Biomedical subjects

Zhen Zhang

Publications and source records attributed to Zhen Zhang.

At least 55 records · Page 3Linked to original sources

PubMed2006

[Rational evolutionary design and modification of gene: a short-path of direct evolution].

Directed evolution is a powerful means in academic study and industrial application to modified and functionally improved proteins. There have been significant and impressive advances using the artificial method. Rational Evolutionary Design utilizes structural and sequence alignment information to create new genes and proteins. Rational Evolutionary Design has recently emerged as an attractive approach for studying function of proteins. The review mainly introduced four aspects: preferred codon usage, structural and sequence alignment, key domain analysis, site-directed recombination.

Directed Molecular Evolution↗

PubMed2006

[Involved-field three-dimensional conformal radiation treatment for stage III non-small-cell lung].

OBJECTIVE: To investigate the feasibility of involved-field irradiation (IFI ) for stage III non-small cell lung cancer (NSCLC). METHODS: From September 1997 to November 2001, 200 stage-III NSCLC patients were randomly divided into two groups-- IFI and ENI (elective node irradiation). The IFI group was irradiated by 3DCR to a dose of 68-74 Gy/34-37f/7-9 w including the primary tumor and the lymph nodes of > or = 10 mm in short axis. The ENI group was irradiated to a dose of 60-64 Gy/30-32f/6-7.5 w including the primary tumor, ipsilateral hilum, subcarinal and mediastinal lymph nodes, even the supraclavicular area when the lymph nodes of superior mediastinum were involved. RESULTS: The overall response (CR + PR) rates were 90.0% in IFI group and 79.0% in ENI group. Radiation pneumonitis developed in 29.0% of the patients in ENI group and 17.0% in IFI group (P = 0.04). The 1-year primary tumor failure rate in IFI group (13.0%) was lower than that (23.0%) in ENI group. The 1-year involved nodal failure rate was 20.0% in ENI group and 10.0% in IFI group (P = 0.048). The 1-year elective node failure rate was 16.0% in ENI group versus 21.0% in IFI group (P = 0.39). The 1-, 2-and 3-year overall survival rate was 67.2% , 38.7% , 27.3% , respectively, in IFI group; versus 59.7% , 25.6% , 19.2% in ENI group, with a difference significant in the 2-year overall survival rate between IFI and ENI group (P = 0.048). CONCLUSION: Involved-field 3D-CRT for stage-III non-small cell lung cancer is well tolerated. It does not increase the rate of lymph node failure in the elective node irradiation field, and may improve the survival due to dose escalation.

Adult↗

PubMed2006

[Research of contour analysis on endoscopy navigation].

In this paper, a new method of contour analysis is presented to find path in endoscopy navigation. At first, edge detection is used to endoscope image. Then we do skeleton extraction and connect the edge skeleton to contours by grouping criteria. At last we can get navigation information by analyzing the contours. Experiments show that this algorithm has good precision and stability.

Algorithms↗

PubMed2005

Irradiated mouse testes efficiently support spermatogenesis derived from donor germ cells of mice and rats.

Testicular cell transplantation has been widely used to investigate the biology of spermatogonial stem cells, production of transgenic animals, and restoration of fertility in rodent models. One critical step in successful transplantation is the preparation of the recipient testes. Busulfan has been widely used, but irradiation has been often suggested as an alternative. There have only been limited reports of the use of irradiated animals as transplant recipients for studying differentiation of transplanted cells, and there has been no direct comparison of irradiation and busulfan as preparation methods. Mouse testes treated with local fractionated irradiation (1.5 + 12 Gy) were compared with busulfan-treated testes as recipients using mouse-to-mouse and rat-to-mouse germ cell transplantation. The fractionated irradiation schedule resulted in depletion of endogenous spermatogenesis similar to that produced by busulfan doses of 50-55 mg/kg. When immature mouse or rat testicular germ cells were transplanted into the irradiated testes, donor cells derived from either rat or mouse spermatogonial stem cells were able to form colonies of differentiated spermatogenic cells 10-13 weeks after transplantation with similar efficiencies as in busulfan-treated testes. Locally irradiated testes could be considered as an alternative to busulfan treatment for animal recipients of germ cell transplants that cannot endure the systemic toxicity of busulfan.

