Biosynthetic concatenated labeled peptides are useful alternatives to whole labeled proteins: human serum albumin as a case study

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	NIMH	PROTEOM-2

Authors

• J Cole
• D Nanavati
• C Chen
• B Martin
• A Makusky
• G Csako
• S Markey

Abstract

We and others have reported on concatenated signature peptides as alternatives to whole labeled proteins as internal standards for protein quantification through proteolytic peptide measurement. We reported peptide yield and quantification for full length human serum albumin and 15N-HSA with a concatemer standard containing 15N, 13C-Lys/Arg. Based on that work, we have extended our study to development of a sensitive assay for HSA in human urine, demonstrating equivalence for the benchmark isotopic dilution assay reported using 15N-HSA, and comparison with an established clinical laboratory immunoturbidometric assay. For this study, an external calibration curve was constructed using the area ratio of the HSA to the internal standard concatemer after both were added to a urine matrix and digested with trypsin. The HSA peptide sequences LVNEVTEFAK, SLHTLFGDK, and FQNALLVR were analyzed on a triple quadrupole analyzer by multiple reaction monitoring following several parent-to-product ion transitions per peptide. These labeled concatemers are of potential value in clinical chemistry where multiple proteins could be assayed with single reference labeled concatemer standards. We will present data on 37 urine samples in which the concentration was determined by using our LC-MS/MS MRM method as well as an established immunoturbidometric assay performed in a clinical laboratory.

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