Subhasish Purkayastha

Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents

We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Δ and xrn1Δ mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5′ to 3′ decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.

LC-ESI-MS/MS analysis of testosterone at sub-picogram levels using a novel derivatization reagent.

Testosterone analysis by LC-MS/MS is becoming the analytical method of choice over immunoassays due to its specificity and accuracy. However, neutral steroid hormones possess poor ionization efficiency in MS/MS, resulting in insufficient sensitivity for analyzing samples with trace concentrations of the hormones. The method presented here utilizes a derivatization step involving a novel, permanently charged, quaternary aminooxy (QAO) reagent or MS-tag that reacts to the ketone functionality of testosterone and significantly enhances its ESI-MS/MS sensitivity. This derivatization method enabled quantitation of total testosterone in human serum (200 μL) with a lower limit of quantitation (LLOQ) of 1 pg/mL (3.47 pmol/L), total testosterone in dried blood spots (8-10 μL) with a LLOQ of 40 pg/mL, and free testosterone in serum ultrafiltrate (400 μL) with a LLOQ of 0.5 pg/mL. The linearity of each of the high sensitivity applications was maintained over a broad dynamic range of 1-5000 pg/mL for the serum samples and 40-10,000 pg/mL for the dried blood spots (DBS) with R(2) >0.998. The %CV at the LLOQ was <15 for all applications. The QAO derivatization and sample preparation workflows are quick, simple, and robust. Comparison of the derivatization method with an LC-ESI-MS/MS nonderivatization method yielded high correlation and agreement. The derivatization reagent is universal and reacts with other compounds containing ketone or aldehyde functionality.

Quantitative protein determination for CYP induction via LC‐MS/MS

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel‐free, high‐throughput LC‐MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope‐labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC‐MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC‐MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays