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Quantification of Sulfotransferases 1A1 and 1A3/4 in Tissue Fractions and Cell Lines by Multiple Reaction Monitoring Mass Spectrometry

[ Vol. 11 , Issue. 1 ]

Author(s):

Sho Yoshitake, Melissa McKay-Daily, Masaki Tanaka and Zeqi Huang*   Pages 35 - 47 ( 13 )

Abstract:


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.

Keywords:

LC-MRM/MS, SULT, quantitative proteomics, stable isotope labeled peptides, absolute quantification, S9 fractions, cell lines.

Affiliation:

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

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