SELDI-TOF ProteinChip Research Articles

Effect of steroids on transcription and secretion of Gal-1 by the human trophoblast cell line in vitro

Galectin-1 (Gal-1) is a lectin with recently documented pro-invasive function in trophoblasts in vitro, whose regulation is currently insufficiently known. The potential involvement of steroid hormones, synthetic glucocorticoid dexamethasone (DEX), the sex steroid progesterone (PRG) and mifepristone (RU486) in the regulation of Gal-1 in the trophoblast-derived cell line HTR-8/SVneo was investigated. Gal-1 mRNA levels were assessed by real-time PCR. The effect on secretion of Gal-1 into the culture media was followed using the SELDI-TOF protein chip array. We present evidence that DEX and RU486 significantly reduced Gal-1 in the HTR-8/SVneo cell line at the mRNA level. In addition, trophoblast-derived HTR-8/SVneo cells were shown to secrete detectable Gal-1 protein, which was only slightly increased by PRG. The potential clinical relevance of these findings remains to be determined.

Using SELDI-TOF MS to identify serum biomarkers in NSCLC

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide with poor differential diagnosis of the disease and with low response rates to standard chemotherapy. Individuals classified as Stage I by the present clinical-based scoring system may display survival characteristics ranging from 10 to 90 %. Identification of reliable biomarkers to detect NSCLC at early stages is therefore a subject of extensive research. Surface-enhanced laser-desorption-ionization time-of-flight mass spectrophotometry (SELDI-TOF-MS) combines chromatographic surfaces with solid-state TOF-MS for the high throughput analysis of proteins in diverse clinical samples such as serum, urine, cerebrospinal fluids and has overcome the limitations of earlier proteomic approaches. The method has been widely used for biomarker discovery. Therefore, in this study we used the SELDI-TOF protein Chip system to achieve a stratification based on the protein expression profile and to identify differentially expressed proteins in sera from 50 patients with NSCLC and 50 healthy controls. IMAC 30-Cu protein chips were used to fractionate the proteins according to their metal binding affinity. The arrays were analyzed using the Ciphergen Protein Chip Reader PB IIa. The spectra were normalized to the total ion current of the mass to charge between 3000 — 10 000 Da. Protein peak clustering and classification analysis were performed using the Express 3.0 Biomarker Pattern Analysis Software. Several proteins with masses ranging from 2000-20000 Da were differentially expressed between NSCLC patients and healthy controls. Among the peak clusters revealing differences we selected two specific clusters as prospective candidate biomarkers in sera of the patients. High expression of one peak and underexpression of the other with respect to the healthy control sera were evaluated in a blind test in terms of the ability to classify a separate group of patients and controls.

Detection and identification of specific serum biomarkers in papillary thyroid cancer

To detect and identify the potential specific serum biomarkers for diagnosis of papillary thyroid cancer. Samples of 35 patients with papillary thyroid carcinoma, 40 patients with benign thyroid nodule and 34 healthy individuals were analyzed using the SELDI-TOF ProteinChip System and bioinfomation technology to find the differential peaks which were separated by HPLC and then further analyzed by LC-MS/MS. The protein sequences were analyzed by SEQUEST software and searched in Bioworks database. The top six mass-to-charge ratio (M/Z) peaks with the smallest P value were 6651, 6452, 7653, 7932, 15 106 and 15 848 Da, respectively. The 6651 and 6452 Da proteins were weakly expressed in papillary thyroid carcinoma but highly expressed in benign thyroid nodules and healthy individuals. The differences had statistical significance (P < 0.01). The 7653, 7932, 15 106, 15 848 Da proteins were highly expressed in papillary thyroid carcinoma but weakly expressed in benign thyroid nodules and healthy individuals. The differences were statistically significant (P < 0.01). Combination of these six proteins, using the method of leave-one-out to make crossing detection, the specificity of discriminating papillary thyroid carcinoma and non-cancer was 88.0%, and its sensitivity was 92.5%. The 6651 and 6452 Da proteins were identified as apolipoprotein C-I and apolipoprotein C-III, respectively. The 7653 and 15 106 Da proteins were identified as the same protein-alpha-globin, and the 7932 and 15,848 Da proteins were identified as the same protein-beta-globin. The detection of differentially expressed apolipoprotein C-I, apolipoprotein C-III, alpha-globin, and beta-globin may have utility for diagnosis of papillary thyroid carcinoma and are worthy of further investigation.

