Antigens secreted from Mycobacterium tuberculosis: identification by proteomics approach and test for diagnostic marker

Tuberculosis caused by mycobacteria, mainly Mycobacterium tuberculosis, is a major infectious disease of the respiratory system. An early diagnosis followed by chemotherapy is the major control strategy. In an effort to identify the antigens suitable for immunodiagnosis and vaccines, the proteins secreted in a culture medium from the M. tuberculosis K-strain, which is the most prevalent among the clinical isolates in Korea and belongs to the Beijing family, were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and compared with those from the M. tuberculosis H37Rv and CDC1551 strains. Eight proteins, Rv0652, Rv1636, Rv2818c, Rv3369, Rv3865, Rv0566c, MT3304, and Rv3160, were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and found to be relatively abundant in the culture medium from the M. tuberculosis K-strain but less so from the CDC1551 or H37Rv strains. In addition, Rv3874 (CFP-10), Rv-0560c and Rv3648c, which were expressed increasingly in the K and CDC1551 strains, were also identified using the same proteomics technology. All proteins were prepared by molecular cloning, expression in Escherichia coli followed by affinity purification. Among them, three proteins, rRv3369, rRv0566c, and rRv3874, were selected by prescreening and examined for their potential as serodiagnostic antigens using an enzyme-linked immunosorbent assay. When 100 sera from tuberculosis patients and 100 sera from the healthy controls were analyzed, rRV3369, rRv3874, and rRv0566c showed a sensitivity of 60%, 74%, and 43%, and a specificity of 96%, 97%, and 84%, respectively. These results suggest that the rRv3369 and rRv3874 proteins, which were expressed more abundantly in the more recently obtained clinical isolates of M. tuberculosis than in the laboratory-adapted H37Rv strain, are promising for use in the serodiagnosis of tuberculosis.     

Computational approach for identification and characterization of GPI-anchored peptides in proteomics experiments

Genes that encode glycosylphosphatidylinositol anchored proteins (GPI-APs) constitute an estimated 1-2% of eukaryote genomes. Current computational methods for the prediction of GPI-APs are sensitive and specific; however, the analysis of the processing site (omega- or omega-site) of GPI-APs is still challenging. Only 10% of the proteins that are annotated as GPI-APs have the omega-site experimentally verified. We describe an integrated computational and experimental proteomics approach for the identification and characterization of GPI-APs that provides the means to identify GPI-APs and the derived GPI-anchored peptides in LC-MS/MS data sets. The method takes advantage of sequence features of GPI-APs and the known core structure of the GPI-anchor. The first stage of the analysis encompasses LC-MS/MS based protein identification. The second stage involves prediction of the processing sites of the identified GPI-APs and prediction of the corresponding terminal tryptic peptides. The third stage calculates possible GPI structures on the peptides from stage two. The fourth stage calculates the scores by comparing the theoretical spectra of the predicted GPI-peptides against the observed MS/MS spectra. Automated identification of C-terminal GPI-peptides from porcine membrane dipeptidase, folate receptor and CD59 in complex LC-MS/MS data sets demonstrates the sensitivity and specificity of this integrated computational and experimental approach.     

