以SELDI芯片进行细胞标本蛋白分析的方法学研究

目的:探讨以SELDI芯片技术进行细胞标本蛋白分析的最适方法及条件,筛选细胞标本蛋白表达差异。方法:对细胞标本分别用超声裂解法,U9细胞裂解缓冲液配方和自配细胞裂解液提取蛋白,以BCA法测定蛋白浓度;分别以磁珠活化后点样和生物芯片处理器点样使蛋白样品与芯片结合;并对提取蛋白进行检测,比较不同蛋白浓度梯度点样及WCX2,SAX2,IMAC-Cu,H50芯片捕获蛋白差异,用WCX2芯片筛选蛋白差异表达。结果:相同培养条件细胞以上述三种不同蛋白提取方法获得的蛋白浓度分别为:0.25±0.034μg/μl,0.6±0.06μg/μl,1.02±0.077μg/μl;生物芯片处理器点样法操作简单,要求样本量较少,点样时间短;SELDI芯片蛋白质峰图谱与蛋白浓度呈较好的正相关;WCX2,SAX2,H50,IMAC-Cu芯片捕获的蛋白质种类有较大区别;在分子量1000～300000Da范围内,以WCX2芯片共检测到87个差异蛋白峰,其中17个呈趋势变化。结论:上述三种方法比较,选用自配的细胞裂解液提取蛋白的浓度较高且更适于芯片研究;生物芯片处理器能较好地使蛋白与芯片结合;SELDI芯片能准确定位蛋白,且其蛋白质峰与被测蛋白浓度呈正相关变化;SELDI各芯片捕获蛋白类型不同,选择适宜芯片或联合运用芯片检测更易获得较理想蛋白差异表达结果。     

Proteomic approaches to biomarker discovery in lung cancers by SELDI technology

The purpose of the present work is to identify protein profiles that could be used to discover specific biomarkers in serum and discriminate lung cancer. Thirty serum samples from patients with lung cancer (15 cases of primary brochogenic carcinoma, 9 cases of metastasis lung cancer and 6 cases of lung cancer after chemotherapy) and twelve from healthy individuals were analyzed by SELDI (Surfaced Enhanced Laser Desorption/Ionization) technology. Anion-exchange columns were used to fractionate the sera with 6 designated pH washing solutions. Two types of protein chip arrays, IMAC-Cu and WCX2, were employed. Protein chips were examined in PBSII ProteinChip Reader (Ciphergen Biosystems Inc.) and the resulting profiles between cancer and normal were analyzed with Biomarker Wizard System. In total, 15 potential lung cancer biomarkers, of which 6 were up-regulated and 9 were down-regulated, were discovered in the serum samples from patients with lung cancer. 5 of 15 these biomarkers were able to be detected on both WCX2 and IMAC-Cu protein chips. The sensitivities provided by the individual markers range from 44.8% to 93.1% and the specificities were 85.0%–94.4%. Our results suggest that serum is a capable resource for detection of lung cancer with specific biomarkers. Moreover, protein chip array system was shown to be a useful tool for identification, as well as detection of disease biomarkers in sera.     

Proteomic approaches to biomarker discovery in lung cancers by SELDI technology

Lung cancer is one of the most common tumors all over the world and one of those with higher mortality in clinic. For instance, 169500 new cases of lung cancer were estimated in the United States for 2001[1]. In recent years, both morbidity and mortality of lung cancer were reported gradually increasing in our country. Therefore, it has become an urgent task to search and discover specific biomarkers for lung cancer. In tumor genesis, certain cellular proteins must have changed their express…     

ProteinChip technology: a new and facile method for the identification and measurement of high-density lipoproteins apoA-I and apoA-II and their glycosylated products in patients with diabetes and cardiovascular disease

This paper describes a ProteinChip technology for the identification and quantification of apolipoprotein profiles in crude biological samples. Expression levels of apoA-I and apoA-II and their glycosylated products were accomplished using single 1 microL plasma samples. In the present studies, strong anionic and weak cationic exchanger ProteinChips (SAX2 and WCX2 chip surfaces) were tested, and the WCX2 chip was found to be selective for specific apolipoproteins. Using the WCX2 chip and analysis via surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS), apoA-I and apoA-II were separated as sharp peaks at 28 and 17 kD and did not overlap with other serum protein peaks. Since these assays can be completed on a large number of clinical samples in approximately 1 h, further development of this technique will facilitate both epidemiological studies and therapeutic trials in assessing the role of the apolipoproteins and their glycosylated products in atherosclerosis.     

