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Rossi C.,Centro Studi Sullinvecchiamento Cesi | Rossi C.,University of Chieti Pescara | Calton L.,Waters Corporation | Hammond G.,Waters Corporation | And 5 more authors.
Clinica Chimica Acta | Year: 2010

Background: Diagnosis of Congenital Adrenal Hyperplasia (CAH) is based on the quantification of 17-hydroxyprogesterone (17-OHP), usually by immunoassay. During the neonatal period the specificity of screening for CAH by blood spot 17-OHP immunoassay is low. High false-positive rates result in a relatively high demand for a second-tier serum confirmation test. A robust, specific and selective method for measurement of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstene-3,17-dione (A4) and 17-OHP in serum has been developed. The method involves a simple extraction procedure and a fast analysis using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS). Methods: The steroids were extracted from 50 μl of serum using methyl-tert-butyl-ether. Analysis was performed on a UPLC tandem quadrupole mass spectrometer system in positive mode electrospray ionization and multiple reaction monitoring acquisition. Results: The assay was linear over each analyte concentration range with all correlation coefficients (r2) > 0.996. Inter- and intra-day CVs were ≤ 10% across the analytical range. In addition simultaneous measurement of the full range of steroids on the pathway to cortisol allows confirmation of the affected steroidogenic enzyme. Conclusions: A second-tier test for the confirmation of CAH has been developed. The method allows for detection and quantification of 5 steroids related to CAH over the range of the clinical assay with good linearity, sensitivity and precision. © 2009 Elsevier B.V. All rights reserved.

Ciavardelli D.,Centro Studi Sullinvecchiamento Cesi | Ciavardelli D.,University of Chieti Pescara | Sacchetta P.,University of Chieti Pescara | Federici G.,Cervello | And 5 more authors.
Talanta | Year: 2010

Molecular mass spectrometry (MS) analysis of protein phosphorylation is partially limited by the molecular specie specificity of the analytical responses that might impair both qualitative and quantitative performances. Elemental MS, such as inductively coupled plasma mass spectrometry (ICP-MS) can overcome these drawbacks; in fact, analytical performance is theoretically independent of the molecular structure of a target analyte naturally containing the elements of interest. Nevertheless, isobaric interferences derived from sample matrix and laboratory environment can hinder the quantitative determination of both phosphorus (P) and sulfur (S) as 31P+ and 32S+ by inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) under standard plasma conditions. These interferences may be overcome by quantifying P and S as oxide ions 31P16O+ and 32S16O+, respectively. In this study, we present a systematic investigation on the effect of plasma instrumental conditions on the oxide ion responses by a design of experiment approach for the simultaneous ICP-QMS determination of P and S (31P16O+ and 32S16O+, respectively) in protein samples without the use of dynamic reaction, collision reaction cells or pre-addition of oxygen as reactant gas in the torch. The proposed method was evaluated in terms of limit of detection, limit of quantification, linearity, repeatability, and trueness. Moreover, detection and quantification capabilities of the optimized method were compared to the standard plasma mode for determination of 31P+ and 34S+. Spectral and non-spectral interferences affecting the quantification of 31P+, 31P16O+ and 32S16O+ were also studied. The suitability of inorganic elemental standards for P and S quantification in proteins was assessed. The method was applied to quantify the phosphorylation stoichiometry of commercially available caseins (bovine β-casein, native and dephosphorylated α-casein) and results were confirmed by Matrix Assisted Laser Desorption Ionization Time of Flight MS analysis. We demonstrate that ICP-QMS, by quantifying P and S as oxide ions, was able to accurately calculate the degree of phosphorylation of β-casein and α-casein and to detect specific partial enzymatic dephosphorylation. The collected results might lead to further development of ICP-QMS interfaces optimized for protein phosphorylation studies and for proteomics investigations. © 2009 Elsevier B.V. All rights reserved.

Mantini D.,CNR Institute of Biomedical Technologies | Mantini D.,University of Chieti Pescara | Mantini D.,Centro Studi Sullinvecchiamento Cesi | Mantini D.,Catholic University of Leuven | And 19 more authors.
Journal of Proteomics | Year: 2010

Background: Mass spectrometry (MS) is becoming the gold standard for biomarker discovery. Several MS-based bioinformatics methods have been proposed for this application, but the divergence of the findings by different research groups on the same MS data suggests that the definition of a reliable method has not been achieved yet. In this work, we propose an integrated software platform, MASCAP, intended for comparative biomarker detection from MALDI-TOF MS data. Results: MASCAP integrates denoising and feature extraction algorithms, which have already shown to provide consistent peaks across mass spectra; furthermore, it relies on statistical analysis and graphical tools to compare the results between groups. The effectiveness in mass spectrum processing is demonstrated using MALDI-TOF data, as well as SELDI-TOF data. The usefulness in detecting potential protein biomarkers is shown comparing MALDI-TOF mass spectra collected from serum and plasma samples belonging to the same clinical population. Conclusions: The analysis approach implemented in MASCAP may simplify biomarker detection, by assisting the recognition of proteomic expression signatures of the disease. A MATLAB implementation of the software and the data used for its validation are available at http://www.unich.it/proteomica/bioinf. © 2009 Elsevier B.V. All rights reserved.

Pieragostino D.,Centro Studi Sullinvecchiamento Cesi | Pieragostino D.,University of Chieti Pescara | Pieragostino D.,Cervello | Petrucci F.,Centro Studi Sullinvecchiamento Cesi | And 23 more authors.
Journal of Proteomics | Year: 2010

Serum proteome investigations have raised an incredible interest in the research of novel molecular biomarker, nevertheless few of the proposed evidences have been translated to the clinical practice. One of the limiting factors has been the lack of generally accepted guidelines for clinical proteomics studies and the lack of a robust analytical and pre-analytical ground for the proposed classification models. Pre-analytical issues may results in a deep impact for biomarker discovery campaign. In this study we present a systematic evaluation of sample storage and sampling conditions for clinical proteomics investigations. We have developed and validated a linear MALDI-TOF-MS protein profiling method to explore the low protein molecular weight region (5-20 kDa) of serum samples. Data normalization and processing was performed using optimise peak detection routine (LIMPIC) able to describe each group under investigation. Data were acquired either from healthy volunteers and from multiple sclerosis patients in order to highlight ex vivo protein profile alteration related to different physio-pathological conditions. Our data showed critical conditions for serum protein profiles depending on storage times and temperatures: 23 °C, 4 °C, - 20 °C and - 80 °C. We demonstrated that upon a - 20 °C short term storage, characteristic degradation profiles are associated with different clinical groups. Protein signals were further identified after preparative HPLC separation by peptide sequencing on a nanoLC-Q-TOF TANDEM mass spectrometer. Apolipoprotein A-IV and complement C3 protein fragments, transthyretin and the oxidized isoforms in different apolipoprotein species represent the major molecular features of such a degradation pattern. © 2009 Elsevier B.V. All rights reserved.

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