Serum acute phase response (APR)-related proteome of loach to trauma

Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-DE PAGE) together with mass spectrometry was applied to screen acute phase response (APR)-related proteins in serum from loach following injury. Six APR-related proteins were identified, in which apolipoprotein, cathepsin, C-reactive protein (CRP) were known APP, while signal recognition protein (SRP), gastrin 71 and parvalbumin were new APR-related proteins.     

Proteome profile of human urine with two-dimensional liquid phase fractionation

Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.     

短乳杆菌DM9218核苷酸代谢过程中的蛋白组学分析

目的探讨短乳杆菌DM9218在核苷酸代谢过程中的蛋白表达差异。方法分别提取DM9218菌株与底物(肌苷+鸟苷)反应前后的菌体蛋白,利用蛋白双向凝胶电泳(2-DE)技术,找出该菌株与底物反应前后的差异蛋白质点,选取其中差异变化较大的蛋白点进一步做蛋白质谱分析。结果 2-DE分析显示两样品蛋白点主要分布在等电点4~9和分子量11~90 kD范围内,将所得的蛋白点结合其蛋白得率、浓度、储存蛋白含量进行比较,得到匹配的蛋白点数为732个。从中选取14个差异显著的蛋白点进行质谱分析,质谱结果显示所选取蛋白质点主要与物质代谢、能量转换及基因水平转录和翻译等生物学功能密切相关。结论本研究为后期分析研究短乳杆菌DM9218在核苷酸代谢过程中蛋白的表达奠定了基础。     

Comparative proteomics analysis of serum proteins in ulcerative colitis patients

In the present study, we investigated the differentially expressed proteins associated with ulcerative colitis (UC) using proteomic methods. Two-dimensional electrophoresis (2-DE) technology was performed to separate the total proteins of ulcerative tissues from those of the normal tissues of UC patients. PDQuest software was applied to analyze the obtained 2-DE images. Candidate protein spots between the two groups were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and bioinformatics analysis. The well resolution and reproducible 2-DE patterns of UC and normal tissues were established. Of the 12 differentially expressed proteins, 9 were successfully identified, of which 6 proteins were up-regulated including apolipoprotein C-III, haptoglobin, receptor tyrosine kinase, aldehyde reductase, pericentriolar material 1, and heat shock factor protein 2, and 3 were down-regulated including keratin, filamin A-interacting protein 1, and tropomyosin 3. These identified proteins were related to hormonal modulation, immune response, oxidative stress, and signal conduction. The 2-DE protein expression profile of the UC tissues displays an obvious difference from that of the normal controls. Various proteins may be involved in the occurrence of UC.     

Proteomic analysis of differential protein expression in platelets of septic patients

Sepsis is one of the major health problems all over the world. Early diagnostic of sepsis is an attractive strategy to decrease the mortality of septic patients. However, an effective biomarker that fulfills all the necessary requirements for the accurate characterization of sepsis is still unavailable until now. In this study, the 2-DE technique followed by mass spectrometry and a database search was used for searching and identifying the differential expressed proteins in platelets between septic patients and paired healthy controls. Platelet 2-DE profiles of septic patients and paired healthy controls with high resolution and reproducibility were obtained. Differential platelet 2-DE profiles between septic patients and paired healthy controls were established. Differential protein spots between normal healthy volunteers and septic patients from platelet 2-DE profiles were identified by 2-DE followed with mass spectrometry and a database search. Five proteins with increased expression were identified between septic patients and healthy controls from platelet samples. These up-expressed proteins were EF-hand calcium-binding domain-containing protein 7, actin, interleukin-1β, glycoprotein IX, and glycoprotein IIB. Sepsis induces a complex regulation of platelet protein changes. Our study highlights the important role of these differential expressed proteins in sepsis, which deserve further research as potential candidates for therapeutic strategies. Furthermore, our research is beneficial for the future developments of sepsis diagnosis and therapy.     

