蛋白质组学技术及其在生物医学上的应用

蛋白质组学部分承用了创立于二十多年前的二维电泳技术。基于其高分辩能力 ,二维电泳主要用于分离和检测复杂混合物中的蛋白质。虽然没有获得更多的改进 ,但是二维电泳结合了通过质谱测定蛋白质的最新进展而成为蛋白质组学中的一项重要技术。随着人类基因组计划项目的完成及由此而产生的大量基因数据库和使用这些数据的生物信息技术 ,科学家们的下一个目标是解析生物体的完整蛋白质组 ,把蛋白质组学数据与基因组学数据关联起来并有机地结合而成为一项有力的工具以阐明病理学中的蛋白质功能、衰老的过程及发现新药目标蛋白质和疾病标识物等。文章综述了蛋白质组学技术的最新知识及其在生物医学研究中的潜在应用     

Binding of copper (II) ion to an Alzheimer's tau peptide as revealed by MALDI-TOF MS, CD, and NMR

The tau protein plays an important role in some neurodegenerative diseases including Alzheimer's disease (AD). Neurofibrillary tangles (NFTs), a biological marker for AD, are aggregates of bundles of paired helical filaments (PHFs). In general, the alpha-sheet structure favors aberrant protein aggregates. However, some reports have shown that the alpha-helix structure is capable of triggering the formation of aberrant tau protein aggregates and PHFs have a high alpha-helix content. In addition, the third repeat fragment in the four-repeat microtubule-binding domain of the tau protein (residues 306-336: VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ, according to the longest tau protein) adopts a helical structure in trifluoroethanol (TFE) and may be a self-assembly model in the tau protein. In the human brain, there is a very small quantity of copper, which performs an important function. In our study, by means of matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy, the binding properties of copper (II) ion to the R3 peptide derived from the third repeat fragment (residues 318-335: VTSKCGSLGNIHHKPGGG) have been investigated. The results show that copper ions bind to the R3 peptide. CD spectra, ultraviolet (UV)-visible absorption spectra, and MALDI-TOF MS show pH dependence and stoichiometry of Cu2+ binding. Furthermore, CD spectra and NMR spectroscopy elucidate the copper binding sites located in the R3 peptide. Finally, CD spectra reveal that the R3 peptide adopts a mixture structure of random structures, alpha-helices, and beta-turns in aqueous solutions at physiological pH. At pH 7.5, the addition of 0.25 mol eq of Cu2+ induces the conformational change from the mixture mentioned above to a monomeric helical structure, and a beta-sheet structure forms in the presence of 1 mol eq of Cu2+. As alpha-helix and beta-sheet structures are responsible for the formation of PHFs, it is hypothesized that Cu2+ is an inducer of self-assembly of the R3 peptide and makes the R3 peptide form a structure like PHF. Hence, it is postulated that Cu2+ plays an important role in the aggregation of the R3 peptide and tau protein and that copper (II) binding may be another possible involvement in AD.     

蛋白质组研究中肽质量指纹谱鉴定方法的建立及应用

建立了用肽质量指纹谱和数据库检索方法鉴定凝胶电泳分离蛋白南的方法。用标准蛋白质对胶上蛋白５质原位酶切制备肽谱的方法进行了讨论。分析了实际细胞蛋白质样品,获得双向电泳分离的人肺癌细胞蛋白质谱中三个蛋白质点的肽指纹谱。并通过数据７库检索分别鉴定为甘油醛－３－磷酸脱氢酶－２,测在蛋白羟基末端水解同工酶和丙糖磷酸异构酶。     

Comparative Analysis of the Endosperm Proteins Separated by 2-D Electrophoresis for Two Cultivars of Hybrid Rice （Oryza sativa L,）

Liangyoupeijiu is a two-parental-line, and Shanyou63 is a three-parental-line hybrid rice （Oryza sativa L.）. Although both belong to the indica subspecies, they have obvious differences with respect to morphology, physiology and grain quality. Variations in endosperm protein compositions were studied by comparing the 2-D electrophoresis （2-DE） maps for these two cultivars of hybrid rice. After matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF/MS） analysis, a 21-kDa precursor of 19- kDa globulin was identified as the major storage protein for both cultivars. Some isoforms of peroxiredoxin and seed maturation protein were found to only exist in Shanyou63, whereas aldose reductase and starch granule-bound starch synthase were only detected in Liangyoupeijiu. These data might provide a foundation for further comparative studies of these two cultivars of hybrid rice.     

