INTRODUCTIONThe proteins found in tears play an important role in maintaining the ocular surface and changes in tear protein components may reflect changes in the health of the ocular surface. Proteomics provides a comprehensive approach for cataloguing all the proteins of the tear proteome, which will help to elucidate disease pathogenesis, make clinical diagnoses and evaluate the influence of medications on the structure, composition and secretion of tear proteins. In this study, an alternative proteomic strategy was investigated to explore the human tear proteome.

MATERIALS AND METHODSTear samples were obtained from patients who had pterygium and were collected on the first day and third day after pterygium surgery. Tears pooled from 6 patients were used in the analysis. Reverse-phase high-pressure liquid chromatograph (RPHPLC) was used as the first step to separate intact proteins into 21 peaks. Each fraction was then tryptic-digested and analysed by nanoLC-nano-ESI-MS/MS to characterise the protein components in each fraction.

RESULTSIn total, 60 tear proteins were identified with high confidence, including well-known abundant tear proteins, and tear-specific proteins such as lacritin and proline-rich proteins. Among them, proline-rich protein 5 was found for the first time in tear fluid. A large number of plasma proteins were also observed in tear fluid.

CONCLUSIONSThe results showed that the proteomic strategy used in this study was successfully applied to analyse tear proteome.

Eye Proteins ; analysis ; Humans ; Mass Spectrometry ; methods ; Proteome ; Tears ; chemistry

Bacterial Proteins ; analysis ; China ; Proteome ; analysis ; Proteomics ; Yersinia pestis ; chemistry ; genetics

The different sera proteomic components between uremia patients and normal subjects were studied through two-dimensional gel electrophoresis technique. Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2DE), silver staining, ImageMaster 2D 5.0 analysis software, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS) and IPI human database searching were used to separate and identify the proteome of the sera from the patients with uremia. The results showed that satisfactory 2DE patterns of the serum proteins were obtained. Twenty-six protein spots showed significant difference in quantity in uremia patients, and 20 protein spots were identified by MALDI-TOF-TOF-MS. It was concluded that good reproducibility could be obtained by applying immobilized pH gradient 2DE to separate and identify the proteome in serum, which provided the foundation for the further study on uremia toxins pertaining to protein.
Blood Protein Electrophoresis ; Blood Proteins/*analysis ; Case-Control Studies ; *Electrophoresis, Gel, Two-Dimensional/methods ; Proteome/*analysis ; Proteome/chemistry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods ; Uremia/*blood

An overview of the background for proteomics and a description of the present state of art are given with a description of the main strategies in proteomics. The advantages and limitations of the two major strategies, 2D-gel based and LC-MS based, are discussed and a combination for the two, CeLC-MS is described. A number of challenging problems which have been solved using different proteomics strategies including the advantage of organell enrichment or modifications specific peptide isolation to get deeper into the proteome are described. Finally the present status and future needs discussed.
Chromatography, High Pressure Liquid ; Electrophoresis, Gel, Two-Dimensional ; Mass Spectrometry ; Peptides ; analysis ; chemistry ; Phosphorylation ; Protein Isoforms ; analysis ; Proteome ; analysis ; Proteomics

Data-independent acquisition (DIA) is a high-throughput, unbiased mass spectrometry data acquisition method which has good quantitative reproducibility and is friendly to low-abundance proteins. It becomes the preferred choice for clinical proteomic studies especially for large cohort studies in recent years. The mass-spectrometry (MS)/MS spectra generated by DIA is usually heavily mixed with fragment ion information of multiple peptides, which makes the protein identification and quantification more difficult. Currently, DIA data analysis methods fall into two main categories, namely peptide-centric and spectrum-centric. The peptide-centric strategy is more sensitive for identification and more accurate for quantification. Thus, it has become the mainstream strategy for DIA data analysis, which includes four key steps: building a spectral library, extracting ion chromatogram, feature scoring and statistical quality control. This work reviews the peptide-centric DIA data analysis procedure, introduces the corresponding algorithms and software tools, and summarizes the improvements for the existing algorithms. Finally, the future development directions are discussed.
Humans ; Proteomics/methods* ; Reproducibility of Results ; Peptides/chemistry* ; Software ; Algorithms ; Tandem Mass Spectrometry/methods* ; Proteome/analysis*

The purpose of this study was to explore the effect of acute hypoxia on urine proteome in rats. In this study, rats were placed in a hypoxic chamber simulating a plateau environment at an altitude of 5 000 m for 24 hours. Urine samples were collected at 0, 12, and 24 h after hypoxia. Urinary proteins were profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Compared with the control (before hypoxia), a total of 144 differentially expressed proteins were identified in the hypoxia 12 h group, and 129 differentially expressed proteins were identified in the hypoxia 24 h group. Functional annotation analysis revealed that these differentially expressed proteins were involved in a series of biological pathways related to hypoxic stress, such as anti-oxidative stress, glycolysis, complement and coagulation cascade. Our results suggest that the urinary proteome can reflect significant changes upon acute hypoxic stimulation. These findings may provide an approach to judge the hypoxia state of the body and help to assist the detection of hypoxia state.
Animals ; Rats ; Proteome/analysis* ; Chromatography, Liquid/methods* ; Tandem Mass Spectrometry/methods* ; Proteomics/methods* ; Hypoxia