Strain-dependent expression of metabolic proteins in the mouse hippocampus

Individual mouse strains differ significantly in terms of behavior and cognitive function. Strain-specific variation of metabolic protein levels in the hippocampus among various commonly used mouse strains, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry [high capacity ion trap (HCT)] has been chosen to address this question by determining strain-dependent levels of metabolic proteins in hippocampal tissue of four inbred and one outbred mouse strain. Statistical analysis of protein spots on 2-DE gels of the individual strains (n = 10) revealed significant strain-dependent differences in densities of 39 spots. Subsequent HCT analysis led to the identification of 22 different metabolic proteins presenting with differential protein levels among the five mouse strains investigated. Among those are proteins concerned with the metabolism of amino acid, nucleic acid, carbohydrate and energy. Moreover, proteins known to play a pivotal role in the processes of learning and memory, such as calcium/calmodulin-dependent protein kinase type II alpha chain, were found to present with significant inter-strain variability, which is also in agreement with our previous reports. Strain-specific protein levels of metabolic proteins in the mouse hippocampus may provide some insight into the molecular underpinnings and genetic determination of strain-dependent neuronal function. Furthermore, data presented herein emphasize the significance of the genetic background for the analysis of metabolic pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments.     

Identification of tumor-associated plasma biomarkers using proteomic techniques: from mouse to human

In an effort to identify tumor-associated proteins from plasma of tumor-bearing mice that may be used as diagnostic biomarkers, we developed a strategy that combines a tumor xenotransplantation model in nude mice with comparative proteomic technology. Five human cancer cell lines (SC-M1, HONE-1, CC-M1, OECM1, GBM 8401) derived from stomach, nasopharyngeal, colon, oral and brain cancers were subcutaneously inoculated into nude mice and compared to control nude mice injected with phosphate-buffered saline. One month later, plasma from mice inoculated with cancer cells was collected for proteomic analysis using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Comparison of plasma 2-DE maps from tumor-bearing mice with those produced from control mice revealed the overexpression of several mouse acute phase proteins (APPs) such as haptoglobin. Another APP, serum amyloid A (SAA), was found only in mice bearing tumors induced by the stomach cancer cell line SC-M1, which has not previously been demonstrated in xenotransplatation experiment. Furthermore, by using immunohistochemistry, SAA and haptoglobin were found to originate from the mouse hosts and not from the human cancer cell line donors. The protein alterations were further confirmed on patients with stomach cancers where up-regulated levels of SAA were also observed. These results indicate that APPs may be used as nonspecific tumor-associated serum markers. SAA in particular may serve as a potential marker for detecting stomach cancer. Taken together, the combination of the xenotransplatation model in nude mice and proteomics analysis provided a valuable impact for clinical applications in cancer diagnostics. In addition, our findings demonstrate that a panel of APPs might serve as screening biomarkers for early cancer detection.     

柔嫩艾美耳球虫地克株利抗药株与敏感株孢子化卵囊的蛋白质差异分析

利用双向电泳技术,对本实验室诱导保存的柔嫩艾美耳球虫地克株利抗药株与敏感株的蛋白质表达图谱进行差异比较和分析,发现两者之间差异有5个蛋白质斑点,利用MALDI_TOF_TOF质谱技术对其中4个差异明显的蛋白质斑点进行分析鉴定,获得4个明确的肽质量指纹图谱,通过在NCBInr数据库中检索分析,确定了其中2个蛋白质分别为球虫子孢子表面抗原TA4和热休克蛋白Hsp70 ,另外两种为真核细胞的功能蛋白。上述蛋白的鉴定将对球虫的抗药性产生机理和柔嫩艾美耳球虫地克株利抗药株的分子标志物提供了研究方向。     

橡胶树死皮病胶乳C-乳清差异表达蛋白质的筛选与鉴定

橡胶树死皮病(Tapping Panel Dryness,TPD)在世界各橡胶种植园普遍发生,给橡胶种植业带来严重的危害。为了更好地了解和阐明死皮病发生、发展的分子机制,本研究应用双向凝胶电泳技术（2-DE）比较橡胶树死皮株与健康株胶乳C-乳清蛋白质组表达的差异。采用固相pH梯度双向凝胶电泳分离橡胶树死皮株与健康株C-乳清的总蛋白质,凝胶经考染显色后,用PDQuest7.40图像分析软件进行比较分析,识别差异表达的蛋白质。这些点经过胶内酶切后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质指纹图谱(PMF),Mascot软件搜索SWISS-PROT和NCBInr数据库鉴定蛋白质。结果：①橡胶树死皮株与健康株C-乳清凝胶的平均蛋白质点数分别为1075±35和1134±27,其平均匹配的点数分别为982±38和1008±22,组内图像匹配率达91.89﹪和88.72﹪。②橡胶树死皮株与健康株C-乳清组间的平均匹配蛋白点数为970±25。利用MALDI-TOF-MS质谱技术对40个差异明显的蛋白点进行分析鉴定,通过查询数据库鉴定了27个蛋白质。本研究建立了分辨率高且重复性较好的橡胶树死皮株与 健康株胶乳C-乳清的双向凝胶电泳图谱,并应用质谱技术鉴定了其中表达差异的蛋白质点,这些差异表达的蛋白质可能参与了死皮发生和发展的过程。     