Comprehensive profiling of the low molecular weight proteins and peptides in weak cation exchange beads human serum retentate

Mass spectrometric profiling using ProteinChip and magnetic beads has rapidly grown over the past years, particularly to generate serum profiles for cancer diagnosis. The molecular weights of these distinguishing peaks are usually under 30 kDa. To identify those low molecular weight proteins and peptides is important for specific assays to be developed and increases biological insight. In this study, low molecular weight proteins and peptides from serum were purified by a combination of weak cation exchange magnetic beads and high performance liquid chromatography. The purified proteins and peptides were analyzed by 1D SDS PAGE, SELDI and LC-MS/MS. 246 proteins were identified from the HPLC fractions by LC-MS/MS. 95(38.62%) proteins were first identified in serum compare with Sys-BodyFluid database. 11(11/96) proteins were documented cancer associated proteins. We also observed about 109 proteins/peptides in SELDI mass spectrum, and 13 of the SELDI features were identified.     

Proteomic analyses of arsenic-induced cell transformation with SELDI-TOF ProteinChip technology

In this study, we demonstrated that low levels (1.5 microM) of arsenite induces B[a]P-treated lung cell transformation. We then used a proteomic approach to identify protein expression by ProteinChips, which could potentially be important for transformation induced by this toxic metal. Most of the protein peaks in cell extracts of all samples, including the control, B[a]P-treated, and B[a]P + As-treated cells are identical. However, surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) analysis with Cu-ProteinChips and WCX-ProteinChips revealed several dramatically different protein peaks that appeared in lung cells after being transformed by a treatment of 1.5 microM arsenite for 12 weeks. SAX2 ProteinChip also identified a prominent protein peak that was preferentially expressed in control cells. Interestingly, by using a SAX2 chip, we were able to detect several protein peaks that increased their expression in lung epithelial cells (LEC) treated with only B[a]P. Identification and characterization of these proteins may reveal the molecular basis of As-induced cell transformation and provide insight into the mechanisms by which arsenic induces carcinogenesis.     

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphophate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.     

Detection and identification of virulence factors in Yersinia pestis using SELDI ProteinChip system

A rapid method for the detection, purification, and identification of proteins in bacterial extracts was developed using surface enhanced laser desorption/ionization (SELDI) ProteinChip technology. The effectiveness of this technique for monitoring the expression and identification of temperature- and calcium-regulated virulence factors of Yersinia pestis, the bacterium that causes human plague, is demonstrated. Y. pestis infection of its mammalian host is thought to be accompanied by rapid up-regulation of a number of genes following a shift from 26 degrees C (the temperature of the flea vector) to 37 degrees C (the temperature of the mammalian host). To model this process, Y. pestis cells were grown at 26 degrees C and 37 degrees C in a Ca(2+)-deficient medium. Through an initial protein profiling of the crude bacterial extract on strong anion exchange and copper affinity, ProteinChip arrays detected five proteins that were up-regulated and three proteins that were down-regulated at 37 degrees C. Two of the proteins predominately expressed at 37 degrees C were semi-purified in less than two days. The two proteins were identified as catalase-peroxidase and Antigen 4. Aside from its speed, a salient feature of the SELDI technique is the microgram amounts of crude sample required for analysis.     

Profiling of amyloid beta peptide variants using SELDI Protein Chip arrays

The profile of amyloid beta (A beta) peptide variants secreted into the media of human cultured cells that express the amyloid precursor protein was examined by Surface Enhanced Laser Desorption/Ionization (SELDI) ProteinChip technology from Ciphergen Biosystems using biologically active ProteinChip Arrays. An anti-A beta polyclonal antibody (anti-NTA4) was used to capture and purify multiple immunoreactive A beta fragments from a single microliter of media onto the ProteinChip Array. Fragments retained on the surface of the ProteinChip Array were detected directly by mass in the ProteinChip System to provide detailed information on the identity of different A beta variants secreted from the cultured cells. We discuss existing and potential applications of this immunoassay for the detection and relative quantitation of A beta species from both cultured cell systems and clinical samples.     