三种前处理在基质辅助激光解析电离飞行时间质谱鉴定假丝酵母菌属中的应用比较

目的 比较3种前处理方法在基质辅助激光解析电离飞行时间质谱(MALDI TOF MS)鉴定假丝酵母菌属中的结果可靠性。 方法 以ITS测序鉴定结果为金标准,对临床分离的66株假丝酵母分别采用传统直涂法、改良直涂法和甲酸-乙腈蛋白提取法进行前处理,MALDI TOF MS鉴定,比较3种方法的Biotyper Log值,分析质谱图的差异。 结果 传统直涂法、改良直涂法和甲酸-乙腈提取法对66株假丝酵母的属水平鉴定率分别为48.5%、50.0%和97.0%,Biotyper Log均值分别为1.628、1.674和2.010,其中甲酸-乙腈提取法对66株假丝酵母的种水平鉴定率为53.0%。甲酸-乙腈提取法得到的质谱图比另2种方法的质谱图离子峰更加密集,图像更复杂,鉴定结果可信度更高。 结论 甲酸-乙腈蛋白提取法对假丝酵母菌属的鉴定成功率和可靠性明显高于传统直涂法和改良直涂法,对临床假丝酵母菌病的准确诊断具有重要价值。     

Pten^＋/＋MEFs与Pten^-/-MEFs细胞系的差异表达蛋白质组分析

目的：研究PTEN缺失细胞的蛋白表达规律。方法：用双向电泳技术比较Pten^＋/＋MEFs与Pten^-/-EFs细胞的蛋白表达差异;用基质辅助激光解吸／电离飞行时间质谱（MALDI-TOF-MS）对差异蛋白进行质谱分析;用肽质量指纹图谱检索数据库对差异蛋白进行鉴定;用Northern印迹和Western印迹验证蛋白的差异表达。结果：与Pten^＋/＋MEFs细胞相比。Pten^-/-MEFs细胞有明显的差异性蛋白表达谱,Pten^-/-MEFs细胞中表达下降的蛋白有铜锌超氧化物歧化酶、过氧化物氧化还原酶5和6等,表达增加的蛋白有低分子量蛋白酪氨酸磷酸酶和丝切蛋白（cofflin）1。结论：PTEN的缺失引起细胞内多种蛋白表达改变,这些表达改变的蛋白可能与PTEN缺失后细胞癌变相关。     

Improvements for the visualization of low-molecular weight protein and peptides of human tears using MALDI

Many low-molecular weight proteins and peptides in human tears are potentially bioactive proteins but the range and number of these is yet to be fully characterized. A number of different sample preparation techniques were used to maximize the visualization of peptides from reflex tears. Samples were pretreated using precipitation and filtration techniques prior to analyses using MALDI-TOF MS. Peptides were searched for between 700 to 4000 m/z. Sample dilution in several different buffer systems followed by filtration with a 30-kDa cutoff filter and C18 reverse phase microcolumn purification produced significantly (p = 0.049) more peaks in tears than other methods used to prepare tears prior to MALDI-TOF MS. This study has established a technique for optimizing the visualization of naturally occurring peptides in tears.     

铜绿假单胞菌的MALDI-TOF-MS检测方法的建立

目的 建立利用基质辅助激光解吸电离飞行时间质谱仪( MALDI-TOF-MS)对铜绿假单胞菌的快速检测方法.方法 通过MALDI-TOF-MS法对铜绿假单胞菌进行检测分析,并与生化鉴定方法相比较.结果 MALDI-TOF-MS对铜绿假单胞菌的检测后得到肽指纹图片及相关质谱数据,建立MALDI-TOF-MS对铜绿假单胞菌的快速检测方法.结论 MALDI-TOF-MS方法检测铜绿假单胞菌准确快速、操作简单等特点,可发展成为食品检验铜绿假单胞菌的重要(辅助)工具.     

Use of diethyl squarate for the coupling of oligosaccharide amines to carrier proteins and characterization of the resulting neoglycoproteins by MALDI-TOF mass spectrometry

The 8-methoxycarbonyloctyl glycosides of GlcNAc, Gal1-4Glc, Fuc1-2Fuc1-3GalNac and Fuc1-2Gal1-3[Fuc1-4]GlcNac were converted to primary amines by reaction with neat ethylenediamine and then coupled to bovine serum albumin (BSA) using diethyl squarate as the connector. The average degree of incorporation of the sugar onto the protein, as well as the molecular weight distribution, could be conveniently determined using matrix assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry thus avoiding cumbersome structure-dependent colour-tests or analysis of cleaved ligand. The present coupling method has the advantages of proceeding under very mild conditions, yielding controlled incorporation values and can reliably be used for the coupling of very small amounts (mg) of oligosaccharide.This paper is dedicated to Sen-itiroh Hakomori on the occasion of his 65th birthday     

Chemical derivatization of phosphoserine and phosphothreonine containing peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS analysis

