Protein ubiquitination is one of the most important and widely exist protein post-translational modifications in eukaryotic cells, which takes the ubiquitin and ubiquitin chains as signal molecules to covalently modify other protein substrates. It plays an important roles in the control of almost all of the life processes, including gene transcription and translation, signal transduction and cell-cycle progression, besides classical 26S protesome degradation pathway. Varied modification sites in the same substrates as well as different types of ubiquitin linkages in the same modification sites contain different structural information, which conduct different signal or even determine the fate of the protein substrates in the cell. Any abnormalities in ubiquitin chain formation or its modification process may cause severe problem in maintaining the balance of intracellular environment and finally result in serious health problem of human being. In this review, we discussed the discovery, genetic characteristics and the crystal structure of the ubiquitin. We also emphasized the recent progresses of the assembly processes, structure and their biological function of ubiquitin chains. The relationship between the disregulation and related human diseases has also been discussed. These progress will shed light on the complexity of proteome, which may also provide tools in the new drug research and development processes.
Humans ; Proteome ; Ubiquitin ; chemistry ; Ubiquitination

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

The systematic and in-depth study of phosphoproteome rely on highly reproducible and specific phosphopeptide enrichment methods. At present, a variety of enrichment methods have been developed based on different principles, and these methods often display different selectivity and specificity. It is therefore very important to select the most suitable enrichment method according to different research purposes. This review summarized the phosphopeptide enrichment based on affinity chromatography, immunoprecipitation, chemical derivatization, chromatography and other newly developed methods. The advantages and disadvantages of these methods, as well as the related optimization and improvement strategies, were discussed in detail. In addition, we also briefly summarized the progress of the combination of phosphopeptide enrichment and fractionation methods developed in recent years.
Phosphopeptides/metabolism* ; Proteomics/methods* ; Titanium/chemistry* ; Chromatography, Affinity ; Proteome ; Phosphorylation

Reversible phosphorylation plays a crucial role in regulating protein activities and functions. Sexual reproduction directly affects yield of most agricultural crops. As the male reproductive organ, anther generates microspores (pollen), delivering gametes (sperms) to complete double fertilization in higher plants. Here, we took the advantage of Nano UHPLC-MS/MS to analyze maize (Zea mays, B73) early anthers at proteomic and phosphoproteomic levels, to explore the protein and phosphorylation modification regulatory networks controlling maize anther development. Our proteomic analysis identified 3 016 unique peptides, belonging to 1 032 maize proteins. MapMan analysis revealed variously potential proteins associated with maize anther development, such as receptor-like kinases (GRMZM2G082823_P01 and GRMZM5G805485_P01). Using phospho-peptides enriched by TiO2 affinity chromatography, our phosphoproteomic analysis detected 257 phospho-peptides from 210 phosphoproteins, discovering 223 phosphosites. Compared to the 86 maize phosphoproteins collected in the Plant Protein Phosphorylation Data Base (P3DB), we found that 203 phosphoproteins and 218 phosphosites were not revealed before. Further bioinformatics analysis revealed that phosphorylation of 14-3-3 proteins, kinases, phosphatases, transcription factors, cell cycle and chromatin structure related proteins might play important roles in regulating normal anther development in maize. Our findings not only enlarged the maize phosphoproteome data, but also provided information for analyzing the molecular mechanism controlling maize anther development at genetic and biochemical levels.
Crops, Agricultural ; chemistry ; Phosphoproteins ; chemistry ; Phosphorylation ; Plant Proteins ; chemistry ; Pollen ; chemistry ; Proteome ; Tandem Mass Spectrometry ; Zea mays ; chemistry

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 establish an efficient and high resolution 2-DE (two-dimensional electrophoresis) protocol for root tuber proteome analysis of Rehmannia glutinosa.

METHODProteins from root tuber of R. glutinosa were extracted by using five different methods and their productivity and profiles were assessed by means of SDS-PAGE and two-dimensional electrophoresis.