Molecular profiling of the human nasal epithelium: A proteomics approach

A comprehensive proteomic profiling of nasal epithelium (NE) is described. This study relies on simple subcellular fractionation used to obtain soluble- and membrane-enriched fractions followed by 2-dimensional liquid chromatography (2D-LC) separation and tandem mass spectrometry (MS/MS). The cells were collected using a brushing technique applied on NE of clinically evaluated volunteers. Subsequently, the soluble- and the membrane-protein enriched fractions were prepared and analyzed in parallel using 2D-LC-MS/MS. In a set of 1482 identified proteins, 947 (63.9%) proteins were found to be associated to membrane fraction. Grand average hydropathy value index (GRAVY) analysis, the transmembrane protein mapping and annotations of primary location deposited in the Human Protein Reference Database (HPRD) confirmed an enrichment of hydrophobic proteins on this dataset. Ingenuity Pathway Analysis (IPA) of soluble fraction revealed an enrichment of molecular and cellular functions associated with cell death, protein folding and drug metabolism while in membrane fraction showed an enrichment of functions associated with molecular transport, protein trafficking and cell-to-cell signaling and interaction. The IPA showed similar enrichment of functions associated with cellular growth and proliferation in both soluble and membrane subproteomes. This finding was in agreement with protein content analysis using exponentially modified protein abundance index (emPAI). A comparison of our data with previously published studies focusing on respiratory tract epithelium revealed similarities related to identification of proteins associated with physical barrier function and immunological defence. In summary, we extended the NE molecular profile by identifying and characterizing proteins associated to pivotal functions of a respiratory epithelium, including the control of fluid volume and ionic composition at the airways' surface, physical barrier maintenance, detoxification and immunological defence. The extent of similarities supports the applicability of a less invasive analysis of NE to assess prognosis and treatment response of lung diseases such as asthma, cystic fibrosis and chronic obstructive pulmonary disease.     

Discovery of biomarkers for gastric cancer: a proteomics approach

Gastric cancer is the second leading cause of cancer-related deaths worldwide. Although many treatment options exist for patients with gastric tumors, the incidence and mortality rate of gastric cancer are on the rise. The early stages of gastric cancer are non-symptomatic, and the treatment response is unpredictable. This situation is further aggravated by a lack of diagnostic biomarkers that can aid in the early detection and prognosis of gastric cancer and in the prediction of chemoresistance. Moreover, clinical surgical specimens are rarely obtained, and traditional biomarkers of gastric cancer are not very effective. Many studies in the field of proteomics have contributed to the discovery and establishment of powerful diagnostic tools (e.g., ProteinChip array) in the management of cancer. The evolution in proteomic technologies has not only enabled the screening of a large number of samples but also enabled the identification of pathologically significant proteins, such as phosphoproteins, and the quantitation of difference in protein expression under different conditions. Multiplexed assays are used widely to accurately fractionate various complex samples such as blood, tissue, cells, and Helicobacter pylori-infected specimens to identify differentially expressed proteins. Biomarker detection studies have substantially contributed to the areas of secretome, metabolome, and phosphoproteome. Here, we review the development of potential biomarkers in the natural history of gastric cancer, with specific emphasis on the characteristics of target protein convergence.     

Expression of the human salivary alpha-amylase gene in yeast and characterization of the secreted protein

Recombinant plasmids were constructed in which the human salivary alpha-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the alpha-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary alpha-amylase. Thus, the human salivary alpha-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.     

Extracellular matrix composition and remodeling in human abdominal aortic aneurysms: a proteomics approach

Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-liquid chromatography-tandem MS and label-free quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein, periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.     

Cancer cells responsible for humoral hypercalcemia express mRNA encoding a secreted form of ODF/TRANCE that induces osteoclast formation

A novel cDNA encoding a secreted form of osteoclast differentiation factor/tumor necrosis factor-related activation-induced cytokine (sODF/TRANCE, GenBank Accession No. AB037599) was sequenced from 5' RACE cDNA clones of squamous cell carcinoma cell lines, SCC-4 and T3M-1 Cl.2, of which parental malignant tissues had caused severe humoral hypercalcemia. The sODF/TRANCE cDNA was composed of unknown 5' end sequence followed by the 100% identical sequence of the ODF/TRANCE extracellular domain-coding region. The longest open reading frame (ORF) of the novel cDNA completely matched the 3' end of the ORF of the ODF/TRANCE cDNA encoding C-terminal amino acid residues (74-318) in the extracellular region. The corresponding protein that reacted with the antibody specific for the extracellular domain of ODF/TRANCE was detected in the culture media conditioned by the cancer cells. Furthermore, human promyeloblastic leukemia cells, HL60, differentiated into osteoclast-like cells (OCLs) when cultured in the media conditioned by SCC-4 and T3M-1 Cl. 2 cells. The differentiation of HL60 cells into OCLs was inhibited by the anti-ODF/TRANCE antibody. These results strongly suggest that sODF/TRANCE plays an important role in enhanced bone-resorption in humoral hypercalcemia of malignancy.     

