A two-dimensional protein map of Caenorhabditis elegans

A protein map of Caenorhabditis elegans was constructed by using two-dimensional gel electrophoresis followed by peptide mass fingerprinting. A whole worm extract of a mixed population was separated on immobilized pH gradient strips 4-7 L, 3-10 NL, 6-11 L and 12% sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) gels. Gels were stained with colloidal Coomassie blue and 286 spots representing 152 proteins were subsequently identified by matrix-assisted laser desorption/ionization-mass spectrometry after in-gel digestion with trypsin. Most of the identified proteins with known cellular function were enzymes related to carbohydrate and lipid metabolism, or structural proteins with subcellular locations in the cytoplasm, mitochondria or cytoskeleton.     

Separation and identification of photosynthetic antenna membrane proteins by high- performance liquid chromatography electrospray ionization mass spectrometry

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.     

Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis

Peptide mass fingerprinting (PMF) is a powerful tool for identification of proteins separated by two-dimensional electrophoresis (2-DE). With the increase in sensitivity of peptide mass determination it becomes obvious that even spots looking well separated on a 2-DE gel may consist of several proteins. As a result the number of mass peaks in PMFs increased dramatically leaving many unassigned after a first database search. A number of these are caused by experiment-specific contaminants or by neighbor spots, as well as by additional proteins or post-translational modifications. To understand the complete protein composition of a spot we suggest an iterative procedure based on large numbers of PMFs, exemplified by PMFs of 480 Helicobacter pylori protein spots. Three key iterations were applied: (1) Elimination of contaminant mass peaks determined by MS-Screener (a software developed for this purpose) followed by reanalysis; (2) neighbor spot mass peak determination by cluster analysis, elimination from the peak list and repeated search; (3) re-evaluation of contaminant peaks. The quality of the identification was improved and spots previously unidentified were assigned to proteins. Eight additional spots were identified with this procedure, increasing the total number of identified spots to 455.     

Assessing matrix assisted laser desorption/ ionization-time of flight-mass spectrometry as a means of rapid embryo protein identification in rice.

Rice embryo proteins were separated by two-dimensional gel electrophoresis (2-DE). A total of 105 spots were digested with trypsin and the resultant peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Raw mass spectra were fully-automatically processed and searched with selected monoisotopic masses against SWISS-PROT/TrEMBL and NCBInr databases. High quality mass spectra were obtained from 53 spots, of which 36 spots were identified including 29 not registered in databases. Fifty percent of the rice embryo proteins resolved in 2-DE could not be identified, indicating more efficient sample preparation techniques need to be developed in the future. At least four to five matching peptides were found to be essential for unambiguous identification of rice embryo proteins; peptide matching of less than four lead to ambiguous results. The suitability of peptide mass fingerprinting method as a means of rapid embryo protein identification in rice was discussed.     

中国小型猪骨髓间充质干细胞的蛋白质组研究

报道了骨髓间充质干细胞(MSCs)的蛋白质组表达研究。从体外培养的MSCs提取细胞蛋白,经二维电泳分离后用银染方法可检出蛋白点约1600个,选取48个蛋白点进行胶内酶解及质谱分析,经数据库检索成功鉴定了37个蛋白,并对蛋白功能进行初步分析。本实验数据为进一步分析MSCs增殖、分化或凋亡的分子机理提供相关信息。     

Protein profile of the HeLa cell line

HeLa cells are widely used for all kinds of in vitro studies in biochemistry, biology and medicine. Knowledge on protein expression is limited and no comprehensive study on the proteome of this cell type has been reported so far. We applied proteomics technologies to analyze the proteins of the HeLa cell line. The proteins were analyzed by two-dimensional (2D) gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MS) on the basis of peptide mass fingerprinting, following in-gel digestion with trypsin. Approximately 3000 spots, excised from six two-dimensional gels, were analyzed. The analysis resulted in the identification of about 1200 proteins that were the products of 297 different genes. The HeLa cell database includes proteins with important functions and unknown functions, representing today one of the largest two-dimensional databases for eukaryotic proteomes and forming the basis for future expressional studies at the protein level.     