Prefractionation of protein samples for proteome analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

To isolate high molecular weight (HMW) or low-abundance proteins we exploited the high resolving power provided by the molecular sieves of polyacrylamide gel matrices. Rice-leaf protein extracts were applied to a single well of an SDS-polyacrylamide gel with prestained molecular size markers at both ends. After electrophoresis, the gel was cut into 4 segments according to size, and each segment was ground in extraction buffer. The eluted proteins were separated from the gel matrix by centrifugation followed by acetone precipitation, and the precipitated proteins were subjected to SDS-PAGE and 2-DE. The SDS-PAGE-based prefractionation method provided non-overlapping discrete sample pools. About 27% more protein spots were detected in the fractionated samples than in the unfractionated samples, and 17% were enhanced. The improvement was especially prominent in the case of HMW proteins. Well-separated HMW proteins were analyzed by MALDI-TOF mass spectrometry. The molecular masses of the identified proteins in the > 48 kDa gel segment were distributed between 50 and 112 kDa, thus validating this prefractionation method. Identified HMW proteins with similar mass but different pI were mostly isoforms. Thus SDS-PAGE-based size prefractionation provides improved separation and detection of HMW proteins.     

Proteome analysis on an early transformed human bronchial epithelial cell line,BEP2D,after alpha-particle irradiation

To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.     

Comparative proteomic analysis of longan (Dimocarpus longan Lour.) seed abortion

Two-dimensional gel electrophoresis (2-DE), coupled with mass spectroscopy, was used to study seed abortion in Dimocarpus longan Lour. (cv. Minjiao 64-1) by comparing normal and aborted seeds at three developmental stages. More than 1,000 protein spots were reproducibly detected in 2-DE gels, with 43 protein spots being significantly altered in their intensity between normal and aborted seeds at least at one stage. Thirty-five proteins were identified by matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF-MS/MS) analysis and protein database searching. Most of the identified proteins were associated with a variety of functions, including energy and metabolism (30%), programed cell death (9%), antioxidative processes (14%), chaperonin (23%), cell division, amino acid metabolism, secondary metabolism, and other functional classes. Furthermore, the expression patterns of HSP70 and cytosolic ascorbate peroxidase (cAPX) were validated by immunoblotting analysis. This study provides a novel, global insight into proteomic differences between normal and aborted seeds in longan. We anticipate that identification of the differentially expressed proteins may lead to a better understanding of the molecular basis for seed abortion in longan.     

Characterisation of rice anther proteins expressed at the young microspore stage

In combination with two-dimensional polyacrylamide gel electrophoresis (2-DE) protein mapping and mass spectrometry analysis, the pattern of gene expression in specific tissues at a specific stage can be displayed and characterised. We used this approach for rice (Oryza sativa L. cultivar Doongara) to display and assign identity to proteins in the anthers at the young microspore stage. Over 4000 anther proteins in the pI range of 4-11 and molecular mass range of 6-122 kDa were reproducibly resolved after silver staining, representing about 10% of the estimated total genomic output of rice. Two hundred and seventy-three protein spots have been extracted either from polyninylidene diffluoride membrane blots or from colloidal Coomassie blue stained 2-DE gels and analysed by N-terminal sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS) analysis or tandem MS sequencing. This enabled identification of 53 anther protein spots representing 43 different proteins. Using the publicly available rice expressed sequence tag (EST) database at the National Centre for Biotechnology Information, a further 37 protein spots were matched to ESTs. After BLAST searching with these ESTs, we were able to predict the identity of 22 of these protein spots. Proteome reference maps of rice anthers have been constructed according to the SWISS-2DPAGE standards and are available for public access at http://semele.anu.edu.au/2d/2d.html.     