Animals↗

PubMed2005

The active form of the Saccharomyces cerevisiae ribonucleotide reductase small subunit is a heterodimer in vitro and in vivo.

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y*) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (beta2) and Y4 (beta'2). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (betabeta') is thought to be the active form, several rnr4delta strains are viable. To resolve this paradox, N-terminally epitope-tagged beta and beta' were expressed in E. coli or integrated into the yeast genome. In vitro exchange studies reveal that when apo-(His6)-beta2 ((His)beta2) is mixed with beta'2, apo-(His)betabeta' forms quantitatively within 2 min. In contrast, holo-betabeta' fails to exchange with apo-(His)beta2 to form holo-(His)betabeta and beta'2. Isolation of genomically encoded tagged beta or beta' from yeast extracts gave a 1:1 complex of beta and beta', suggesting that betabeta' is the active form. The catalytic activity, protein concentrations, and Y* content of the rnr4delta and wild type (wt) strains were compared to clarify the role of beta' in vivo. The Y* content of rnr4delta is 15-fold less than that of wt, consistent with the observed low activity of rnr4delta extracts (<0.01 nmol min(-1) mg(-1)) versus wt (0.06 +/- 0.01 nmol min(-1) mg(-1)). (FLAG)beta2 isolated from the rnr4delta strain has a specific activity of 2 nmol min(-1) mg(-1), similar to that of reconstituted apo-(His)beta2 (10 nmol min(-1) mg(-1)), but significantly less than holo-(His)betabeta' (approximately 2000 nmol min(-1) mg(-1)). These studies together demonstrate that beta' plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is betabeta'.

Amino Acid Sequence↗

PubMed2005

Identification of BCOX1, a novel gene overexpressed in breast cancer.

The identification of tumor-associated antigens, which are specifically expressed in cancer tissues, is of utmost important for immunotherapy of breast cancer. We have combined in silico screening and experimental expression analysis to identify genes that are differentially expressed in breast carcinomas compared with their corresponding normal tissues. Using these approaches, we identified a novel gene, BCOX1, with overexpression in breast carcinoma. BCOX1 was highly homologous to KIAA0100, a hypothetical gene located on chromosome 17q11.2. RNA in situ hybridization shows that BCOX1 mRNA signal is mainly located in the cytoplasm of breast carcinoma epithelial cells, but not in those of normal epithelial cells, stroma cells and lymphocytes. Furthermore, mRNA expression of BCOX1 was moderately elevated in ductal in situ carcinoma (DCIS), peaked in invasive breast carcinoma (IBC) and metastatic breast carcinoma cells (MET) whereas absent in benign ductal epithelial cells. The predicted BCOX1 open reading frame of 666 bp encodes a putative protein of 222 amino acid residues with a calculated molecular weight of 2,4920 Da and a PI of 5.86. Computational analyses predict that the putative BCOX1 protein is a cytoplasmic protein. The functional relevance of this novel gene is yet to be determined. This study warrants further investigations to explore the molecular functions of BCOX1, and to determine its potential diagnostic and therapeutic applications for breast cancer.

Antigens, Neoplasm↗

PubMed2005

Using proteomic approaches to identify new biomarkers for detection and monitoring of ovarian cancer.

