in peak detection are reduced because the retention time is based on each LC/MS/MS run and actual precursor m/z. (ii) Detection sensitivity is increased because protein quantification is based on the combination of peptide count, ion intensity, and peak area. (iii) Peak intensity and peak area are calculated in each LC/MS/MS run for all slices from 1D PAGE for every single identified protein and visualized as a Western blot image. The sensitivity and accuracy of this algorithm were demonstrated by using standard curves (17.4 fmol to 8.7 pmol), complex protein mixtures (30 fmol to 1.16 pmol) of known composition, and spiked protein (34.8 fmol to 17.4 pmol) in complex proteins. We studied the feasibility of this approach using the secretome of angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs). From the VSMC-conditioned medium, 629 proteins were identified including 212 putative secreted proteins. 26 proteins were differently expressed in control and Ang II-stimulated VSMCs, including 18 proteins not previously reported. Proteins related to cell growth (CYR61, protein NOV, and clusterin) were increased, whereas growth arrest-specific 6 (GAS6) and growth/differentiation factor 6 were decreased by Ang II stimulation. Ang II-stimulated changes of plasminogen activator inhibitor-1, GAS6, cathepsin B, and periostin were validated by Western blot. In conclusion, a novel label-free quantitative analysis of 1D PAGE-LC/MS/MS-based proteomics has been successfully applied to the identification of new potential mediators of Ang II action and may provide an alternative to traditional protein staining methods.     

A careful disorderliness in the proteome: sites for interaction and targets for future therapies

The community of scientists interested in studying intrinsically unstructured (or disordered) proteins has emerged in recent years. What began as a controversial idea has become an established phenomenon. The new, greater focus on proteins that are in some way normally unstructured promises to provide a greater understanding of protein function, particularly with respect to protein-protein interactions. These regions also offer new possibilities into how interactions can be targeted by small molecules.     

An accurate quantitative LC/ESI-MS/MS method for sirolimus in human whole blood

Sirolimus is a widely used immunosuppressant that requires therapeutic drug monitoring (TDM). We optimized a preanalytical procedure that allows for the accurate quantiation of sirolimus in whole blood by LC/ESI-MS/MS with minimal matrix effects. Sirolimus is highly lipophilic, and solvents containing greater than 50% methanol were required to maintain sirolimus recovery. The final pretreatment procedure developed consists of a zinc sulfate protein precipitation, an extraction using octadecyl silyl-silica gel for eliminating water-soluble and hydrophilic compounds, and HybridSPE cartridge treatment to eliminate phospholipids. Using this procedure prior to LC/ESI-MS/MS led to the accurate and reproducible quantitation of sirolimus in human whole blood. The linear range of detection was 0.5-50 ng/mL, a range appropriate for TDM, and the method demonstrated good repeatability and intermediate precision within this quantitative range. In order to investigate the quantitative performance of this method, we compared it to two commercially available sirolimus immunoassays and our previously reported LC/ESI-MS/MS method. The immunoassays gave consistently greater values for the sirolimus concentration, and this may be related to antibody cross-reactivity with sirolimus metabolites and/or other matrix effects. Although our procedure is too long to support real-time TDM for outpatients, it can serve as reference method to assess the performance of other analytical methods that are currently available or may be developed in the future.     

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.     

Serial changes in urinary proteome profile of membranous nephropathy: implications for pathophysiology and biomarker discovery

Membranous nephropathy is one of the most common causes of primary glomerular diseases worldwide. The present study adopted a gel-based proteomics approach to better understand the pathophysiology and define biomarker candidates of human membranous nephropathy using an animal model of passive Heymann nephritis (PHN). Clinical characteristics of Sprague-Dawley rats injected with rabbit anti-Fx1A antiserum mimicked those of human membranous nephropathy. Serial urine samples were collected at Days 0, 10, 20, 30, 40, and 50 after the injection with anti-Fx1A (number of rats = 6; total number of gels = 36). Urinary proteome profiles were examined using 2D-PAGE and SYPRO Ruby staining. Quantitative intensity analysis and ANOVA with Tukey post-hoc multiple comparisons revealed 37 differentially expressed proteins among 6 different time-points. These altered proteins were successfully identified by MALDI-TOF MS and classified into 6 categories: (i) proteins with decreased urinary excretion during PHN; (ii) proteins with increased urinary excretion during PHN; (iii) proteins with increased urinary excretion during PHN, but which finally returned to basal levels; (iv) proteins with increased urinary excretion during PHN, but which finally declined below basal levels; (v) proteins with undetectable levels in the urine during PHN; and (vi) proteins that were detectable in the urine only during PHN. Most of these altered proteins have functional significance in signaling pathways, glomerular trafficking, and controlling the glomerular permeability. The ones in categories (v) and (vi) may serve as biomarkers for detecting or monitoring membranous nephropathy. After normalization of the data with 24-h urine creatinine excretion, changes in 34 of initially 37 differentially expressed proteins remained statistically significant. These data underscore the significant impact of urinary proteomics in unraveling disease pathophysiology and biomarker discovery.     

Characterization of the human salivary proteome by capillary isoelectric focusing/nanoreversed-phase liquid chromatography coupled with ESI-tandem MS

Saliva is a readily available body fluid with great diagnostic potential. The foundation for saliva-based diagnostics, however, is the development of a complete catalog of secreted and "leaked" proteins detectable in saliva. By employing a capillary isoelectric focusing-based multidimensional separation platform coupled with electrospray ionization tandem mass spectrometry (MS), a total of 5338 distinct peptides were sequenced, leading to the identification of 1381 distinct proteins. A search of bacterial protein sequences also identified many peptides unique to several organisms and unique to the NCBI nonredundant database. To the best of our knowledge, this proteome study represents the largest catalog of proteins measured from a single saliva sample to date. Data analysis was performed on individual MS/MS spectra using the highly specific peptide identification algorithm, OMSSA. Searches were conducted against a decoyed SwissProt human database to control the false-positive rate at 1%. Furthermore, the well-curated SwissProt sequences represent perhaps the least redundant human protein sequence database (12,484 records versus the 50,009 records found in the International Protein Index human database), therefore minimizing multiple protein inferences from single peptides. This combined bioanalytical and bioinformatic approach has established a solid foundation for building up the human salivary proteome for the realization of the diagnostic potential of saliva.