Protein phosphorylation is one of the most important and common ways of regulating protein function in cells. However, phosphopeptides are difficult to analyse, ionising poorly under standard MALDI conditions. Several methods have been developed to deal with the low sensitivity and specificity of phosphopeptide analysis. Here, we show an approach using a simple one-step beta-elimination/Michael addition reaction for the derivatization of phosphoserine and phosphothreonine. The substitution of the negatively charged phosphate group by a positively charged S-ethylpyridyl group greatly improves the ionisation of the modified peptides, especially in MALDI MS, increasing the sensitivity of the analysis. The modification allows the formation of a unique fragment ion at m/z 106 under mild collisional activation conditions, which can be used for parent (precursor) ion scanning in order to improve both the sensitivity and the selectivity of the analysis. The optimisation of the approach is described for a standard model peptide and protein and then applied to phosphorylation analysis in two biologically derived proteins purified from different experimental systems.     

Differential proteomic analysis during the vegetative phase change and the floral transition in Malus domestica

In order to identify the proteomic changes of apple (Malus domestica Borkh.) during the vegetative phase change and the floral transition, leaf protein of juvenile, adult vegetative and reproductive phase in a seedling ('Jonathan' × 'Golden Delicious') was extracted and analyzed by 2-D electrophoresis and Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Seventy two gel spots with significant expression differences between ontogenetic phases were obtained. Five protein spots were only detected in leaves of juvenile phase and 11 were not; 17 spots were found exclusively in adult vegetative leaves; and only one spot solely appeared in reproductive leaves while 12 did not. Twenty six of the differentially expressed proteins identified were involved in photosynthesis. Seven enzymes were related to respiration and carbohydrate metabolism. Fifteen other proteins also presented qualitative or quantitative differences among developmental phases. The spatial distribution of one differentially expressed protein, serine hydroxymethyltransferase, was confirmed by enzyme linked immunosorbent assay and immunohistochemistry. These results strongly support the idea that the vegetative phase change and the floral transition are regulated independently during developmental process.     

正常与重金属铅注射的兔脑蛋白质双向电泳图谱比较与鉴定

重金属污染对人类健康的威胁日益受到关注,为了了解大量重金属摄入对脑蛋白质的影响,对比研究了正常兔脑组织蛋白质与重金属铅腹腔注射2周后的兔脑组织在蛋白质双向电泳图谱中的差异,分析重金属注射对脑蛋白质表达的可能影响.通过对脑组织蛋白质的提取,分离出水溶性的蛋白质组分,经双向电泳图谱比较正常与注射重金属铅的兔子在脑蛋白质表达上的差异,其中3个蛋白质斑点经提取,反相高效液相色谱(RP-HPLC)分离,基质辅助激光解析电离质谱(MALDI-TOF MS)确定了分子质量,并利用肽质量指纹图谱检索数据库确定蛋白质的归属.实验结果表明正常兔脑与金属铅注射的兔脑在水溶性蛋白质的表达上具有显著性差异.     

Proteomic comparison between interphase and metaphase of isolated chloroplasts of Cyanidioschyzon merolae (Cyanidiophyceae,Rhodophyta)

With the completion of the Cyanidioschyzon merolae genome project, detailed analysis of organelle proteins with mass spectrometry has now become possible. Chloroplasts of the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei et Varano (Rhodophyta, Cyanidiophyceae) were isolated from synchronized culture and the chloroplast lysates of both interphase and metaphase cells were prepared and subjected to 2D‐polyacrylamide gel electrophoresis. A total of 355 spots (170 identical spots) were recognized and quantified and then analyzed using mass spectrometry. A total of 105 proteins were identified, including 18 proteins for posttranslational functions, 17 photosynthesis‐related proteins, 10 carbohydrate‐related proteins, 15 proteins of unknown functions, and eight proteins predicted to be contaminated from other organelles. On the basis of spot quantity, photosynthesis‐related proteins were most dominant (45.3% in interphase and 56.4% in metaphase). In particular, the proteins forming phycobilisomal complexes were abundant. Comparison of interphase with metaphase revealed that CMG086C (aminomethyltransferase) notably increased in interphase. CMN235C (similar to chlorophyll a/b‐binding protein, CP24) increased in metaphase in agreement with a previously performed microarray analysis. Both CMQ295C (cell division protein FtsH) and CMS004C (plastid division protein FtsZ) increased in interphase. Seven proteins were detected to be interphase‐specific, and 12 proteins were metaphase‐specific. Proteins of unknown functions were poorly characterized by homology search, although thioredoxin‐like domains were predicted in several proteins.     

