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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Bender, Gunes; Schexnaydre, Erin E; Murphy, Robert C et al. (2016) Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart: IMPLICATIONS FOR MURINE MODELS OF ATHEROSCLEROSIS. J Biol Chem 291:19413-24
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Guo, Lili; Worth, Andrew J; Mesaros, Clementina et al. (2016) Diisopropylethylamine/hexafluoroisopropanol-mediated ion-pairing ultra-high-performance liquid chromatography/mass spectrometry for phosphate and carboxylate metabolite analysis: utility for studying cellular metabolism. Rapid Commun Mass Spectrom 30:1835-45
Xiang, Yaozu; Hwa, John (2016) Regulation of VWF expression, and secretion in health and disease. Curr Opin Hematol 23:288-93
Dong, Liang; Zou, Hechang; Yuan, Chong et al. (2016) Interactions of 2-O-arachidonylglycerol ether and ibuprofen with the allosteric and catalytic subunits of human COX-2. J Lipid Res 57:1043-50

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