OBJECTIVES: Early detection and monitoring the treatment remain the most important factors in improving long-term survival of ovarian cancer patients. New biomarkers that individually or in combination improve the diagnostic performance of existing tumor markers are critically needed. This study uses proteomic approaches to identify new biomarkers for detection and monitoring of ovarian cancer. METHODS: We analyzed protein profiles of three sets of sera using surface enhanced laser desorption and ionization time-of-light mass spectroscopy (SELDI-TOF-MS) on IMAC ProteinChip arrays and ProPeak software for bioinformatics data analysis. The first set of patients included 21 ovarian cancers, 18 benign diseases, and 20 normal patients. The second set included 32 ovarian cancers, 30 benign ovarian diseases, and 30 age-matched healthy controls. The third set included samples collected before and after chemotherapy from 18 ovarian cancer patients. All samples were collected at the Gynecology and Obstetrics Hospital of Fudan University in Shanghai, China. The datasets from low-intensity and high-intensity spectra were analyzed separately. RESULTS: Seven peaks were selected for their contribution to the separation of ovarian cancers from controls using the first and second set of samples. The same dysregulation trends were confirmed for six of the seven peaks in independent validation using the third set of samples. CONCLUSIONS: Using SELDI-TOF analysis of 195 unique specimens, we discovered with preliminary validation six distinct peaks that may potentially be useful in the detection and monitoring of ovarian cancer. Additional studies are required to determine the protein identities of these peaks and to further validate their performance as biomarkers.

Adult↗

PubMed2005

Independent validation of candidate breast cancer serum biomarkers identified by mass spectrometry.

BACKGROUND: We previously selected a panel of 3 breast cancer biomarkers (BC1, BC2, and BC3) from serum samples collected at a single hospital based on their collective contribution to the optimal separation of breast cancer patients and noncancer controls by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The identities and general applicability of these markers, however, were unknown. In this study, we performed protein expression profiling on samples obtained from a second hospital, included a greater number of ductal carcinoma in situ (DCIS) cases, and performed purification and identification of the 2 confirmed markers. METHODS: Using a case-control study design, we performed protein expression profiling on serum samples from the National Cancer Institute (Milan, Italy). The validation sample cohort consisted of 61 women with locally invasive breast cancer, 32 with DCIS, 37 with various benign breast diseases (including 13 atypical), and 46 age-matched apparently healthy women (age range, 44-68 years). Validated biomarkers were purified and identified with serial chromatography, 1-dimensional gel electrophoresis, in-gel ASP-N digestion, peptide mass fingerprinting, and tandem mass peptide sequencing. RESULTS: The BC3 and BC2 expression patterns in this sample set were consistent with the first study sample set. BC3 and BC2 were identified to be complement component C3a(desArg) and a C-terminal-truncated form of C3a(desArg), respectively. CONCLUSIONS: Evaluation of biomarkers in independent sample sets can help determine the broader utility of candidate markers, and protein identification permits understanding of their molecular basis. C3a(desArg) appears to lack specificity among patients with benign diseases, limiting its utility as a stand-alone tumor marker, but it may still be useful in a multimarker panel for early detection of breast cancer.

Adolescent↗

PubMed2005

Both testosterone and follicle-stimulating hormone independently inhibit spermatogonial differentiation in irradiated rats.

Simultaneous suppression of both testosterone and FSH with GnRH antagonists (GnRH-ant) reverses the radiation-induced block in spermatogonial differentiation in F1 hybrids of Lewis and Brown-Norway rats. Although addition of exogenous testosterone restores the block, it also raises FSH, and hence it had not been possible to conclusively determine which hormone was inhibiting spermatogonial differentiation. In the present study, we establish the relative roles of testosterone and FSH in this inhibition using three different approaches. The first approach involved the treatment of irradiated rats, in which differentiation was stimulated by GnRH-ant plus flutamide, with FSH for 2 wk; the FSH reduced the percentage of tubules that were differentiated (TDI) by about 2-fold, indicating that FSH does have an inhibitory role. The second approach involved treatment of irradiated, hypophysectomized rats with exogenous testosterone for 10 wk; testosterone also reduced the TDI, demonstrating that testosterone had a definite inhibitory effect, independent of pituitary hormones. Furthermore, in this protocol we showed that TDI in the hypophysectomized testosterone-treated group, which had higher intratesticular testosterone levels but lacked FSH, was slightly higher than the TDI in a GnRH-antagonist-testosterone-treated group of irradiated rats, which had normal physiological levels of FSH; this result supports a role for endogenous FSH in suppressing spermatogonial differentiation in the latter group. The third approach involved injection of an active anti-FSH antibody for 10 d in untreated, GnRH-ant plus flutamide-treated, or GnRH-ant plus testosterone-treated irradiated rats. This was not sufficient to increase the TDI. However, flutamide given in a similar treatment schedule did increase the TDI in GnRH-ant plus testosterone-treated rats. We conclude that both testosterone and FSH individually inhibit spermatogonial differentiation after irradiation, but testosterone is a more highly potent inhibitor than is FSH.