Predicting left ventricular remodeling after a first myocardial infarction by plasma proteome analysis

Recent improvements in therapeutic strategies did not prevent left ventricular remodeling (LVR), which remains a common event (30%) after acute myocardial infarction (AMI). We report the use of a systematic approach, based on comparative proteomics, to select circulating biomarkers that may be associated with LVR. We selected 93 patients enrolled in a prospective study. These patients with anterior wall Q-wave AMI underwent echocardiographic follow-up at hospitalization, 3 months and 1 year after AMI. They were divided into three groups (no, low, or high remodeling). Plasma samples of these patients (day 5 of hospitalization) were processed and stored at -80 degrees C within 2 h and analyzed using SELDI-TOF protein chip technology. This systematic approach allowed to select candidate proteins modulated by LVR: post-translational variants of alpha1-chain of haptoglobin (Hpalpha1) corresponding to m/z 9493, 9565, and 9623, which were more elevated in remodeling patients. The peak 9493 m/z was shown having a receiving-operating characteristic (ROC) value of 0.71 between non- and remodeling patients. SELDI-TOF approach may lead to the identification of circulating proteins associated with LVR. Whether these candidate proteins will help to identify patients who are at high risk of heart failure after AMI will have to be tested in future studies.

Serum proteomic profiling of obese patients: correlation with liver pathology and evolution after bariatric surgery

Objective:Chronic liver diseases, including cirrhosis, may develop in obese patients. Steatosis and non-alcoholic steatohepatitis (NASH) are risk factors for progression to fibrosis. To date, diagnosis of steatosis and NASH relies...

SERUM PROTEOMIC PROFILING BY SURFACE-ENHANCED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY IN TESTICULAR GERM CELL CANCER PATIENTS

Objectives: Surface-enhanced laser desorption ionization mass spectrometry (sELDI-TOF MS) incorporates time-of-flight protein mass spectrometry and chip-based chromatographic protein selection and allows rapid protein profiling of complex biological samples. In this study, serum protein profiles were examined in testicular germ cell cancer patients and healthy controls with the aim of predicting cancer patients by a molecular test combining on-chip chromatography and mass spectrometry. Methods: Serum samples were taken from 49 patients with histologically proven seminomas. Forty-nine healthy subjects served as controls. Mass-spectrometry-based SELDI-TOF ProteinChip technology was used for serum proteomic analysis. The samples were fractionated and bound to the IMAC-Cu+ and CM10 ProteinChip. Proteomic spectra were generated by the ProteinChip system and analyzed by the proteomic platform ?proteomic.net?. Serum samples were also examined for beta-human chorionic gonadotropin levels. An artificial intelligence learning algorithm was used to differentiate between cancer and control subjects. Results The classification algorithm correctly identified the pattern in 90.4% of the samples (95% confidence interval of 82.6%-95.5%). Decision trees predicted seminomas with 91.5% sensitivity and 89.4% specificity. Seminoma patients with normal beta-human chorionic gonadotropin serum levels (n=38 of 49) were correctly predicted with 80% sensitivity and 70% specificity. Conclusion Our study is the first to demonstrate that SELDI-TOF MS protein profiles of cancer and control subjects differ to a highly significant degree in testicular germ cell cancer patients. Validation of these findings may enable proteomic profiling to become a valuable tool, especially for aftercare.

SELDI-TOF-MS Profiling of serum for detection of liver cirrhosis and hepatocellular carcinoma in chronic hepatitis C

The aim of the present study was to establish protein profiles using surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) for identification of liver cirrhosis and hepatocellular carcinoma (HCC) in patients with chronic hepatitis C. Serum samples of 40 patients with F1/F2 fibrosis, 44 patients with F4 fibrosis, 34 patients with HCC were applied to CM 10 cation exchange protein chips and analysed using the SELDI-TOF ProteinChip System (PBS-IIc; Ciphergen Biosystems) after anion-exchange fractionation. Fibrosis stage and HCC had been histologically confirmed in all patients. Data were analysed for protein patterns by multivariate statistical techniques and artificial neural networks. A 4 peptide/protein multimarker panel correctly identified HCCs with a sensitivity of 100% and specificity of 84% in the HCV-cirrhosis versus HCV-HCC training sample set. Sensitivity and specificity for HCC were 68% and 80% for an internal cross validated test sample set. Cirrhotic patients could be discriminated against patients with F1 or F2 fibrosis using a 5 peptide/protein multimarker pattern with a specificity of 100% and a sensitivity of 85%. Whereas the internal cross validation test set displayed sensitivity and specificity for liver cirrhosis of 80% and 67%, respectively. SELDI-TOF-MS technology combined with protein pattern analysis seems a valuable approach for the identification of liver cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis C. Due to the suitability of the SELDI-TOF-MS/ProteinChip technology for mass application it might become a tool for screening HCV patients for cirrhosis and HCC.

