Analysis of expression and comparative profile of normal placental tissue proteins and those in preeclampsia patients using proteomic approaches

Preeclampsia (PE) is a complex and serious condition of pregnancy. Trophoblasts in human placenta can be separated and collected by laser capture micro-dissection (LCM). Protein in trophoblasts have been extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), finally 962 unique proteins are identified by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Comparison of differential expressed proteins in normal and those in PE are investigated. Two-dimensional electrophoresis (2DE) and MS were used to identify differential expressed proteins. 13 differential expressed proteins include signal transduction protein, molecular chaperone, cell skeleton proteins are identified, in which 3 proteins are down-regulated and 10 proteins are up-regulated. They might be correlated with the cause of PE.     

A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis

Study of the complexome - all the protein complexes of the cell - is essential for a better understanding and more global vision of cell function. Using two-dimensional blue native/SDS-PAGE (2-D BN/SDS-PAGE) technology, the cytosolic and membrane protein complexes of Escherichia coli were separated. Then, the different partners of each protein complex were identified by LC-MS/MS. In this report, 306 protein complexes were separated and identified. Among these protein complexes, 50 heteromultimeric and 256 homomultimeric protein complexes were found. Among the 50 heteromultimeric protein complexes, 18 previously described protein complexes validate the technology. In this study, 109 new protein complexes were found, providing insight into the function of previously uncharacterized bacterial proteins.     

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.     

Down-regulation of Vinculin in Human Colorectal Carcinoma Identified by Proteomics Analysis

In the present study, we aimed to globally profile the proteins involved in colorectal carcinoma(CRC), in order to find clues to the pathological process of CRC. Pairs of malignant tissues and their adjacent healthy tissues from patients with colorectal cancer were subject to differential proteomics analysis. Two dimensional electrophoresis coupled with mass spectrometry(2-DE/MS) was used to identify differentially expressed proteins between pairs of tissue samples. A list of proteins relevant to the progression of colorectal tumor was identified by two dimensional gel electrophoresis(2-DE)-based proteomics approach. Among the identified proteins, vinculin was found to be remarkably down-regulated in colorectal carcinoma tissues. In addition, three phosphorylation modifications within the isolated vinculin were identified by in-depth liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis. Our results provide a basis for further understanding the pathological significance of vinculin in the regulation of carcinogenesis, invasion and metastasis of colorectal tumors.     

Development of an integrated approach for evaluation of 2-D gel image analysis: impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow

With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through a comparison of protein identifications using direct MALDI-TOF/TOF and LC-ESI-MS/MS analyses of 2-D gel separated proteins from cauliflower florets, we have developed an integrated approach to improve the accuracy and reliability of comparative 2-D electrophoresis. From 46 spots of interest, we identified 51 proteins by MALDI-TOF/TOF analysis and 108 proteins by LC-ESI-MS/MS. The results indicate that 75% of the analyzed spots contained multiple proteins. A comparison of hit rank for protein identifications showed that 37 out of 43 spots identified by MALDI matched the top-ranked hit from the ESI-MS/MS. By using the exponentially modified protein abundance index (emPAI) to determine the abundance of the individual component proteins for the spots containing multiple proteins, we found that the top-hit proteins from 40 out of 43 spots identified by MALDI matched the most abundant proteins determined by LC-MS/MS. Furthermore, our 2-D-GeLC-MS/MS results show that the top-hit proteins in 44 identified spots contributed on average 81% of the spots' staining intensity. This is the first quantitative measurement of the average rate of false assignment for direct MALDI analysis of 2-D gel spots using a new integrated workflow (2-D gel imaging, "2-D GeLC-MS/MS", and emPAI analysis). Here, the new approach is proposed as an alternative to traditional gel-based quantitative proteomics studies.     

Murine fecal proteomics: A model system for the detection of potential biomarkers for colorectal cancer

Tumor related products shed into the feces offer a potential source of biomarkers for the detection of colorectal cancer (CRC). Using SDS-PAGE followed by nanoflow reversed-phased LC–MS/MS to analyse fecal samples from Apc^Min/+ mice (that develop spontaneous multiple intestinal neoplasia with age) we have identified 336 proteins (115 proteins of murine origin, 201 from fecal bacteria, 18 associated with food intake and 2 of apparent parasitic origin). 75% of the murine proteins identified in this study are predicted to be extracellular or associated with the cell plasma membrane. Of these proteins, a number of the murine homologues of colorectal cancer associated proteins (CCAP) such as hemoglobin, haptoglobin, hemopexin, alpha-2-macroglobulin and cadherin-17 have been identified, demonstrating the potential of fecal proteomics for detecting potential biomarkers and paving the way for subsequent MS/MS based biomarker studies on similar human samples.     