Identification of collagen-binding proteins in Lactobacillus spp. with surface-enhanced laser desorption/ionization-time of flight ProteinChip technology

Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)-time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions.     

ApoC-I and ApoC-III as potential plasmatic markers to distinguish between ischemic and hemorrhagic stroke

Early diagnosis and immediate therapeutic interventions are crucial factors to reduce the damage extent and the risk of death. Currently, the diagnosis of stroke relies on neurological assessment of the patient and neuro-imaging techniques including computed tomography and/or magnetic resonance imaging scan. An early diagnostic marker of stroke, ideally capable to discriminate ischemic from hemorrhagic stroke would considerably improve patient acute management. Using surface-enhanced laser desorption/ionization (SELDI) technology, we aimed at finding new early diagnostic plasmatic markers of stroke. Strong anionic exchange (SAX) SELDI profiles of plasma samples from 21 stroke patients were compared to 21 samples from healthy controls. Seven peaks appeared to be differentially expressed with significant p values (p < 0.05). Proteins were stripped from the SAX chips, separated on a one-dimensional electrophoresis (1-DE) gel and stained using mass spectrometry (MS)-compatible silver staining. Following in-gel tryptic digestion, the peptides were analyzed by MS. Four candidate proteins were identified as apolipoprotein CI (ApoC-I), apolipoprotein CIII (ApoC-III), serum amyloid A (SAA), and antithrombin-III fragment (AT-III fragment). Assessment of ApoC-I and ApoC-III levels in plasma samples using a sandwich enzyme-linked immunosorbent assay (ELISA) allowed to distinguish between hemorrhagic (n = 15) and ischemic (n = 16) stroke (p < 0.001). To the best of our knowledge, ApoC-I and ApoC-III are the first reported plasmatic biomarkers capable to accurately distinguish between ischemic and hemorrhagic stroke in a small number of patients. It requires further investigation in a large cohort of patients.     

Identification of Collagen-Binding Proteins in Lactobacillus spp. with Surface-Enhanced Laser Desorption/Ionization–Time of Flight ProteinChip Technology

Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)–time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions.     

大肠埃希菌蛋白指纹图谱的建立

目的建立大肠埃希菌（Escherichia coli,E．coli）蛋白指纹图谱,为Ecoli感染快速诊断奠定基础。方法收集临床分离E.coli88株,提取细菌DNA,PCR检测Ecoli 16S rRNA。蛋白提取液提取细菌蛋白,干化学法测蛋白浓度,应用表面增强激光解析电离飞行时间质谱技术（SELDI-TOF-MS）检测Ecoli蛋白,采用Ciphergen Pro-teinchip软件自动采集数据。重复测定20次Ecoli混合标本,评价SELDI检测Ecoli蛋白分子量的重复性。结果E．coil标准菌株ATCC 25922和临床分离株均可检出16S rRNA。AU芯片能捕获近30个E．coli蛋白峰,其中19个蛋白峰构成E．coli特征性蛋白指纹图谱,各蛋白峰在临床分离E．coli间分子量变异系数≤0．2％。SELDI重复检测20次E．coli混合标本显示同一蛋白峰的分子量变异系数≤0．05％。结论E．coli在分子量3～20kD范围内具有特征性蛋白指纹图谱,为快速诊断E.coli感染提供了新思路。     

Discovery of laryngeal carcinoma by serum proteomic pattern analysis

Laryngeal carcinoma is the most common malignancy among head and neck tumors. The purpose of this study is to find biomarkers for laryngeal carcinoma in patient blood serum using the Surface Enhanced Laser Desorption/Ionization (SELDI) technique. Serum samples from 33 laryngeal carcinoma (12 cases of glottis, 18 of supraglottis and 3 of subglottis) patients and 31 age- and sex-matched healthy people were analyzed by SELDI-TOF on a ProteinChip reader, PBSII-C. Protein profiles were generated using WCX2 protein chips. Protein peak clustering and classification analyses were performed utilizing the Biomarker Wizard and Biomarker Pattern software packages, respectively. The results showed that sixteen peaks had significant difference between laryngeal cancer patients and healthy group, eight of which were up-regulated in the patient samples, and the others were down-regulated. Two protein peaks 8153 Da and 2035 Da were automatically chosen for the system training and development of a classification tree. The analysis yielded a correct percentage of 96.9% for patients and 96.7% for control. The results suggest that serum is a useful resource for the detection of specific biomarkers for laryngeal carcinoma. Proteinchip Array System was a useful tool for a high throughput screening of large-sized serum samples to discover potential biomarkers for carcinoma.     