Animals↗

PubMed2005

Isolation and characterization of a cDNA encoding two novel heat-shock factor OsHSF6 and OsHSF12 in Oryza sativa L.

As a crucial transcription factor family, heat-shock factors were mainly analyzed and characterized in tomato and Arabidopsis. In this study, we isolated two putative heatshock factors OsHSF6 and OsHSF12 that interact specifically with heat-shock element (HSE) from Oryza sativa L by yeast one-hybrid method. The full-length cDNA of OsHSF6 and OsHSF12 have 1074bp and 920bp open reading frame (ORF), respectively. Analysis of the deduced amino acid sequences revealed that OsHSF6 was a class A heat shock factor (HSF) with all the conserved sequence elements characteristic of heat stress transcription factor, while OsHSF12 was a class B HSF with C-terminal domain (CTD) lacking of AHA motif. Bioinformatic analysis showed that the sequences and structures of two HSFs' DNA binding domain (DBD) had a high similarity with LpHSF24. The results of RT-PCR indicated OsHSF6 gene was expressed immediately after rice plants exposure to heat stress, and the transcription of OsHSF6 gene accumulated primarily in immature seeds, roots and leaves. However, we did not find the transcription of OsHSF12 gene in different organs and growth periods. Our results implied that OsHSF6 might be function as a HSF regulating early expression of stress genes in response to heat shock, and OsHSF12 might be act as a synergistic factor to regulate the expression of down-stream genes.

Amino Acid Sequence↗

PubMed2005

Dissecting phenotypic variation among AIS patients.

We have created genital skin fibroblast cell lines directly from three patients in a Chinese family affected by androgen insensitivity syndrome (AIS). All patients in the family share an identical AR Arg840Cys mutant but show different disease phenotypes. By using the cell lines, we find that the mutation has not influenced a normal androgen-binding capacity at 37 degrees C but has reduced the affinity for androgens and may cause thermolability of the androgen-receptor complex. The impaired nuclear trafficking of the androgen receptor in the cell lines is highly correlated with the severity of donors' disease phenotype. The transactivity of the mutant is substantially weakened and the extent of the reduced transactivity reflects severity of the donors' disease symptom. Our data reveal that although etiology of AIS is monogenic and the mutant may alter the major biological functions of its wild allele, the function of the mutant AR can also be influenced by the different genetic backgrounds and thus explains the divergent disease phenotypes.

Adult↗

PubMed2005

C-H stretching vibrations of methyl, methylene and methine groups at the vapor/alcohol (N = 1-8) interfaces.

In IR and Raman spectral studies, the congestion of the vibrational modes in the C-H stretching region between 2800 and 3000 cm(-1) has complicated spectral assignment, conformational analysis, and structural and dynamics studies, even with quite a few of the simplest molecules. To resolve these issues, polarized spectra measurement on a well aligned sample is generally required. Because the liquid interface is generally ordered and molecularly thin, and sum frequency generation vibrational spectroscopy (SFG-VS) is an intrinsically coherent polarization spectroscopy, SFG-VS can be used for discerning details in vibrational spectra of the interfacial molecules. Here we show that, from systematic molecular symmetry and SFG-VS polarization analysis, a set of polarization selection rules could be developed for explicit assignment of the SFG vibrational spectra of the C-H stretching modes. These polarization selection rules helped assignment of the SFG-VS spectra of vapor/alcohol (n = 1-8) interfaces with unprecedented details. Previous approach on assignment of these spectra relied on IR and Raman spectral assignment, and they were not able to give such detailed assignment of the SFG vibrational spectra. Sometimes inappropriate assignment was made, and consequently misleading conclusions on interfacial structure, conformation and even dynamics were reached. With these polarization rules in addition to knowledge from IR and Raman studies, new structural information and understanding of the molecular interactions at these interfaces were obtained, and some new spectral features for the C-H stretching modes were also identified. Generally speaking, these new features can be applied to IR and Raman spectroscopic studies in the condensed phase. Therefore, the advancement on vibrational spectra assignment may find broad applications in the related fields using IR and Raman as vibrational spectroscopic tools.

