Sho Yoshitake, Melissa McKay-Daily, Masaki Tanaka and Zeqi Huang* Pages 35 - 47 ( 13 )
Background: Within the sulfotransferase (SULT) superfamily of metabolic enzymes, SULT1A1 and 1A3/4 isoforms are of particular interest, due to their abilities to catalyze the sulfation of phenolic endobiotics and xenobiotics. Although the difference in their substrate specificity is well documented, an isoform-specific quantification method is still not available.
Objective: To detect and quantify SULT1A1 and 1A3/4 in S9 fractions and cell lines using targeted mass spectrometry-based proteomics.
Method: Samples were tryptically digested, and signature peptides were quantified using liquid chromatography- multiple reaction monitoring mass spectrometry (LC-MRM/MS). Stable isotopelabeled (SIL) peptides were used as internal and calibration standards. SULT1A1 and SULT1A3/4 were quantified in various S9 fractions and cell line samples.
Results: Intraday and interday variabilities were low for relative quantification in S9 and cell line matrices (<8%). Expression profiles were validated using Western blot analysis of S9 fractions and lentiviral transduced SULT1A-overexpressing cell lines.
Conclusion: A reproducible method for simultaneous quantification of SULT1A1 and SULT1A3/4 in S9 fractions and cell line samples was established and validated.
LC-MRM/MS, SULT, quantitative proteomics, stable isotope labeled peptides, absolute quantification, S9 fractions, cell lines.
Otsuka Maryland Medicinal Laboratories, Rockville, MD 20850, Otsuka Maryland Medicinal Laboratories, Rockville, MD 20850, Otsuka Maryland Medicinal Laboratories, Rockville, MD 20850, Otsuka Maryland Medicinal Laboratories; 9900 Medical Center Drive, Rockville, MD