Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.     

Identification of stool proteins in C57BL/6J mice by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry

Gastrointestinal disease is a major cause of mortality in humans and animals, and the detection of disease-associated protein in stool is an established diagnostic method in this context. Yet, no data currently exists about the protein composition of mammalian faeces. Using a newly developed two-dimensional (2D) gel method, 28 of the most abundant proteins in murine faeces were identified. Mammalian faeces contains protein from multiple species (from the individual, from gastrointestinal bacteria, from food, etc.). Yet, it was found that the majority of mouse stool proteins were of mouse origin, with a minority of proteins being derived from food (in particular soybean glycinin and conglycinin) and bacteria (flagellin). Most mouse proteins were proteases and saccharidases derived from the exocrine pancreas. In addition, two unexpected mouse proteins were identified: one was a newly described mucin-like protein from intestinal goblet cells (FcγBP); the other was the secreted form of carbonic anhydrase (type VI) from salivary gland. The data suggest that 2D analysis of faecal protein is likely to provide meaningful information about the physiological stage of the gastrointestinal tract. Compared with studies based on biopsies, faecal protein analysis may reduce the number of laboratory animals, and might also allow quicker bridging from animal studies to humans, where biopsy material is more difficult to obtain and is less relevant for general practice use.     

Identification of p21Cip1 binding proteins by gel electrophoresis and capillary liquid chromatography microelectrospray tandem mass spectrometry

Proteins bound to a glutathione-S-transferase-p21Cip1 affinity column were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using tandem mass spectrometry. Capillary liquid chromatography coupled to microelectrospray tandem mass spectrometry (capLC-microESI MS/MS) in an ion trap allowed identification of the proteins present in the gel bands. Of eleven bands analyzed, fifty-three proteins were identified. More than one hundred tryptic peptides were detected on-line, automatically fragmented and used for protein characterization in databases. Samples were also analyzed by off-line nanospray and matrix-assisted laser desorption/ionization mass spectrometry. CapLC-microESI MS/MS was the most efficient technique for the analysis of these protein mixtures.     

Identification of dysregulation of atrial proteins in rats with chronic obstructive apnea using two‐dimensional polyacrylamide gel electrophoresis and mass spectrometry

Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide and has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. Here, we used two‐dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We found enzymes involved in the glycolysis, beta‐oxidation, electron transport chain and Krebs cycle to be down‐regulated. The data suggested that the dysregulated proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia. Our results are consistent with our previous 1D‐PAGE and nanoLC‐MS/MS study (Channaveerappa et al, J Cell Mol Med. 2017), where we found that some aerobic and anaerobic glycolytic and Krebs cycle enzymes were down‐regulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents (NADH). The 2D‐PAGE study not only complements our current study, but also advances our understanding of the OSA. The complete mass spectrometry data are available via ProteomeXchange with identifier PXD011181.     

Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.     

Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL(2) and HDL(3), were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL(2), and apo L and a glycosylated apo A-II were identified in HDL(3). Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.     

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.     

Lipoproteomics I: mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.     

家蚕催青前期胚胎蛋白质双向电泳图谱分析

为了探讨家蚕Bombyx mori胚胎蛋白质整体变化,以多化性品种P50为材料,采用蛋白质双向电泳技术及图像分析技术分析了催青前期胚胎（戊₃以前）各个时期蛋白质图谱及其变化情况。研究发现:从临界Ⅱ期（丙_２）胚胎到缩短期(戊₂)胚胎蛋白质双向电泳图谱基本稳定,存在于临界Ⅱ期胚胎的蛋白斑点在催青前期的4个胚胎中消失的个数较少,仅占22.80％,而在催青的最后2个胚胎中消失的蛋白质斑点却占48.18％;在神经沟出现（丁₁）、腹肢突起（丁₂）、上唇突起（戊₁）和缩短期（戊₂）胚胎的双向电泳图谱中能够检测到100个特异蛋白质斑点,这些特异蛋白质斑点大多在随后邻近的胚胎发育中消失,暗示了这些特异蛋白可能与相应胚胎的形体特征发育有关。     

Scrapie prion protein structural constraints obtained by limited proteolysis and mass spectrometry

Elucidation of the structure of scrapie prion protein (PrP^Sc), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrP^Sc. Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in β-strands. We treated PrP^Sc isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrP^Sc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and limited proteolysis after partial unfolding of PrP^Sc confirmed that only the mentioned cleavage sites at the N-terminal side of the PrP^Sc are susceptible to PK. Our results indicate that besides the “classic” amino-terminal PK cleavage points, PrP^Sc contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short β-sheet stretches connected by loops and turns.     