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.     

Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.     

Differential Proteins of the Optic Ganglion in Octopus vulgaris Under Methanol Stress Revealed Using Proteomics

An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.     

Structure and dynamics of photosystem II light-harvesting complex revealed by high-resolution FTICR mass spectrometric proteome analysis

Structure and dynamics of membrane-bound light-harvesting pigment-protein complexes (LHCs), which collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identified and characterized using high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LHCs from photosystem II (LHCII) were isolated from the thylakoid membrane of Arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel-filtration into LHCII subcomplexes. Using reversed-phase high-performance liquid chromatography and two-dimensional (2D) gel electrophoresis, the LHCII proteins, Lhcb1-6 and fibrillins, were efficiently separated and identified by FTICR-MS. Some of the LHCII subcomplexes were shown to migrate from photosystem II to photosystem I as a result of short-term adaptation to changes in light intensity. In the mobile LHCII subcomplexes, decreased levels of fibrillins and a modified composition of LHCII protein isoforms were identified compared to the tightly bound LHCII subcomplexes. In addition, FTICR-MS analysis revealed several oxidative modifications of LHCII proteins. A number of protein spots in 2D gels were found to contain a mixture of proteins, illustrating the feasibility of high-resolution mass spectrometry to identify proteins that remain unseparated in 2D gels even upon extended pH gradients.     

Capillary electrophoresis/tandem mass spectrometry for analysis of proteins from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis

Capillary electrophoresis/time-of-flight mass spectrometry(CE/TOFMS) has been used for analysis of in-gel digests of protein spots excised from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). An off-line purification and preconcentration procedure with a Zip Tip is used before CE/TOFMS analysis which allows for detection of protein spots with <1 picomole of material from 2-D gels. The off-line procedure provides sufficient purification for analysis while maintaining the quality of the CE separation. Using this procedure, several proteins from Coomassie Blue and zinc negatively stained gels are identified by the peptide maps generated and database searching. CE/TOF tandem mass spectrometry is used for the confirmation of database searching results and structural analysis of peptides that do not match the expected peptide maps obtained from the database in order to identify structural modifications. Several modifications were pinpointed and identified by this method.     

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO red staining

The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies.     

Efficient peptide mapping and its application to identify embryo proteins in rice proteome analysis

Using direct N-terminal analysis, only 31 N-terminally unblocked proteins out of 100 rice embryo proteins could be identified. To obtain protein sequence information for the remaining 69 blocked proteins, we developed a simple, efficient and rapid method. Using this method, we determined the peptide maps of 20 proteins per day in 10 pmol amounts. Applying this method to rice proteome analysis, we determined the internal sequences of all 69 blocked proteins. A total of 28 proteins out of 100 analyzed showed sequence similarity to the proteins with known functions in the SWISS-PROT and NCBI databases. Alternatively, we also used peptide mass fingerprinting determined by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to identify the rice proteins separated by two-dimensional electrophoresis (2-DE). Although peptide-mass fingerprinting is a high-throughput method, we could not easily identify all the rice proteins or genes by this method, because the complete database information on rice, is not yet available and many proteins are post-translationally modified. Therefore, at present, the improved peptide mapping method as we report here is considered to be very useful in rice proteome analysis, especially for blocked proteins.     