Proteome analysis of tunicamycin-induced ER stress

Endoplasmic reticulum (ER) stress occurs upon increased levels of unfolded proteins and results in activation of cellular responses such as the unfolded protein response (UPR) and ER-associated protein degradation (ERAD). To examine ER stress, we performed a quantitative proteome analysis of human neuroblastoma cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with SDS-PAGE and LC-MS/MS. Proteins associated with the ER were overrepresented in the dataset of altered proteins. In particular, ER chaperones responsible for protein folding were significantly upregulated in response to ER stress. The important ER stress regulator 78 kDa glucose-regulated protein (GRP-78 or BiP) was highly upregulated together with several proteins that have been found to form a multiprotein complex with BiP including cyclophilin B, DnaJ homolog subfamily B member 11, endoplasmin, hypoxia upregulated protein 1, protein disulfide isomerase and protein disulfide isomerase A4 upon tunicamycin-induced ER stress. Furthermore, seven aminoacyl-tRNA synthetases and five proteins belonging to the Sec61 complex were increased in response to tunicamycin-induced ER stress.     

用于相对和绝对定量的等量异位标签-二维液相色谱-串联质谱法分析雌二醇和血清剥夺对乳腺癌细胞内蛋白质含量的影响

雌激素和血清中的激素及各种生长因子在乳腺癌的发生、发展过程中发挥着重要的作用。研究雌激素和血清对乳腺癌细胞中蛋白质组成和含量的影响对于阐明雌激素和血清对乳腺癌细胞影响的分子机理具有重要的意义。利用四重用于相对和绝对定量的等量异位标签(isobaric tags for relative and absolute quantification, iTRAQ)标记结合二维液相色谱-串联质谱(two-dimensional liquid chromatography-tandem mass spectrometry, 2D-LC-MS/MS)对雌二醇(17β-oestradiol, E2)和血清各自以及共同作用对MCF7乳腺癌细胞内蛋白质表达的影响进行了比较,共鉴定到置信度在95%以上的蛋白质576种,其中各组相对于正常培养组的细胞共找到26种差异在1倍以上的蛋白质,其中10种上调,16种下调。研究发现E2和血清可显著影响细胞内与蛋白质合成相关的蛋白质的水平。实验结果表明: iTRAQ-LC-MS/MS是进行多个样品差异蛋白组比较的一种有效方法。     

Development of an effective sample preparation approach for proteomic analysis of silkworm eggs using two-dimensional gel electrophoresis and mass spectrometry

Sample preparation is still the first and important step toward successful two-dimensional gel electrophoresis (2DE) and identification in proteomics study. The 2DE profiling of eggs of silkworm species by using conventional one-step extraction, however, is unsatisfactory because high-abundance proteins such as egg-specific protein (ESP) and No 30 family (30 KP) in the extract lead to difficulties in detecting most of biologically relevant proteins. Based on the tendency of these abundant proteins to be soluble in Tris-HCl buffer, we report herein a robust approach in which the extract enriched in ESP and 30 KP was fractionationed and mixed with the re-extract of residual pellet in an optimal proportion. In comparison with the one-step method, the 2DE pattern was improved by this new method with over one-third enhancement in spots. A total of 48 unique proteins obtained have been furthermore identified by mass spectrometry (MS) and MS/MS. The identified proteins are found to include heat shock proteins families, ribosomal proteins, disulfide isomerase proteins, Glutathione S-transferase, and elongation factor, etc., which are mainly involved in some important processes. To our knowledge, this is the first time that the several proteins have been detected in silkworm eggs by proteomics means. This simple and reproducible approach would raise the opportunity of discovering and identifying more biomarkers and determining their possible roles in further studies.     

Proteomics Analysis of Up-regulated NPM1 Protein in Colorectal Cancer

In order to identify potential protein targets involved in colorectal cancer(CRC), we used a liquid chromatography coupled with mass spectrometry(LC-MS)/MS-based proteomics approach to characterize global protein expression patterns in malignant tissues and their adjacent healthy tissues from Dukes C stage CRC patients. A total number of 34 differentially expressed proteins were detected and identified by LC-MS/MS and database searching, which are supposed to be relevant to progression of colorectal tumor. Among these proteins, nucleophosmin 1(NPM1) was found to be remarkably up-regulated in colorectal carcinoma tissues, as compared with that in their normal counterparts. The results presented here could provide clues to elucidate the pathological significance of NPM1 in regulation of carcinogenesis of Dukes C stage colorectal tumors.     

