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Lopez M.F.,ThermoFisher Scientific BRIMS | Rezai T.,ThermoFisher Scientific BRIMS | Sarracino D.A.,ThermoFisher Scientific BRIMS | Prakash A.,ThermoFisher Scientific BRIMS | And 8 more authors.
Clinical Chemistry | Year: 2010

BACKGROUND: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminalPTHvariants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84. METHODS: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. RESULTS: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R2 = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%-12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%-9%. CONCLUSIONS: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH. © 2009 American Association for Clinical Chemistry.

Lopez M.,ThermoFisher Scientific BRIMS | Kuppusamy R.,ThermoFisher Scientific BRIMS | Sarracino D.,ThermoFisher Scientific BRIMS | Prakash A.,ThermoFisher Scientific BRIMS | And 6 more authors.
Journal of Proteome Research | Year: 2011

The accurate diagnosis of Trisomy 21 requires invasive procedures that carry a risk of miscarriage. The current state-of-the-art maternal serum screening tests measure levels of PAPP-A, free bhCG, AFP, and uE3 in various combinations with a maximum sensitivity of 60-75% and a false positive rate of 5%. There is currently an unmet need for noninvasive screening tests with high selectivity that can detect pregnancies at risk, preferably within the first trimester. The aim of this study was to apply proteomics and mass spectrometry techniques for the discovery of new putative biomarkers for Trisomy 21 in first trimester maternal serum coupled with the immediate development of quantitative selective reaction monitoring (SRM) assays. The results of the novel workflow were 2-fold: (1) we identified a list of differentially expressed proteins in Trisomy21 vs Normal samples, including PAPP-A, and (2) we developed a multiplexed, high-throughput SRM assay for verification of 12 new putative markers identified in the discovery experiments. To narrow down the initial large list of differentially expressed candidates resulting from the discovery experiments, we incorporated receiver operating characteristic (ROC) curve algorithms early in the data analysis process. We believe this approach provides a substantial advantage in sifting through the large and complex data typically obtained from discovery experiments. The workflow efficiently mined information derived from high-resolution LC-MS/MS discovery data for the seamless construction of rapid, targeted assays that were performed on unfractionated serum digests. The SRM assay lower limit of detection (LLOD) for the target peptides in a background of digested serum matrix was approximately 250-500 attomoles on column and the limit of accurate quantitation (LOQ) was approximately 1-5 femtomoles on column. The assay error as determined by coefficient of variation at LOQ and above ranged from 0 to 16%. The workfloweveloped in this study bridges the gap between proteomic biomarker discovery and translation into a clinical research environment. Specifically, for Trisomy 21, the described multiplexed SRM assay provides a vehicle for high-throughput verification of these, and potentially other, peptide candidates on larger sample cohorts. ©2011 American Chemical Society.

Prakash A.,Thermo Fisher Scientific | Rezai T.,Thermo Fisher Scientific | Krastins B.,Thermo Fisher Scientific | Sarracino D.,Thermo Fisher Scientific | And 14 more authors.
Journal of Proteome Research | Year: 2010

Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Four independent laboratories in North America, using identical triple-quadrupole mass spectrometers (Quantum Ultra, Thermo), were provided with standard protocols and instrumentation settings to analyze unknown samples and internal standards in a digested plasma matrix to quantify 51 peptides from 39 human proteins using a multiplexed SRM assay. The interlaboratory coefficient of variation (CV) was less than 10% for 25 of 39 peptides quantified (12 peptides were not quantified based upon hydrophobicity) and exhibited CVs less than 20% for the remaining peptides. In this report, we demonstrate that previously developed research platforms for SRM assays can be improved and optimized for deployment in clinical research environments. © 2010 American Chemical Society.

Lopez M.F.,Thermo Fisher Scientific | Sarracino D.A.,Thermo Fisher Scientific | Prakash A.,Thermo Fisher Scientific | Athanas M.,VAST Scientific | And 9 more authors.
Proteomics - Clinical Applications | Year: 2012

Purpose: Typically, apolipoproteins are individually measured in blood by immunoassay. In this report, we describe the development of a multiplexed selected reaction monitoring (SRM) based assay for a panel of apolipoproteins and its application to a clinical cohort of samples derived from acute stroke patients. Experimental Design: An SRM assay for a panel of nine apolipoproteins was developed on a triple quadrupole mass spectrometer. Quantitative data for each apolipoprotein were analyzed to determine expression ratio and receiver operating characteristic (ROC) values for ischemic versus hemorrhagic stroke. Results: The optimized SRM assay was used to interrogate a small cohort of well-characterized plasma samples obtained from patients with acute ischemic and hemorrhagic strokes. The ROC analyses demonstrated good classification power for several single apolipoproteins, most notably apoC-III and apoC-I. When a novel multi-marker ROC algorithm was applied, the ischemic versus hemorrhagic groups were best differentiated by a combination of apoC-III and apoA-I with an area under the curve (AUC) value of 0.92. Conclusions and clinical relevance: This proof-of-concept study provides interesting and provocative data for distinguishing ischemic versus hemorrhage within first week of symptom onset. However, the observations are based on one cohort of patient samples and further confirmation will be required. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Riter L.S.,Monsanto Corporation | Jensen P.K.,Monsanto Corporation | Ballam J.M.,Monsanto Corporation | Urbanczyk-Wochniak E.,Monsanto Corporation | And 6 more authors.
Analytical Methods | Year: 2011

The application of a label-free, LC-MS/MS based proteomics method for analysis of plant tissues was evaluated using both a spike study and case study in corn (Zea mays) leaf tissue. The spike study was utilized to establish a label-free proteomics protocol for corn leaf tissue, with focus on the assessment of sensitivity and accuracy. The data from this spike study indicated that this protocol had quantitative accuracy within ±20% of the true values and was able to differentiate 1.5 fold changes in protein abundance in a corn leaf matrix. Furthermore, the applicability of this protocol as a useful tool for answering biologically relevant questions was tested in a case study of the response of the proteome to night-to-day transition in corn leaf tissue. The label-free proteomics approach detected 136 differentially abundant proteins (FDR = 0.01 with an absolute log fold change ≥ 0.8) and 313 proteins whose abundance did not change in response to the diurnal cycle using ANOVA fixed effects model analysis. Identified proteins were mapped to their Gene Ontology (GO) biological processes and compared with expected diurnal biology. Many observed changes, including an increase in photosynthetic processes, were consistent with anticipated biological responses to the night-to-day transition. The results from the spike and case studies show that the label-free method can reliably provide a means to detect changes in protein abundance in plant tissue. © 2011 The Royal Society of Chemistry.

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