2-Propanol↗

PubMed2005

Classification of cancer types by measuring variants of host response proteins using SELDI serum assays.

Protein expression profiling has been increasingly used to discover and characterize biomarkers that can be used for diagnostic, prognostic or therapeutic purposes. Most proteomic studies published to date have identified relatively abundant host response proteins as candidate biomarkers, which are often dismissed because of an apparent lack of specificity. We demonstrate that 2 host response proteins previously identified as candidate markers for early stage ovarian cancer, transthyretin and inter-alpha trypsin inhibitor heavy chain 4 (ITIH4), are posttranslationally modified. These modifications include proteolytic truncation, cysteinylation and glutathionylation. Assays using Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS) may provide a means to confer specificity to these proteins because of their ability to detect and quantitate multiple posttranslationally modified forms of these proteins in a single assay. Quantitative measurements of these modifications using chromatographic and antibody-based ProteinChip array assays reveal that these posttranslational modifications occur to different extents in different cancers and that multivariate analysis permits the derivation of algorithms to improve the classification of these cancers. We have termed this process host response protein amplification cascade (HRPAC), since the process of synthesis, posttranslational modification and metabolism of host response proteins amplifies the signal of potentially low-abundant biologically active disease markers such as enzymes.

Algorithms↗

PubMed2005

The D-methyl group in beta-lactamase evolution: evidence from the Y221G and GC1 mutants of the class C beta-lactamase of Enterobacter cloacae P99.

The beta-lactam antibiotics act through their inhibition of D-alanyl-D-alanine transpeptidases (DD-peptidases) that catalyze the last step of bacterial cell wall synthesis. Bacteria resist beta-lactams by a number of mechanisms, one of the more important of which is the production of beta-lactamases, enzymes that catalyze the hydrolysis of these antibiotics. The serine beta-lactamases are evolutionary descendants of DD-peptidases and retain much of their structure, particularly at the active site. Functionally, beta-lactamases differ from DD-peptidases in being able to catalyze hydrolysis of acyl-enzyme intermediates derived from beta-lactams and being unable to efficiently catalyze acyl transfer reactions of D-alanyl-D-alanine terminating peptides. The class C beta-lactamase of Enterobacter cloacae P99 is closely similar in structure to the DD-peptidase of Streptomyces R61. Previous studies have demonstrated that the evolution of the beta-lactamase, presumably from an ancestral DD-peptidase similar to the R61 enzyme, included structural changes leading to rejection of the D-methyl substituent of the penultimate D-alanine residue of the DD-peptidase substrate. This seems to have been achieved by suitable placement of the side chain of Tyr 221 in the beta-lactamase. We show in this paper that mutation of this residue to Gly 221 produces an enzyme that more readily hydrolyzes and aminolyzes acyclic D-alanyl substrates than glycyl analogues, in contrast to the wild-type beta-lactamase; the mutant is therefore a more efficient DD-peptidase. Molecular modeling showed that the D-alanyl methyl group fits snugly into the space originally occupied by the Tyr 221 side chain and, in doing so, allows the bound substrate to assume a conformation similar to that on the R61 DD-peptidase, which has a hydrophobic pocket for this substituent. Another mutant of the P99 beta-lactamase, the extended spectrum GC1 enzyme, also has space available for a D-alanyl methyl group because of an extended omega loop. In this case, however, no enhancement of activity against D-alanyl substrates with respect to glycyl was observed. Accommodation of the penultimate D-alanyl methyl group is therefore necessary for efficient DD-peptidase activity, but not sufficient.

Binding Sites↗

PubMed2005

NELIN, a new F-actin associated protein, stimulates HeLa cell migration and adhesion.