Defining salivary biomarkers using mass spectrometry-based proteomics: a systematic review

Recent advancements in mass spectrometric proteomics provide a promising result in utilizing saliva to explore biomarkers for diagnostic purposes. However, the issues of specificity or redundancy of disease-associated salivary biomarkers have not been described. This systematic review was therefore aimed to define and summarize disease-related salivary biomarkers identified by mass spectrometry proteomics. Peer-reviewed articles published through July 2009 within three databases were reviewed. Out of 243 articles, 21 studies were selected in this systematic review with conditions including Sj?gren's syndrome, squamous cell carcinoma, dental caries, diabetes, breast cancer, periodontitis, gastric cancer, systemic sclerosis, oral lichen planus, bleeding oral cavity, and graft-versus-host disease. The sample size ranged from 3-41 in both diseased and control subjects, with no consensus on sample collection protocol. One hundred eighty biomarkers were identified in total; 87 upregulated, 63 downregulated, and 30 varying based on disease. Except for Sj?gren's syndrome, the majority of studies with the same disease produce inconsistent biomarkers. Larger sample size and standardization of sample collection/treatment protocol may improve future studies.     

Positional proteomics: preparation of amino-terminal peptides as a strategy for proteome simplification and characterization

We describe a protocol for selective extraction of the amino (N)-terminal-most peptide of a protein or a mixture of proteins after proteolysis. The first stage of the protocol blocks the free amino groups alpha and epsilon (the latter being lysyl residues) on the intact proteins by acetylation. In the second stage, proteolysis of the acetylated proteins yields a mixture of N-terminally acetylated (true N-terminal) and non-acetylated (internal and carboxy-terminal) peptides. Affinity capture of peptides bearing free amino groups using an immobilized amine-reactive reagent removes internal peptides from the mixture. The unbound fraction is highly enriched in N-terminal peptides, which can be analyzed without further treatment. This method is compatible with a range of proteolytic enzymes and fragmentation methods, and should take 2 d to complete. The N-terminal peptides can then be analyzed by mass spectrometry. This low cost, rapid method is readily adopted using off the shelf reagents.     

Metabolites secreted by human atherothrombotic aneurysms revealed through a metabolomic approach

Abdominal aortic aneurysm (AAA) is perma-nent and localized dilation of the abdominal aorta. Intraluminal thrombus (ILT) is involved in evolution and rupture of AAA. Complex biological processes associated with AAA include oxidative stress, proteolysis, neovascularization, aortic inflammation, cell death, and extracellular matrix breakdown. Biomarkers of growth and AAA rupture could give a more nuanced indication for surgery, unveil novel pathogenic pathways, and open possibilities for pharmacological inhibition of growth. Differential analysis of metabolites released by normal and pathological arteries in culture may help to find molecules that have a high probability of later being found in plasma and start signaling processes or be useful diagnostic/prognostic markers. We used a LC-QTOF-MS metabolomic approach to analyze metabolites released by human ILT (divided into luminal and abluminal layers), aneurysm wall (AW), and healthy wall (HW). Statistical analysis was used to compare luminal with abluminal ILT layer, ILT with AW, and AW with HW to select the metabolites exchanged between tissue and external medium. Identified compounds are related to inflammation and oxidative stress and indicate the possible role of fatty acid amides in AAA. Some metabolites (e.g., hippuric acid) had not been previously associated to aneurysm, others (fatty acid amides) have arisen, indicating a very promising line of research.     