慢性阻塞性肺疾病与非慢性阻塞性肺疾病吸烟者肺组织蛋白质组学比较分析

吸烟是导致慢性阻塞性肺疾病（COPD）发生发展的主要原因之一.但吸烟者是否发生COPD存在个体差异,其机制尚未完全阐明.蛋白质组学研究能够高效率发现疾病相关蛋白并为深入研究疾病的发病机制提供线索.本研究运用二维凝胶电泳（2-DE）和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)蛋白质组学研究方法结合生物信息学数据,对吸烟COPD患者和非COPD吸烟者肺组织表达的差异蛋白进行筛选和鉴定,共鉴定出24种差异蛋白,涉及基本代谢酶类、氧化应激相关酶类、凝血/纤溶相关蛋白、蛋白降解相关酶以及细胞生长分化相关蛋白等.本研究结果为进一步探索COPD的发病机制提供了新的线索.     

Identification of differentially expressed proteins in poplar leaves induced by Marssonina brunnea f. Sp. Multigermtubi

Black spot disease in poplar is a disease of the leaf caused by fungus. The major pathogen is Marssonina brunnea f. sp. multigermtubi. To date, little is known about the molecular mechanism of poplar （M. brunnea） interaction. In order to identify the proteins related to disease resistance and understand its molecular basis, the clone ＂NL895＂ （P. euramericana CL＂NL895＂）, which is highly resistant to M. brunnea f. sp. multigermtubi, was used in this study. We used two-dimensional gel electrophoresis （2-DE） and mass spectrometry （MS） to identify the proteins in poplar leaves that were differentially expressed in response to black spot disease pathogen, M. brunnea f. sp. multigermtubi. Proteins extracted from poplar leaves at 0, 12, 24, 48, and 72 h after pathogen-inoculation were separated by 2-DE, About 500 reproducible protein spots were detected, of which 40 protein spots displayed differential expression in levels and were subjected to Matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS） followed by database searching. According to the function, the identified proteins were sorted into five categories, that is, protein synthesis, metabolism, defense response and unclassified proteins.     

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.     

基质辅助激光解吸电离飞行时间质谱技术用于常见益生菌鉴定的初步研究

目的建立基质辅助激光解吸电离飞行时间质谱(MADLI-TOF MS)技术鉴定常见益生菌的实验方法并对MADLI-TOF MS技术的适用性进行初步评价。方法对MADLI-TOF MS技术鉴定常见益生菌过程中各影响因素进行考察,筛选出最佳的实验条件。利用19株供试菌株所得的蛋白指纹图谱对MADLI-TOF MS技术的适用性进行研究。结果建立了MADLI-TOF MS技术鉴定常见益生菌的最佳实验方法。初步证明MADLI-TOF MS技术具备在属、种、亚种以及菌株水平上鉴定常见益生菌的能力。结论建立的实验方法稳定性高、重复性好,可以作为MADLI-TOF MS技术鉴定常见益生菌的参考方法。MADLI-TOF MS技术可以作为常见益生菌鉴定的方法之一。     

Proteomic analysis of differential protein expression in response to epidermal growth factor in neonatal porcine pancreatic cell monolayers

We have proposed that porcine neonatal pancreatic cell clusters (NPCCs) may be a useful alternative source of cells for islet transplantation, and that monolayer cultures might provide an opportunity to manipulate the cells before transplantation. In addition we previously identified 10 genes up-regulated by epidermal growth factor (EGF) in cultured porcine NPCC monolayers. We have now analyzed the intracellular signaling pathways activated by EGF and searched for proteins differentially expressed following EGF treatment of the monolayers, using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). EGF treatment resulted in phosphorylation of both Erk 1/2 and Akt, as well as increased cell proliferation. Five unknown and 13 previously identified proteins were differentially expressed in response to EGF. EGF treatment increased the expression of several structural proteins of epithelial cells, such as cytokeratin 19 and plakoglobin, whereas vimentin, the intermediate filament protein of mesenchymal cells, and non-muscle myosin alkali chain isoform 1, decreased. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 factor, which promotes epithelial cell proliferation, and hemoglobin alpha I & II also increased, whereas cyclin A1, immunoglobulin heavy chain, apolipoprotein A1, 5,10-ethylenetetrahydrofolated reductase (5,10-MTHFR), angiotensin-converting enzyme 2 (ACE2), co-lipase II precursor, and NAD+ isocitrate dehydrogenase (NAD+ IDH) alpha chain proteins decreased. Our results show that EGF stimulates proliferation of pancreatic epithelial cells by simultaneously activating the MAPK and PI-3K pathways. HnRNP A2/B1, hemoglobin, cyclin A1, and ACE2 may play roles in the proliferation of epithelial cells in response to EGF.