Titanocene-catalyzed coupling of aromatic amides in the presence of organosilanes: a novel route to vicinal diamines and a new class of amine-substituted oligomers

The title reaction has been surveyed for a number of substrates with differing substitution patterns. With a few exceptions, the methodology provides a one-pot synthesis of the 1,2-diamines from widely available and inexpensive starting materials, and in high yields. In addition, the coupling of 1,4- and 1,3-bis-(N,N,N',N'-tetraalkyl)arylenediamides is shown, under the same experimental conditions, to yield oligomers: R2NC(O)C6H4CH(NR2)-[CH(NR2)C6H4CH(NR2)]n-CH(NR2)C6H4C(O)-NR2 (R = methyl and ethyl; n = 0 to ca. 5). The chemical structures of these unprecedented oligomers are determined by comparison of NMR and MS spectra to those of vicinal diamines, prepared from the analogous N,N-dialkylbenzamides. The origin of the limitation of oligomer chain length is probably due to a specific effect of the internal benzylic amine group, since the substrate 4-Me2NCH2C6H4C(O)NMe2 was found to be uniquely unreactive compared to the other 4-substituted N,N-dialkylbenzamides investigated. N-Methylphthalimide was briefly studied as a monomer and analysis by MS showed that oligomers are formed. Attempts to fully characterize these polymers were unsuccessful.     

Towards a two-dimensional proteome map of Mycoplasma pneumoniae

A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS). M. pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology. The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems. Proteins were identified using liquid chromatography-electrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS. Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods. So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides. Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes. The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication. Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M. pneumoniae cells grown under modified conditions.     

One-pot method of preparing novel macrocyclic(thio arylene) oligomers

A series of macrocyclic(thio arylene) oligomers were synthesized under high dilution conditions by reacting diphenyl ether, diphenyl, diphenyl disulfide or diphenyl methane with dichloro disulfide in the presence of trace amount of iron powder by a one-step method. The composition of these macrocyclic oligomers was analyzed by MALDI-TOF technology, and the experimental results showed that the cyclics were a mixture with repeating units separated by one to seven sulfur atoms. These macrocyclics can undergo ring-opening polymerization to form linear polymers under mild conditions. The glass transition temperatures of produced polymers varied with the sulfur content.     

Disposable chromatography for a high-throughput nano-ESI/MS and nano-ESI/MS-MS platform

High-throughput proteomics has typically relied on protein identification based on MALDI-MS peptide maps of proteolytic digests of 2D-gel-separated proteins. This technique, however, requires significant sequence coverage in order to achieve a high level of confidence in the identification. Tandem MS data have the advantage of requiring fewer peptides (2) for high confidence identification, assuming adequate MS/MS sequence coverage. MALDI-MS/MS techniques are becoming available, but can still be problematic because of the difficulty of inducing fragment ions of a singly charged parent ion. Electrospray ionization, however, has the advantage of generating multiply charged species that are more readily fragmented during MS/MS analysis. Two electrospray/tandem mass spectrometry-based approaches, nanovial-ESI-MS/MS and LC-MS/MS, are used for high throughput proteomics, but much less often than MALDI-MS and peptide mass fingerprinting. Nanovial introduction entails extensive manual manipulation and often shows significant chemical background from the in-gel digest. LC-MS has the advantages that the chemical background can be removed prior to analysis and the analytes are concentrated during the separation, resulting in more abundant analyte signals. On the other hand, LC-MS can often be time intensive. Here, we report the incorporation of on-line sample clean-up and analyte concentration with a high-throughput, chip-based, robotic nano-ESI-MS platform for proteomics studies.     

基质辅助激光解析飞行时间质谱分析条件及准确度对蛋白质数据库搜索结果的影响

考察了基质辅助激光解析飞行时间质谱获得肽质量指纹谱(PMF)时主要分析条件及准确度对蛋白质数据库搜索结果的影响.将牛血清白蛋白(BSA)经聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、酶解;肽段经质谱分析得PMF数据;用MS-Fit引擎在相同数据库及参数下搜索.得出:(1)激光脉冲击打次数在300次以前时,蛋白序列和肽段覆盖率逐步增加;当击打400次时,覆盖率呈下降趋势;(2)随着激光强度增加,目标蛋白得分、氨基酸序列和肽段的覆盖率逐步增加,但当强度增到1413时,则呈现降低趋势;(3)随延迟时间的增加,肽覆盖率呈递增趋势,但当达260 ns后呈下降趋势.按照优化的分析条件对同一位点重复3次,重复性良好.通过对质谱数据准确度的考察,发现以Mix2为外标,数据跟标准值差异越小时,则经其校正后的肽段数据所搜索目标蛋白的得分越高、排序靠前、检出预选蛋白个数越少,可信度越高;反之,难获得候选蛋白.结果表明:质谱准确度和分析条件对蛋白质数据库搜索影响显著.     