蛋白质组学技术鉴定与分析三七粉诱导海兔神经连索表达的差异蛋白质

RP-HPLC分离三七粉提取液,并鉴定含有Rb1、Rg1、Re、R1等皂甙成分。以蓝斑背肛海兔(Notarcusleachii cirrosus Stimpson,NLCS)为分析模型,三七粉提取液为诱导剂,选用蛋白质组技术研究NLCS神经连索诱导前后所表达的差异蛋白质。通过优化双向凝胶电泳分离NLCS神经连索全蛋白质组技术,获得496个蛋白质斑点。采用肽指纹图谱技术和数据库检索比对法,初步鉴定了NLCS受三七粉提取液诱导前后,其神经连索表达13个差异蛋白质,其中较高的匹配率蛋白质为肌动蛋白、3-羟酯酰辅酶A脱氢酶、ATP结合转运子和甲基转移酶12。选用LOC tree软件对13个差异蛋白质进行亚细胞定位,认为它们在保护神经系统中发挥重要的调节作用。     

Thylakoid membrane at altered metabolic state: challenging the forgotten realms of the proteome

Analysis of the membrane integral proteome is mainly dependent on the ability of protein separation. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique capable of efficient membrane protein separation, so far mainly applied to the mitochondrial oxidative phosphorylation machinery. Applying BN-PAGE to the thylakoid membranes after mild solubilization with digitonin we succeeded in displaying the response of the green algae Chlamydomonas reinhardtii to altered culture conditions. In addition, by peptide mass fingerprinting and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) extremely hydrophobic subunits of the photosystem complexes with 5-11 transmembrane helices were identified, which could not be accessed by in-gel digestion in previous studies.     

Proteome analysis of the Chlamydia pneumoniae elementary body

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniae cultivated in Hep-2 cells were 35S-labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two-dimensional gel electrophoresis. Excised protein spots were in-gel digested with trypsin and peptides were concentrated on reverse-phase chromatographic beads for identification analysis by matrix-assisted laser desorption/ionization-mass spectrometry. In the pH range from 3-11, 263 C. pneumoniae protein spots encoded from 167 genes were identified. These genes constitute 15% of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.     

人脑枕叶区衰老进程的比较蛋白质组学研究

分别从23岁、64岁、72岁、83岁以及94岁无神经性和精神性疾病史个体大脑皮层枕叶区取样．制备蛋白质样晶．进行双向凝胶电泳(2-DE)、考马斯亮蓝染色、凝胶扫描和Image Master 2D Elite软件分析，每张胶上平均可检测到1000个以上蛋白质点．通过软件半定量分析．进一步研究了衰老过程中枕叶蛋白质的差异表达，发现随年龄增长有7种蛋白质有一致的显著上升或下降趋势．应用质谱进行肽质量指纹图谱(PMF)和／或肽序列标签(PST)分析．数据库检索共鉴定了11种蛋白质．其中有5种具有一致的上调或下调性．包括神经元突触结构蛋白低分子量神经丝蛋白(Neurofilament triplet L protein．NF—L)、参与抗氧化反应的硫氧还原蛋白过氧化物酶(Peroxiredoxin)、三羧酸循环关键酶(顺)乌头酸水合酶(Aconitate hydratase)和糖代谢途径中的关键酶烯醇化酶2(Enolase 2)以及分子伴侣蛋白T复合物蛋白l(T-complex protein 1)．首次建立了正常人脑枕叶区的双向电泳蛋白质表达图谱．针对人脑枕叶区蛋白质在衰老过程中的差异表达进行了研究，并对差异表达蛋白在衰老进程中可能的生物学意义做了探讨．     

Proteomic analysis of the Arabidopsis thaliana cell wall

With the completion of the Arabidopsis genome, many hypothetical proteins have been predicted without any information on their expression, subcellular localisation and function. We have performed proteomic analysis of proteins sequentially extracted from enriched Arabidopsis cell wall fractions and separated by two-dimensional gel electrophoresis (2-DE). The proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and genomic database searches. This is part of a targeted exercise to establish the entire Arabidopsis secretome database. We report evidence for new proteins of unknown function whose existence had been predicted from genomic sequences and, furthermore, localise them to the cell wall. In addition, we observed an unexpected presence in the cell wall preparations of proteins whose known biochemical activity has never been associated with this compartment hitherto. We discuss the implications of these findings and present results suggesting a possible involvement of cell wall kinases in plant responses to pathogen attack.     

Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry

Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis. The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis. In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated. A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2. The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1). Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.