Urine profiling by SELDI-TOF/MS: monitoring of the critical steps in sample collection, handling and analysis.

The topic of this study is the impact of several pre-analytical and analytical variables on proteomic profiling of human urine by surface enhanced laser desorption/ionization time of flight-mass spectrometry (SELDI-TOF-MS) in healthy subjects. Urine storage at room temperature caused a progressive degradation of proteins, which was prevented by the addition of protease inhibitors only up to 2 h from the collection. The timing of collection over the day had only a minor impact on protein profile, although influencing the intensity of peaks. Repeated freeze/thaw cycles (up to five) did not affect either the number or the intensity of the peaks. A comparison of the protein profile from eight different healthy individuals showed fairly consistent inter-subject similarities, along with between-subject differences, which were markedly dependent on the sex and the type of ProteinChip array used. The addition of a variety of denaturing agents improved the quality of the spectra with all the chips tested (CM10, Q10 and H50), but not with the copper-coated IMAC-30 chip. Finally, SPA matrix allowed to achieve a better performance of SELDI-TOF/MS spectrum, as compared with CHCA, regardless of the ProteinChip array used and even in the low m/z range (2500-10,000). In conclusion, we suggest that a careful choice of a number of pre-analytical and analytical conditions is required to accomplish and define a unifying protocol for the analysis of human urine by SELDI-TOF/MS, in physiological and in pathological states.

HMGB1 binds to the rs7903146 locus in TCF7L2 in human pancreatic islets.

The intronic SNP rs7903146 in the T-cell factor 7-like 2 gene (TCF7L2) is the common genetic variant most highly associated with Type 2 diabetes known to date. The risk T-allele is located in an open chromatin region specific to human pancreatic islets of Langerhans, thereby accessible for binding of regulatory proteins. The risk T-allele locus exhibits stronger enhancer activity compared to the non-risk C-allele. The aim of this study was to identify transcriptional regulators that bind the open chromatin region in the rs7903146 locus and thereby potentially regulate TCF7L2 expression and activity. Using affinity chromatography followed by Edman sequencing, we identified one candidate regulatory protein, i.e. high-mobility group protein B1 (HMGB1). The binding of HMGB1 to the rs7903146 locus was confirmed in pancreatic islets from human deceased donors, in HCT116 and in HEK293 cell lines using: (i) protein purification on affinity columns followed by Western blot, (ii) chromatin immunoprecipitation followed by qPCR and (iii) electrophoretic mobility shift assay. The results also suggested that HMGB1 might have higher binding affinity to the C-allele of rs7903146 compared to the T-allele, which was supported in vitro using Dynamic Light Scattering, possibly in a tissue-specific manner. The functional consequence of HMGB1 depletion in HCT116 and INS1 cells was reduced insulin and TCF7L2 mRNA expression, TCF7L2 transcriptional activity and glucose stimulated insulin secretion. These findings suggest that the rs7903146 locus might exert its enhancer function by interacting with HMGB1 in an allele dependent manner.

Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS): from sample collection to data analysis.

BACKGROUND: Saliva is one of the most promising and easy-to-collect source of potential biomarkers of oral and systemic disease. We standardized a protocol suitable for pre-analytical treatment and for the analysis of whole normal saliva by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS). METHODS: We evaluated the impact of storage time, freeze/thaw cycles, denaturing agents, glycoproteins depletion, centrifugation, type of matrix and ProteinChip used on the quality of the SELDI protein profile. Moreover, we explored the inter-individual and between-sex differences and the changes in the sample composition over the day. RESULTS: Saliva was qualitatively stable, in the absence of protease inhibitors, for up to 3 h from the collection at room temperature, although the intensity of a number of peaks slightly decreased between 0 and 3 h and the addition of protease inhibitors did not completely revert this trend. The saliva proteome changed during the day and showed relevant between-sex differences. The protein profile remained stable for up to five freeze/thaw cycles. The addition of denaturing solutions and the depletion of glycoproteins improved the quality of the spectra without affecting their reproducibility. CONCLUSIONS: We defined a protocol that improved the quality and the reproducibility of SELDI-TOF/MS analysis, thus potentially supporting the search for putative biomarkers of disease.

Structural study of GCDFP-15/gp17 in disease versus physiological conditions using a proteomic approach.

Gross cystic disease fluid protein (GCDFP-15), also known as prolactin-inducible protein (PIP), is a specific breast tumor marker. GCDFP-15/PIP is also identified as gp17 and/or seminal actin-binding protein (SABP) from seminal vesicles and as extraparotid glycoprotein (EP-GP) from salivary glands. It is an aspartyl proteinase able to specifically cleave fibronectin (FN), suggesting a possible involvement in mammary tumor progression and fertilization. Other functions were attributed to this protein(s) on the basis of its ability to interact with an array of molecules such as CD4, actin, and fibrinogen. We investigated the structure of the protein(s) under disease versus physiological conditions by RP-HPLC chromatography, ProteinChip technology, and QStar MS/MS mass spectrometry. The proteins behaved differently when examined by RP-HPLC chromatography and surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry, suggesting different conformations and/or tissue-specific posttranslational modifications of the proteins, although their primary structure was identical by MS/MS analysis. Both showed a single N-glycosylation site. A different N-linked glycosylation pattern was observed in pathological GCDFP-15/PIP as compared with physiological gp17/SABP protein by coupling enzymatic digestion and ProteinChip technology. Furthermore, taking advantage of ProteinChip technology, we analyzed the interaction of both proteins with CD4 and FN. We observed that the physiological form was mainly involved in the binding to CD4. Moreover, we defined the specific FN binding-domain of this protein. These data suggested that, depending on its conformational state, the protein could differently bind to its various binding molecules and change its function(s) in the microenviroments where it is expressed.

The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension.

An oxygen responsive transcription factor regulating human glutathione peroxidase gene (GPx) through two oxygen responsive elements (ORE I and ORE2) has been purified and characterized by sequence-specific DNA affinity chromatography. The DNA binding activity, termed Oxygen Responsive Element Binding Protein (OREBP), was partially represented by a 77 kD polypeptide (p70) possessing a blocked N-terminus. The p70 subunit co-eluted with an 86 kD subunit (p80) from affinity columns. N-terminal sequencing analysis of the 86 kD component revealed that this protein represented the larger member of the Ku antigen complex. The identity of the purified 77 kD subunit was determined by Western blot analysis using an antibody directed against the p70 protein. In addition to binding the GPx-ORE, the OREBP was itself regulated by oxygen tension. It was found that the abundance of the ORE binding activity was decreased in cells maintained at low oxygen tension (40 mm Hg). Anti-Ku-antibodies specifically supershifted the OREBP-ORE DNA complex. These observations further add to the numerous nuclear roles of the Ku-transcription factor.