In-bone protein digestion followed by LC-MS/MS peptide analysis as a new way towards the routine proteomic characterization of human maxillary and mandibular bone tissue in oral surgery

Proteomic characterization of alveolar bones in oral surgery represents an analytical challenge due to their insoluble character. The implementation of a straightforward technique could lead to the routine use of proteomics in this field. This work thus developed a simple technique for the characterization of bone tissue for human maxillary and mandibular bones. It is based on the direct in-bone tryptic digestion of proteins in both healthy and pathological human maxillary and mandibular bone samples. The released peptides were then identified by the LC-MS/MS. Using this approach, a total of 1120 proteins were identified in the maxillary bone and 1151 proteins in the mandibular bone. The subsequent partial least squares–discrimination analysis (PLS-DA) of protein data made it possible to reach 100% discrimination between the samples of healthy alveolar bones and those of the bone tissue surrounding the inflammatory focus. These results indicate that the in-bone protein digestion followed by the LC-MS/MS and subsequent statistical analysis can provide a deeper insight into the field of oral surgery at the molecular level. Furthermore, it could also have a diagnostic potential in the differentiation between the proteomic patterns of healthy and pathological alveolar bone tissue. Data are available via ProteomeXchange with the identifier PXD026775.     

Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。     

基于不同提取方法的水稻叶片蛋白质组的二维液相色谱分离及高分辨质谱分析

建立了酚法提取-二维液相色谱分离-高分辨质谱分析水稻叶片蛋白质组的方法。水稻叶片蛋白质经过酚法提取,酶解肽段脱盐后用离线反相-反相二维液相色谱分离,然后用线性离子阱/静电场轨道阱组合式高分辨质谱分析,共鉴定到2712种蛋白质。比较了液相色谱分离系统（一维液相色谱与二维液相色谱）和水稻叶片蛋白质提取方法（酚法、十二烷基硫酸钠法（SDS法）和三氯乙酸/丙酮法（TCA/丙酮法））对鉴定蛋白质数量的影响,结果表明：在二维液相色谱条件下,酚法、SDS法和TCA/丙酮法鉴定到的蛋白质数目为2712、2415和1914,分别是一维液相色谱条件下鉴定到的蛋白质数目的2.7、2.5和1.9倍。二维液相色谱条件下,酚法鉴定到的蛋白质数目比SDS法和TCA/丙酮法分别多297和798。与SDS法和TCA/丙酮法相比,酚法不但鉴定到的蛋白质数量多,而且能够鉴定到一些极端蛋白质,如酸性、碱性及高等电点的蛋白质。此外,对二维液相色谱条件下3种蛋白质提取方法提取到的蛋白质进行生物学功能分类,发现3种方法鉴定到的蛋白质的功能存在互补性,但酚法鉴定到的蛋白质功能种类最多。该法为水稻蛋白质组学研究提供了技术支撑,同时也为其他作物的蛋白质组学研究技术提供重要的借鉴。     

Intramolecular disulfide bond between catalytic cysteines in an intein precursor

Protein splicing is a self-catalyzed and spontaneous post-translational process in which inteins excise themselves out of precursor proteins while the exteins are ligated together. We report the first discovery of an intramolecular disulfide bond between the two active-site cysteines, Cys1 and Cys+1, in an intein precursor composed of the hyperthermophilic Pyrococcus abyssi PolII intein and extein. The existence of this intramolecular disulfide bond is demonstrated by the effect of reducing agents on the precursor, mutagenesis, and liquid chromatography-mass spectrometry (LC-MS) with tandem MS (MS/MS) of the tryptic peptide containing the intramolecular disulfide bond. The disulfide bond inhibits protein splicing, and splicing can be induced by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP). The stability of the intramolecular disulfide bond is enhanced by electrostatic interactions between the N- and C-exteins but is reduced by elevated temperature. The presence of this intramolecular disulfide bond may contribute to the redox control of splicing activity in hypoxia and at low temperature and point to the intriguing possibility that inteins may act as switches to control extein function.     

Label-Free Proteomic Identification of Endogenous,Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

Protein–protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein–protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein–protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.