RESULTThe trichloroacetic acid (TCA)-phenol extraction method was found most effective for the extraction with the highest protein yield, the most spots in protein patterns, and the highest resolution of proteins, and the clearest background could be achieved simultaneously. A 1:5 solution of ampholine pH 3-10 and pH 5-8 for a nonlinear gel and the 170 microg of protein loading dosage obtained maps with more protein spots and higher resolution of separation patterns.

CONCLUSIONThis study based on the optimized root tuber proteome preparation and the 2-DE protocol gets a high resolution and reproducibility 2-DE image, which will be expected to have excellent applications in proteomics studies of R. glutinosa tuber root.

Electrophoresis, Gel, Two-Dimensional ; methods ; Plant Proteins ; analysis ; chemistry ; Plant Roots ; chemistry ; Proteome ; chemistry ; isolation & purification ; Rehmannia ; chemistry

OBJECTIVETo establish and optimize the two-dimensional electrophoresis (2-DE) of uterine leiomyoma for the proteome analysis.

METHODSRun immobilized pH gradient (IPG)-isoelectric focusing electrophoresis as the first dimension, then vertical SDS-PAGE electrophoresis as the second dimension. A series of important steps,such as sample solubility, volume of loading, electrophoresis parameters and protocol for staining were optimized.

RESULTSThe 2-DE patterns of uterine leiomyoma and myometrium with good quality were obtained.

CONCLUSIONWith optimal condition the two-dimensional electrophoresis of uterine leiomyoma can be obtained.

Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Leiomyoma ; chemistry ; Myometrium ; chemistry ; Neoplasm Proteins ; analysis ; Proteome ; analysis ; Uterine Neoplasms ; chemistry

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

To demonstrate the fructose metabolism pathway in Bifidobacterium Longum NCC2705 and to construct its fermentation model, we explored the comparative proteome cultivating the strain on glucose or fructose, based on a proteomic reference map of B. longum NCC2705 constructed earlier. Then, we used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and electro-spray ionization tandem mass spectrometry (ESI-MS/MS) for differently expressed proteins identification. Furthermore, with semi-quantitative RT-PCR we determined the distinctively expressed proteins at the level of transcription. Proteomic comparison of glucose- and fructose-grown cells demonstrated much similarity. On the page of fructose there were all the enzymes and proteins that exist during the process of glucose degradation. We observed a greater variation of more than three-fold for the identified 9 spots representing 5 protein entries by MALDI-TOF MS. The sugar-binding protein specific to fructose (BL0033) and an ABC transporter ATP binding protein (BL0034) showed higher expression level from cells grown on fructose. It was also determined by semi-quantitative RT-PCR subsequently. BL0033 time course and concentration experiments showed that the induction time correlated to higher fructose concentration, and increased expression of BL0033. Fructose was catabolized via the same degradation pathway as glucose at the level of proteomics. BL0033 was induced by fructose. All results suggest that the uptake of fructose into the cell may be conducted by a specific ABC transport system, in which BL0033 and BL0034 as components might have played an important role.
Bifidobacterium ; chemistry ; genetics ; metabolism ; Culture Media ; Fermentation ; Fructose ; pharmacology ; Glucose ; pharmacology ; Proteome ; analysis ; genetics ; Proteomics ; methods

The aim of this study was to establish and optimize two-dimensional electrophoresis method for human bone marrow leukemia cells in order to obtain the profiles with high resolution and reproducibility. The total protein was extracted and separated by isoelectric focusing (IEF) and SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with silver nitrate or Coomassie brilliant blue, and then scanned and analyzed with PDQuest 7.4 analysis software. The effects of different protein preparation methods and electrophoresis conditions on the profiles were compared. The results indicated that by optimizing preparation of protein sample and electrophoresis protocols, clear profiles with 780 +/- 73 well separated protein spots on an average were obtained and the match rate was 82 +/- 5% between reproducible gels from leukemia cells of different sub-type. It is concluded that the two-dimensional electrophoresis method of proteome from human bone marrow leukemia cells is established successfully and is suitable for the further comparative proteomic research between leukemia of different types.
Bone Marrow Cells ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; methods ; Humans ; Leukemia ; metabolism ; Proteome ; analysis