Functional characterization of the modified melanocortin peptides responsible for ligand selectivity at the human melanocortin receptors

The melanocortin system plays an important role in energy homeostasis as well as skin pigmentation, steroidogenesis and exocrine gland function. In this study, we examined eight Ac-His-Phe-Arg-Trp-NH(2) tetrapeptides that were modified at the Phe position and pharmacologically characterized their activities at the human MCR wild-types and their mutants. Our results indicate that at the hMC1R, all D stereochemical modified residues at the Phe position of peptides increase cAMP production in a dose-dependent manner. At the hMC3R, the DPhe peptide dose dependently increases cAMP production but all other three tetrapeptides were not. At the hMC4R, both the DPhe and DNal(1') peptides induce cAMP production. However, both DTyr and DNal(2') were not able to induce cAMP production. Further studies indicated that at the hMC1R M128L mutant receptor, the all D-configured tetrapeptides reduce their potencies as compared to that of hMC1R wild-type. However, at the hMC3R and hMC4R L165M and L133M mutant receptors, the DNal(2') and DTyr tetrapeptides possess agonist activity. These findings indicate that DPhe in tetrapeptide plays an important role in ligand selectivity and specific residue TM3 of the melanocortin receptors is crucial for ligand selectivity.     

The coupling of RP-HPLC and ESI-MS in the study of small peptides and proteins secreted in vitro by human salivary glands that are soluble in acidic solution

The aim of this study was the development of a method based on the coupling of RP-HPLC and ESI-MS for identifying and quantifying proteins and peptides secreted by human salivary glands in vitro. Salivary gland specimens, obtained from informed patients undergoing surgical resection, were incubated in an optimized medium. Incubation media of glandular specimens, selected on the basis of cytomorphological and ultrastructural analysis, were investigated by HPLC-MS. Several salivary peptides/proteins, previously recognized in human whole saliva, were searched for along the chromatogram by the selected ion monitoring (SIM) strategy. Analysis of the incubation media of parotid glands revealed the presence of basic PRPs PC, PD, PH, IB-1, II-2, and acidic PRP-1 and PRP-3 in all of the investigated samples. Basic PRPs PB and PA, acidic PRPs, and cystatins SN and S1 were detected in all of the incubation media of submandibular glands, whereas histatin 1 was detected in only one sample. Moreover, the method allowed detection of some post-translational derivatives of known salivary proteins, as well as of several previously unidentified small peptides. The present method represents a sensitive and powerful instrument to detect peptides and proteins secreted by human salivary glands in vitro.     

Dental handpiece contamination: a proteomics and surface analysis approach

Dental handpieces (DHPs) become biofouled internally with patient derived material that is difficult to access for removal and inactivation. This study undertook a quantitative and qualitative investigation of protein contamination of internal components from three different types of DHP: the turbine, slow speed contra-angle and surgical. Eluates from the high speed turbine, low speed spray channels and surgical gear were assayed for protein using an orthophthaldehyde assay. Eluates concentrated by Amicon ultrafiltration were also analysed by SDS-PAGE, mass spectroscopy, Western blotting and ELISA. The surfaces of handpiece components were also investigated by SEM, EFSCAN and EDAX microscopy. Surgical gears contained highest levels of protein (403?μg), followed by low speed spray channels (17.7?μg) and the high speed turbine (<5?μg). Mass spectroscopy of surgical gears demonstrated mostly serum derived proteins. Decontamination of the DHPs using an automated washer disinfector and handpiece irrigator showed a significant reduction in residual protein levels.     

Signal peptides of secreted proteins of the archaeon Sulfolobus solfataricus: a genomic survey

Analysis of the recently completed genome sequence of the thermoacidophilic archaeon Sulfolobus solfataricus reveals that about 4.2% of its proteome consists of putative secretory proteins with signal peptides. This includes members of the four major classes of signal peptides: secretory signal peptides, twin-arginine signal peptides, possible lipoprotein precursors, and type IV pilin signal peptides. The latter group is surprisingly large compared to the size of the groups in other organisms and seems to be used predominately for a subset of extracellular substrate-binding proteins.     

