Quantitative proteomics studies are accomplished by the use of LC-MS analysis of tryptic peptides coupled with tagging with chemical reagents or metabolic labeling of cells in culture. The majority of current chemical tagging techniques label peptides on the N-terminus and epsilon-amino group of lysine using Nhydroxysuccinimide chemistry. Isobaric mass tags are particularly popular for biomarker discovery experiments since the mass difference between labeled forms occur on fragment ions rather than on the parent analyte (3). A limitation to this application, however, is that the isotope label is introduced post-protein extraction, meaning extreme care must be taken to ensure error is not introduced during sample preparation. We have shown the utility of using the SILAC labeled proteins as SILAP standards which can be applied to any source material (1). Proof of principle for the utility of this approach was obtained for cells in culture (4;5) and for serum samples from pancreatic cancer patients (6). In this latter study, the CAPAN-2 pancreatic cancer cell line was labeled by SILAC and secreted proteins from the conditioned media collected. The secreted labeled proteins were then used as a SILAP standard that was added to pooled pancreatic cancer or control serum Over one hundred differentially expressed biomarker candidates were identified. This approach was extended to study biomarkers in the Apcmin mouse, a colon cancer model (7), for cervicovaginal fluid samples in a preterm birth study (8), and differential protein expression in the post-synaptic density from schizophrenic compared with normal brain samples (9). The development of clinically useful biomarkers is a two-stage process. The initial bioanalytical validation stage uses control samples in appropriate biofluids to establish analytical limits of quantification and determine errors introduced by sample handling that are typically used in conventional bioanalytical validation studies (10). This stage is followed by clinical validation using patient samples to determine the assay?s sensitivity to detect the drug response or discrimination of a particular disease. We have focused on the use of LC-MS methodology for the analysis of DNA-adducts, glutathione-adducts, thiols, steroids, folates, and glucuronide and sulfate metabolites because it is applicable to the analysis of a wider range of biomarkers (11-16). The analysis of NSAIDs requires rigorous validation and quality control procedures to ensure that levels are determined with high accuracy and precision. This is accomplished by the use of stable isotope dilution methodology coupled with the implementation of rigorous standard operating procedures (10).

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54HL117798-02
Application #
8516106
Study Section
Special Emphasis Panel (ZGM1-PPBC-0)
Project Start
Project End
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
2
Fiscal Year
2013
Total Cost
$513,713
Indirect Cost
$59,753
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Ocana, Jesus A; Romer, Eric; Sahu, Ravi et al. (2018) Platelet-Activating Factor-Induced Reduction in Contact Hypersensitivity Responses Is Mediated by Mast Cells via Cyclooxygenase-2-Dependent Mechanisms. J Immunol 200:4004-4011
Grosser, Tilo; Naji, Ali; FitzGerald, Garret A (2018) Urinary Prostaglandin Metabolites: An Incomplete Reckoning and a Flush to Judgment. Circ Res 122:537-539
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Hao, Huifeng; Hu, Sheng; Wan, Qing et al. (2018) Protective Role of mPGES-1 (Microsomal Prostaglandin E Synthase-1)-Derived PGE2 (Prostaglandin E2) and the Endothelial EP4 (Prostaglandin E Receptor) in Vascular Responses to Injury. Arterioscler Thromb Vasc Biol 38:1115-1124
Mazaleuskaya, Liudmila L; Salamatipour, Ashkan; Sarantopoulou, Dimitra et al. (2018) Analysis of HETEs in human whole blood by chiral UHPLC-ECAPCI/HRMS. J Lipid Res 59:564-575
Chen, Lihong; Yang, Guangrui; Zhang, Jiayang et al. (2018) Time-Dependent Hypotensive Effect of Aspirin in Mice. Arterioscler Thromb Vasc Biol 38:2819-2826
Song, Wen-Liang; Ricciotti, Emanuela; Liang, Xue et al. (2018) Lipocalin-Like Prostaglandin D Synthase but Not Hemopoietic Prostaglandin D Synthase Deletion Causes Hypertension and Accelerates Thrombogenesis in Mice. J Pharmacol Exp Ther 367:425-432
Wang, Dandan; Hu, Xiaoyue; Lee, Seung Hee et al. (2018) Diabetes Exacerbates Myocardial Ischemia/Reperfusion Injury by Down-Regulation of MicroRNA and Up-Regulation of O-GlcNAcylation. JACC Basic Transl Sci 3:350-362
Di Giovanni, James P; Barkley, Robert M; Jones, David N M et al. (2018) Tandem Mass Spectrometry and Ion Mobility Reveals Structural Insight into Eicosanoid Product Ion Formation. J Am Soc Mass Spectrom 29:1231-1241

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