家蚕催青后期胚胎蛋白质双向电泳图谱分析

采用蛋白质双向电泳技术分析了家蚕Bombyx mori催青后期胚胎蛋白质图谱的变化。研究发现: 在头胸分化期（戊₃）、反转期（己₁）、毛瘤发生期（己₂）、点青期（己₃）、转青期（己₄）和孵化期（己₅）胚胎蛋白质的双向电泳图谱中共检测到209个特异蛋白斑点,其中己₃和己₄两个胚胎出现的特异蛋白斑点数在整个催青期胚胎中为最多,分别达55和77个。与催青前期胚胎出现的特异蛋白斑点变化规律相似,这些特异蛋白斑点大多也是在随后邻近的胚胎发育中消失。推测这些特异蛋白可能与相应胚胎的形体特征发育有关。     

Approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography,gel electrophoresis and mass spectrometry

A combination of separation and identification techniques was used to rapidly and reproducibly analyze influenza vaccine constituents. Size-exclusion HPLC analysis reduced significantly the complexity by providing a constituents profile according to size. Significantly, no sample treatment was required prior to analysis thus eliminating a potential source of artifacts and degradation. Distinct profiles were associated with influenza strains as well as with vaccines from different manufacturers. Samples analyzed over several years allowed evaluation of method performance and provided stability-indicating data relating to the structural integrity of separated components. Collected chromatographic peaks were identified by gel electrophoresis and MALDI/MS of tryptic digests from excised gel bands. The challenge in obtaining high quality analytical data from complex mixtures clearly demonstrated the value of separation steps prior to MS identification. The method presented here is not intended to replace existing methodology; it is intended to provide a product specific profile to be used as a rapid screen for manufacturer, year (for annual influenza vaccines), stability or counterfeit product. It is a new screening method that provides a rapid and robust indication of products which require further investigation as a result of a deviation in their characteristic profile. Until now this tool did not exist.     

Proteome analysis of human hepatocellular carcinoma tissues by two-dimensional difference gel electrophoresis and mass spectrometry

Proteome analysis of human hepatocellular carcinoma tissues was conducted using two-dimensional difference gel electrophoresis coupled with mass spectrometry. Paired samples from the normal and tumor region of resected human liver were labeled with Cy3 and Cy5, respectively while the pooled standard sample was labeled with Cy2. After analysis by the DeCyder software, protein spots that exhibited at least a two-fold difference in intensity were excised for in-gel tryptic digestion and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A total of 6 and 42 proteins were successfully identified from the well- and poorly-differentiated samples, respectively. The majority of these proteins are related to detoxification/oxidative stress and metabolism. Three down-regulated metabolic enzymes, methionine adenosyltransferase, glycine N-methyltransferase, and betaine-homocysteine S-methyltransferase that are involved in the methylation cycle in the liver are of special interest. Their expression levels, especially, methionine adenosyltransferase, seemed to have a major influence on the level of S-adenosylmethionine (AdoMet), a vital intermediate metabolite required for the proper functioning of the liver. Recent work has shown that chronic deficiency in AdoMet in the liver results in spontaneous development of steatohepatitis and hepatocellular carcinoma, and hence the down-regulation of hepatic methionine adenosyltransferase in our hepatocellular carcinoma samples is in line with this observation. Moreover, when a comparison is made between the differentially expressed proteins from our human hepatocellular carcinoma samples and from the liver tissues of knockout mice deficient in methionine adenosyltransferase, there is a fairly good correlation between them.     

Identification of stool proteins in C57BL/6J mice by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry.

Gastrointestinal disease is a major cause of mortality in humans and animals, and the detection of disease-associated protein in stool is an established diagnostic method in this context. Yet, no data currently exists about the protein composition of mammalian faeces. Using a newly developed two-dimensional (2D) gel method, 28 of the most abundant proteins in murine faeces were identified. Mammalian faeces contains protein from multiple species (from the individual, from gastrointestinal bacteria, from food, etc.). Yet, it was found that the majority of mouse stool proteins were of mouse origin, with a minority of proteins being derived from food (in particular soybean glycinin and conglycinin) and bacteria (flagellin). Most mouse proteins were proteases and saccharidases derived from the exocrine pancreas. In addition, two unexpected mouse proteins were identified: one was a newly described mucin-like protein from intestinal goblet cells (FcgammaBP); the other was the secreted form of carbonic anhydrase (type VI) from salivary gland. The data suggest that 2D analysis of faecal protein is likely to provide meaningful information about the physiological stage of the gastrointestinal tract. Compared with studies based on biopsies, faecal protein analysis may reduce the number of laboratory animals, and might also allow quicker bridging from animal studies to humans, where biopsy material is more difficult to obtain and is less relevant for general practice use.     

Protein expression analysis ofChlamydia pneumoniae persistence by combined surface-enhanced laser desorption ionization time-of-flight mass spectrometry and two-dimensional polyacrylamide gel electrophoresis

The aim of this study was to examine the protein expression profiles of persistentChlamydia pneumoniae by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Although 2D PAGE is still the method of choice for separating and detecting components of complex protein mixtures, it has several distinct disadvantages; i.e., being labor-intensive and having a bias toward proteins within the dynamic range of the gel condition. Hence, SELDI-TOF-MS technology was used to complement 2D PAGE.C. pneumoniae-infected HEp2 cells were treated with or without IFN-γ, and protein expression profiles were determined at 48 h postinfection (hpi). Unfractionated monolayers were also used for protein profiling by SELDI-TOF, using two different chip surface types: weak cation exchanger and hydrophobic surface. Under IFN-γ-induced persistence,C. pneumoniae expresses an altered protein expression profile. Twenty chlamydial proteins showed differential regulatory patterns by SELDI-TOF-MS, two of which, HSP-70 cofactor, and a hypothetical protein, were identified by 2D PAGE and mass spectrometry. Two additional proteins, phosphatidylserine decarboxylase and 30S ribosomal protein S17, were exclusively identified by SELDI TOF-MS analysis, as these were not present in sufficient quantity for detection by 2D PAGE. We propose that a combination of 2D-PAGE and SELDI-TOF-MS may complement the disadvantages of each technique alone and may provide a rapid and precise screening technique.