In order to investigate the antioxidant effect of alkylhydroxide peroxidase (ahpC) of Helicobacter pylori (H. pylori) 26695, an ahpC-deficient mutant (H. pylori 26695 ahpC::cat) was generated. ahpC-deficient mutant was grown slowly at lower pressure of oxygen (5% oxygen) compared to the H. pylori 26695. Whole cell proteins isolated form H. pylori 26695 and H. pylori 26695 ahpC::cat were analyzed by MALDI-TOF and tandem-MS. The expression of 15 proteins, including Ppa, HypB, GrpE, Elp, RecA, GroES, Mda66, RibE, NapA, GlnA, BioB, TrxB, Tsf, FumC and Icd, was more than doubled in H. pylori 26695 ahpC::cat. Production of 10 proteins such as UreG, FabE, Adk, Pnp, OorC, AtpA, AtpD, Nqq3, Pfr, and TagD decreased below 50% in H. pylori 26695 ahpC::cat compared to the H. pylori 26695. In microarray analysis, 9 genes including sul1, amiE, frxA, fecA, hyuA, and katA increased in transcription level in H. pylori 26695 ahpC::cat compared to H. pylori 26695. A total of 24 genes, including flaB, protein kinase C inhibitor, cag16, pabC, and sabA, reduced in transcription. 27 genes, including HP0889, showed common expression changes in ahpC, katA, and sodB-deficient mutations. As a result of this study, there were not many genes whose expression was commonly changed by the deletion of each of the three major antioxidant enzymes of H. pylori. These results showed the functions and regulation of the three antioxidant enzymes were different in H. pylori.
Antioxidants ; Helicobacter pylori ; Helicobacter ; Microarray Analysis ; Oxygen ; Peroxidase ; Protein Kinase C ; Proteome ; Ribes

This paper is aimed to establish and optimize proteomic research platform using isobaric tags for relative and absolute quantitation (iTRAQ) so as to facilitate further proteomic research of human peripheral blood mononuclear cells (hPBMC). We collected hPBMC, after protein extraction and trypsin digestion, we labeled the samples with iTRAQ reagents and then subjected to mass spectrometry. In triplicates, thirty precursors were randomly selected and detected; as a result, 26, 28 and 29 peptides were respectively tagged with ITRAQ reporter ions. The labeling efficiencies ranged between 86.7%-96.7%, with no significant difference among the groups (P>0.05). The coefficient of variance for the relative ratios of peptides from different proteins was ranged from 7.6% to 25.5% and there were no significant differences across the groups (P>0.05). The coefficient of variance for the relative ratios of different peptides from the same protein was varied from 9.3% to 19.1% and the differences across groups were not significant (P>0.05). The labeling of iTRAQ combined with tandem mass spectrometry in PBMC was successful with favourable reproducibility and accuracy, which could lay a foundation for further proteomic study of hPBMC in autoimmune disorders.
Humans ; Leukocytes, Mononuclear ; chemistry ; Proteins ; analysis ; chemistry ; Proteome ; Proteomics ; methods ; Staining and Labeling ; Tandem Mass Spectrometry ; methods

OBJECTIVETo identify differential proteins in the seminal plasma of healthy fertile men and non-obstructive azoospermia patients by the shotgun proteomic strategy.

METHODSSix seminal plasma samples from 3 healthy fertile and 3 non-obstructive azoospermia volunteers were collected by Percoll isolation, balanced-mixed, and followed by separation of the mixture by SDS-PAGE. The proteins were subjected to in-gel enzymolysis and isolation of peptide fragments, and then identified by the shotgun proteomic strategy. Then comparative analyses were made between the two groups on the identified proteins with the unique peptide count > or = 2 and = 1 but with the peptide count > or = 4.

RESULTSA total of 213 differential proteins were identified, 133 in the non-obstructive azoospermia patients and 80 in the healthy fertile men. According to the molecular function, these differential proteins mainly fell into the types of signal transduction, cytoskeleton and catalytic activity, especially oxidoreductase activity in the latter type. Eighteen of the differential proteins were found to be of particular significance, including dynein heavy chain, fatty acid synthase, and tubulin alpha-6 chain.

CONCLUSIONThe differential proteins identified in this study were many in number and various in function, which not only demonstrated the value of the shotgun proteomic strategy in protein identification, but also suggested the complicated pathogenesis and varied types of non-obstructive azoospermia. The samples must be selected strictly based on their gene and histological types. Non-obstructive azoospermia was shown to be related with the M phase of the mitotic cell cycle at the protein level, but its specific mechanism remains unknown.

Azoospermia ; metabolism ; physiopathology ; Case-Control Studies ; Humans ; Male ; Proteome ; analysis ; Proteomics ; methods ; Semen ; chemistry ; Sperm Motility

OBJECTIVETo develop a method for extracting cytoskeletons and cytoskeleton-associated proteins for proteomic analysis.

METHODSA subcellular sequential proteome extraction method was exploited. The extraction procedure was optimized and controlled according to observed cell morphology changes and one- and two-dimensional electrophoresis images. The extraction efficiency and selectivity were evaluated by Western blotting and mass spectrometry.

RESULTSFour extracted fractions clearly displayed distinct patterns. Western blotting detected the fraction-marker proteins FAK, integrin-β1, histone H1 and cytokeratin 19 only in their expected fractions. About 90% of the protein spots in the cytoskeleton fraction were identified by mass spectrometry as cytoskeleton and/or its associated proteins.

CONCLUSIONThe subcellular proteome sequential fractionation method facilitates the detection of proteins of low abundance and shows a high reproducibility and selectivity, and thus can serve as an ideal pre-fractionation method prior to two-dimensional electrophoresis.

Cytoskeleton ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; methods ; Humans ; Proteome ; analysis ; Proteomics ; methods ; Subcellular Fractions