A new gene (GenBank Accession No. AF114264) was cloned from umbilical vein wall tissue by using RT-PCR. The gene shares high similarity to the gene encoding F-actin binding protein nexilin, so named as NELIN. A clone of 2737bp contains open reading frame of 1344bp extending from 412 to 1755. NELIN was expressed primarily in the heart and skeletal muscle among eight tested normal tissues. Immunofluorescence and immunoprecipitation demonstrated that NELIN product was associated with F-actin. Stable transfection of NELIN into HeLa cells increased the cell migration by 2.17-fold and the adhesion by 1.67-fold, respectively, compared to cells with the empty vector (P<0.05). The results support that NELIN product is an F-actin associated protein and mediates cell motility.

Actins↗

PubMed2005

Dose-response relationship in locoregional control for patients with stage II-III esophageal cancer treated with concurrent chemotherapy and radiotherapy.

PURPOSE: To evaluate the correlation between radiation dose and locoregional control (LRC) for patients with Stage II-III unresectable esophageal cancer treated with concurrent chemotherapy and radiotherapy. METHODS AND MATERIALS: The medical records of 69 consecutive patients with clinical Stage II or III esophageal cancer treated with definitive chemoradiotherapy at the University of Texas M. D. Anderson Cancer Center between 1990 and 1998 were retrospectively reviewed. Of the 69 patients, 43 had received < or =51 Gy (lower dose group) and 26 >51 Gy (higher dose group). The median dose in the lower and higher dose groups was 30 Gy (range, 30-51 Gy) and 59.4 Gy (range, 54-64.8 Gy), respectively. Two fractionation schedules were used: rapid fractionation, delivering 30 Gy at 3 Gy/fraction within 2 weeks, and standard fractionation, delivering > or =45 Gy at 1.8-2 Gy/fraction daily. Total doses of <50 Gy were usually given with rapid fractionation. Cisplatin and 5-fluorouracil were administrated to 93% of the patients. RESULTS: The patient characteristic that differed between the two groups was that patients in the lower dose group were more likely to have had weight loss >5% (46.2% vs. 23.3%). The lower dose group had more N1 tumors, but the tumor classification and stage grouping were similar in the two groups. The median follow-up time for all patients was 22 months (range, 2-56 months). Patients in the higher dose group had a statistically significant better 3-year local control rate (36% vs. 19%, p = 0.011), disease-free survival rate (25% vs. 10%, p = 0.004), and overall survival rate (13% vs. 3%, p = 0.054). A trend toward a better distant-metastasis-free survival rate was noted in the higher dose group (72% vs. 59%, p = 0.12). The complete clinical response rate was significantly greater in the higher dose group (46% vs. 23%, p = 0.048). In both groups, the most common type of first failure was persistence of the primary tumor. Significantly fewer patients in the higher dose group had tumor persistence after treatment (p = 0.02). No statistically significant difference was found between the two groups in the pattern of locoregional or distant failure. The long-term side effects of chemoradiotherapy were similar in the two groups, although it was difficult to assess the side effects accurately in a retrospective fashion. On multivariate analysis, Stage II (vs. III) disease and radiation dose >51 Gy were independent predictors of improved LRC, and locoregional failure was an independent predictor of worse overall survival. CONCLUSION: Our data suggested a positive correlation between radiation dose and LRC in the population studied. A higher radiation dose was associated with increased LRC and survival in the dose range studied. The data also suggested that better LRC was associated with a lower rate of distant metastasis. A threshold of tumor response to radiation dose might be present, as suggested by the flattened slope in the high-dose area on the dose-response curve. A carefully designed dose-escalation study is required to confirm this assumption.

Adult↗

PubMed2005

Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.

Acetylation↗

PubMed2005

Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry: multi-institution correlation of spectra and identification of biomarkers.

We report on a multicenter analysis of HUPO reference specimens using SELDI-TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at approximately 9200 and approximately 15,950 m/z were present only in select reference specimens. Chromatographic fractionation using anion-exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the approximately 9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high-resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population-based studies in all phases of the HUPO PPP.

Biomarkers↗