The defense response of germinating maize embryos against fungal infection: a proteomics approach

Pathogen attack on plants results in numerous host-specific biochemical responses, the activation of some of them being critical for the ability of the plant to withstand disease. We have used high-resolution two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify proteins that are differentially expressed in response to fungal infection in maize embryos. Differential spots corresponding to induced or repressed proteins were apparent in silver stained 2-DE gels of proteins extracted from sterile and fungal-infected germinating embryos. Selected spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and nanospray ion-trap tandem mass spectrometry. Among the proteins induced in response to infection are proteins involved in protein synthesis, or in protein folding and stabilization, as well as proteins involved in oxidative stress tolerance. Additionally, the accumulation of specific pathogenesis-related proteins in tissues of the fungal-infected germinating embryos was studied by 2-DE and immunoblotting.     

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

慢性应激大鼠海马的比较蛋白质组学研究

慢性应激可造成海马神经细胞丢失、树突萎缩等损伤,但有关其损伤机制仍有很多问题不甚明了.为了寻找应激致海马损伤相关的重要蛋白质、从蛋白质水平揭示应激致海马损伤的分子机制,应用双向凝胶电泳(2-DE)技术分离对照组和束缚应激组大鼠海马组织总蛋白质,图像分析检测差异表达的蛋白质点,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MSS)和数据库检索对差异表达的蛋白质点进行鉴定,并采用半定量的RT-PCR在mRNA水平验证2-DE结果.得到了分辨率较高、重复性较好的对照和束缚应激大鼠海马2-DE图谱,质谱分析和数据库检索鉴定了14个差异表达蛋白质点中的11个蛋白质,大多数差异蛋白的功能涉及能量代谢、信号传递等过程.研究结果为揭示应激致海马损伤的机制、提高机体的应激适应能力提供了理论依据.     

Structural proteome of human colostral fat globule membrane proteins

Milk fat globule membrane (MFGM) contains proteins derived from the apical membrane of secreting epithelial cells of the mammary gland. Between 2-4% of total human milk protein content is associated with the fat globule fraction, as MFGM proteins. While MFGM proteins have very low classical nutritional value, they play important roles in various cell processes and defence mechanisms for the newborn. To date, fewer than 30 human MFGM proteins have been identified and characterized, either by immunological methods or by Edman sequencing and mass spectrometry. This study aimed to update the structural proteome of human colostral MFGM proteins and to create an annotated two-dimensional electrophoresis (2-DE) MFGM protein database available on-line. More than one hundred 2-DE spots derived from human colostral MFGM proteins were investigated by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and proteins were identified by three different software packages available on the web (PeptIdent, MS-Fit and ProFound); uncertain identifications were solved by nanoelectrospray ionization-ion trap mass spectrometry using SEQUEST software.     

Identification of a glucose-6-phosphate isomerase involved in adaptation to salt stress of <Emphasis Type="Italic">Dunaliella salina</Emphasis>

The unicellular green alga Dunaliella salina is a recognized model for studying plant adaptation to high salinity. To isolate some salt-induced proteins at proteomics levels and to identify their expressions at gene levels, algal cells at logarithmic phase cultured in 1.5 and 3.5 M NaCl media were harvested for protein extraction. Solubilized proteins were applied to two-dimensional gel electrophoresis (2-DE) and analyzed by ImageMaster 2D Platinum software. Twenty-one protein spots whose intensities were elevated threefold to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. One salt-induced protein isolated from the 2-DE gels was identified as a glucose-6-phosphate isomerase (GPI) from D. salina (DsGPI). A full-length cDNA of DsGPI was obtained using rapid amplification of cDNA end technique, and it was shown by heterologous expression to encode a protein with a molecular weight consistent with the protein spot in the 2-DE gels. Real-time quantitative RT-PCR demonstrated that the mRNA of DsGPI was induced up to eightfold (P < 0.01) by 2.5 M and 14-fold higher (P < 0.01) by 3.5 M NaCl than by 1.5 M NaCl, respectively. It is concluded that the protein isolated through 2-DE is indeed DsGPI and that the DsGPI gene may be involved in adaptation to high salinity.