New multi protein patterns differentiate liver fibrosis stages and hepatocellular carcinoma in chronic hepatitis C serum samples

To identify a multi serum protein pattern as well as single protein markers using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) for detection and differentiation of liver fibrosis (F1-F2), liver cirrhosis (F4) and hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus (HCV). Serum samples of 39 patients with F1/F2 fibrosis, 44 patients with F4 fibrosis, 34 patients with HCC were applied to CM10 arrays and analyzed using the SELDI-TOF ProteinChip System (PBS-IIc; Ciphergen Biosystems) after anion-exchange fractionation. All patients had chronic hepatitis C and histologically confirmed fibrosis stage/HCC. Data were analyzed for protein patterns by multivariate statistical techniques and artificial neural networks. A 4 peptide/protein multimarker panel (7486, 12,843, 44,293 and 53,598 Da) correctly identified HCCs with a sensitivity of 100% and specificity of 85% in a two way-comparison of HCV-cirrhosis versus HCV-HCC training samples (AUROC 0.943). Sensitivity and specificity for identification of HCC were 68% and 80% for random test samples. Cirrhotic patients could be discriminated against patients with F1 or F2 fibrosis using a 5 peptide/protein multimarker pattern (2873, 6646, 7775, 10,525 and 67,867 Da) with a specificity of 100% and a sensitivity of 85% in training samples (AUROC 0.976) and a sensitivity and specificity of 80% and 67% for random test samples. Combination of the biomarker classifiers with APRI score and alfa-fetopotein (AFP) improved the diagnostic performance. The 6646 Da marker protein for liver fibrosis was identified as apolipoprotein C-I. SELDI-TOF-MS technology combined with protein pattern analysis seems a valuable approach for the identification of liver cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis C. Most probably a combination of different serum markers will help to identify liver cirrhosis and early-stage hepatocellular carcinomas in the future.

Telangiectatic focal nodular hyperplasia: a variant of hepatocellular adenoma

Background & Aims : “Telangiectatic focal nodular hyperplasia” designate atypical lesions considered as variants of focal nodular hyperplasia (FNH). However, because “telangiectatic FNH” share several morphologic patterns with hepatocellular adenomas, classification of such lesions deserve further clarification. Therefore, the aim of the present study was to reconsider the classification of telangiectatic FNH with the help of a molecular approach. Methods : Ten telangiectatic FNH, 6 typical FNH, and 6 hepatocellular adenomas were studied. DNA, RNA, and protein from each lesion were extracted. Clonality was assessed by the study of the X chromosome inactivation pattern (HUMARA assay). Angiopoietin ( ANGPT-1 and ANGPT-2) mRNA, genes the expression of which is typically modified in FNH, were quantified by a real-time RT-PCR procedure. Protein profiles were analyzed by SELDI-TOF PROTEINCHIP (Cyphergen Biosystem, Inc., Fremont, CA) technology. Results : Although all informative cases of FNH (5 of 6) and hepatocellular adenomas (6 of 6) were polyclonal and monoclonal, respectively, clonal analysis showed a nonrandom pattern of X chromosome inactivation consistent with a monoclonal lesion in 6 of 8 cases of telangiectatic FNH. The mean value of the ANGPT-1/ANGPT-2 mRNA ratio was 21.4 in FNH, 2.6 in adenomas, and 2.1 in telangiectatic FNH ( P ≤ 0.001 in telangiectatic FNH vs. FNH). SELDI-TOF PROTEINCHIP profiling and hierarchical clustering analysis showed that all except 1 telangiectatic FNH clustered within the group of hepatocellular adenomas. Conclusions : These results show that telangiectatic FNH display a molecular pattern closer to that of hepatocellular adenomas than to FNH and suggest that these lesions should instead be referred to as “telangiectatic hepatocellular adenomas.”

Proteomic analysis of human tears: defensin expression after ocular surface surgery.

Human tear protein profiles were monitored by surface enhanced laser desorption/ionization-time-of-flight mass spectrometry ProteinChip technology (SELDI-TOF ProteinChip) and liquid chromatography-mass spectrometry (LC-MS). Tears were collected from 21 patients scheduled for surgery to remove an ocular surface neoplasm prior to surgery (day 0) and on days 1, 3, and 30 postoperatively. Using this proteomic approach, we verified that three human alpha-defensins (HNP-1, HNP-2, and HNP-3) were significantly up-regulated in their expression after surgery and that their levels decreased to approximately normal by day 30 by which time healing was complete. Further confirmation of the identity of the alpha-defensins in human tears was made by LC purification, trypsin digestion, and ESI-MS/MS analysis of their tryptic digests. The concentrations of HNP-1 and HNP-2 were determined and shown to be markedly increased after ocular surface surgery. The results of the study suggest that human alpha-defensins HNP-1, HNP-2, and HNP-3 are up-regulated after surgery, and may in addition to their antimicrobial properties have an important role in wound healing.

1229 Identification of a new marker of hepatocellular carcinoma (HCC) by serum protein profiling of cirrhotic patients using SELDI-TOF proteinchip

1229 Identification of a new marker of hepatocellular carcinoma (HCC) by serum protein profiling of cirrhotic patients using SELDI-TOF proteinchip