Proteomic analyzes of copper metabolism in an in vitro model of Wilson disease using surface enhanced laser desorption/ionization-time of flight-mass spectrometry

In Wilson disease, mutations in the ATP7B-gene lead to hepatic accumulation of copper that becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and acute liver failure. Several proteins are probably involved in dealing with the excess copper and oxidative stress. As a first step towards biomarker discovery and analyzes of copper metabolism in Wilson disease patients we characterized copper-induced changes in protein expression in cell lysates and culture media from an in vitro copper-overload model using surface enhanced laser desorption/ionization (SELDI) proteomics technology. HepG2 cells were cultured for 48 h with a physiological (0.5 microM) or a pathological (100 microM) copper concentration. Samples were applied to weak cation exchange (WCX) proteinchip arrays and chips were analyzed by time of flight (TOF)-mass spectrometry. Copper-coated IMAC chips were used to detect copper-binding proteins in cell lysate of copper depleted cells using buffers with increasing imidazole concentrations. Data from the 2 to 50 kDa range indicate that high extra-cellular copper substantially altered both intra-cellular protein expression as well as the composition of the secretome. In the lysate 15 proteins were found up-regulated, while 6 proteins were down-regulated. In culture media 21 proteins were increased while 4 proteins were decreased in abundance. Copper-coated protein chips revealed the presence of 18 high-affinity copper-binding proteins. Further identification is necessary to determine the exact cellular roles of the discovered proteins.     

Proteomic profiling of secreted proteins from CHO cells using Surface-Enhanced Laser desorption ionization time-of-flight mass spectrometry

Chinese hamster ovary (CHO) cells are the most commonly used host cell line for the production of recombinant biopharmaceuticals. These biopharmaceuticals are typically secreted from CHO cells and purified from harvested cell culture media. The purpose of this study was to investigate changes in the secreted proteome of CHO cells over the various stages of the growth cycle using Surface Enhanced Laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Conditioned media samples were collected each day over a 6 day growth period from CHO-K1 cells grown in low serum (0.5% FBS) conditions in monolayer culture. Samples were profiled on a number of ProteinChip arrays with different chromatographic surfaces. From this study, 24 proteins were found to be differentially regulated at different phases of the growth cycle in CHO-K1 cells, when profiled on two chromatographic surfaces, Q10 (anionic) and IMAC30 (metal affinity) ProteinChip arrays.     

Structural analyses of O-glycan sugar chains on IgA1 hinge region using SELDI-TOFMS with various lectins

The aim of the study was to develop a simple and precise method for identifying glycosylation of the IgA hinge region using surface-enhanced laser desorption/ionization (SELDI)-TOFMS with a lectin-coupled ProteinChip array. Serum IgA was isolated using an anti-IgA antibody column. Following reduction, alkylation, and trypsin digestion, the IgA fragments were applied on the ProteinChip coupled with jacalin, peanut agglutinin (PNA), or Vilsa villosa lectin (VVL). The SELDI-TOFMS peaks corresponding to the fragments containing IgA1 hinge glycopeptides trapped by each lectin were compared. The jacalin-, PNA-, and VVL-immobilized ProteinChips detected 13, 4, and 2 peaks, respectively. One major peak was confirmed as a glycopeptide by MS/MS analysis. These results suggest that a lectin-immobilized ProteinChip assay can be used to simplify the procedures for the analyses of the O-glycans in IgA1 hinge. This method potentially makes it possible to identify a disease-specific glycoform by selecting the appropriate ligand-coupled ProteinChip array.     

Comparison of extraction procedures for proteome analysis of Streptococcus pneumoniae and a basic reference map

Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.