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Di Girolamo F.,Multidisciplinary InterInstitutional BioBank BioBIM | Di Girolamo F.,Polytechnic of Milan | Boschetti E.,Polytechnic of Milan | Chung M.C.M.,National University of Singapore | And 2 more authors.
Journal of Proteomics | Year: 2011

The performance of sera pre-treatments for biomarker discovery has been recently assessed as very poor not only for immuno-subtraction, in turn evaluated as a tool unable to look deep into the low-abundance proteome (LAP) and thus incapable to lead to any novel biomarker discovery (J Proteome Res 2010;9:4982-4991), but also for combinatorial peptide ligand libraries (CPLL) (Proteomics 2010;10:1416-1425). The performance of both tools has been given as enabling to barely detect a meagre 25% more as compared to control, untreated sera. Meanwhile, other studies indicated the extreme effectiveness of peptide libraries to enlarge the knowledge of proteome compositions. In this contradictory situation we are here re-evaluating some protocol aspects and report that indeed CPLL is an excellent tool, able to dig really deep into the low-abundance proteome. The problem is that in those reports under-optimized capture and elution protocols had been adopted. With the protocols here reported, namely (a) abandoning the step of adding 150. mM salt to the sample; (b) capture at three different pH values (pH 4.0, 7.0 and 9.3) and (c), most importantly, eluting from CPLL beads in 4% boiling SDS in the presence of 25. mM DTT, we can largely expand the windows of visibility. In particular, it is here shown that a common elution protocol adopted in several reports, in 4. M urea and 1% CHAP, barely elutes about 15% of the captured species. Nevertheless if the CPLL beads thus treated are further eluted with boiling SDS-DTT, an additional 80% is recovered. © 2011 Elsevier B.V. Source

Spila A.,Multidisciplinary InterInstitutional BioBank BioBIM | Ettorre G.M.,San Camillo Hospital | Vennarecci G.,San Camillo Hospital | Santoro R.,San Camillo Hospital | And 2 more authors.
Clinica Chimica Acta | Year: 2011

Background: Vascular endothelial growth factor (VEGF165) is stored, transported and released by platelets. Platelet functional abnormalities have been described in patients with hepatocellular carcinoma (HCC). Thus, this study was designed to investigate the behavior of VEGF165 with respect to platelet activation in HCC. Methods: Plasma and serum VEGF165 and plasma sP-selectin levels were analyzed in patients with HCC (n=70) or cirrhosis (n=45) and control subjects (n=70). Given the thrombocytopenia that characterizes both HCC and cirrhotic patients, plasma VEGF165 and sP-selectin as well as serum VEGF (plt-VEGF165-load) levels were normalized by platelet counts. Results: Median concentrations of plasma VEGF165/platelet (p=0.002) and sP-selectin/platelet (p<0.0001) were higher in HCC or cirrhotic patients compared to controls. Moreover, sP-selectin/platelet was the only independent variable predictive of plasma VEGF165/platelet at multivariate analysis (p<0.0001). Conversely, plt-VEGF165-load correlated with tumor diameter (p<0.05) but not with sP-selectin/platelet and was an independent predictor for 5year overall survival (p=0.012). Conclusions: The results obtained are suggestive for VEGF165 release by tumor in HCC. It is plt-VEGF165-load, but not plasma VEGF165 or serum VEGF165 that is an independent predictor for overall survival of HCC patients. © 2010 Elsevier B.V. Source

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