Characterization of the human salivary basic proline-rich protein complex by a proteomic approach

Thirteen samples of human normal whole saliva were analyzed by RP-HPLC-ESI-MS and MALDI-TOF-MS to investigate the basic proline-rich protein complex. Between known basic-PRPs the P-B, P-C (or IB-8b), P-D (or IB-5), P-E (or IB-9), P-F (or IB-8c), P-H (or IB-4), IB-6, II-2, IB-1, and IB-8a glucosylated were identified, whereas the II-1, IB-7, PA, and D1-A peptides were not detected. Some detected masses not attributable to known basic-PRPs were putatively ascribed to II-2 and IB-1 nonphosphorylated, II-2 and IB-1 missing the C-terminal arginine residue, and the 1-62 fragment of IB-6, named P-J peptide. A correlation matrix analysis revealed a cluster of correlation among all the basic PRPs (apart from the P-B peptide) which is in agreement with their common parotid origin.     

Signal-CF: a subsite-coupled and window-fusing approach for predicting signal peptides

We have developed an automated method for predicting signal peptide sequences and their cleavage sites in eukaryotic and bacterial protein sequences. It is a 2-layer predictor: the 1st-layer prediction engine is to identify a query protein as secretory or non-secretory; if it is secretory, the process will be automatically continued with the 2nd-layer prediction engine to further identify the cleavage site of its signal peptide. The new predictor is called Signal-CF, where C stands for "coupling" and F for "fusion", meaning that Signal-CF is formed by incorporating the subsite coupling effects along a protein sequence and by fusing the results derived from many width-different scaled windows through a voting system. Signal-CF is featured by high success prediction rates with short computational time, and hence is particularly useful for the analysis of large-scale datasets. Signal-CF is freely available as a web-server at http://chou.med.harvard.edu/bioinf/Signal-CF/ or http://202.120.37.186/bioinf/Signal-CF/.     

Platforms for enrichment of phosphorylated proteins and peptides in proteomics

Protein phosphorylation is a complex and highly dynamic process involved in numerous biological events. Abnormal phosphorylation is one of the underlying mechanisms for the development of cancer and metabolic disorders. The identification and absolute quantification of specific phospho-signatures can help elucidate protein functions in signaling pathways and facilitate the development of new and personalized diagnostic and therapeutic tools. This review presents a variety of strategies currently utilized for the enrichment of phosphorylated proteins and peptides before mass spectrometry analysis during proteomic studies. The investigation of specific affinity reagents, allied to the integration of different enrichment processes, is triggering the development of more selective, rapid and cost-effective high-throughput automated platforms.     

Identification of proteins responsible for the development of adriamycin resistance in human gastric cancer cells using comparative proteomics analysis

Resistance to anticancer drugs is a major obstacle in the effective treatment of tumors. To understand the mechanisms responsible for multidrug resistance (MDR), a proteomic approach was used to identify proteins that were expressed in different levels by the adriamycinresistant human gastric cancer cell line, SGC7901/ADR, and its parental cell line, SGC7901. Two-dimensional gel electrophoresis (2-DE) and image analysis was used to determine which protein spots were expressed in different levels by the two cell lines. These spots were then partially identified using ESI-Q-TOF mass spectrometry, and the differential expressional levels of the partially identified proteins were then determined by western blot analysis and real-time RT-PCR. Additionally, the association of Nucleophosmin (NPM1), a protein that was highly expressed by SGC7901/ADR, with MDR was analyzed using siRNA. As a result of this study, well-resolved, reproducible 2-DE patterns of SGC7901/ADR and SGC7901 were established, and 16 proteins that may play a role in the development of thermoresistance were identified. Additionally, suppression of NPM1 expression was found to enhance adriamycin chemosensitivity in SGC7901/ADR. These results provide a fundamental basis for the elucidation of the molecular mechanism of MDR, which may assist in the treatment of gastric cancer.