Natural Flanking Sequences for Peptides Included in Quantification Concatamer Internal Standard

Published

January 20, 2015

Author(s)

Sao F. Cheung, Kyle Anderson, Meiyao Wang, Illarion Turko

Abstract

Quantification by targeted proteomics has largely depended on mass spectrometry and isotope-labeled internal standards. In addition to traditionally used recombinant proteins or synthetic peptides, concatenated peptides (QconCATs) were introduced as a conceptually new source of internal standard. In the present study, we focused on assessing the length of natural flanking sequences, which surround each peptide included in the QconCAT and provide for identical rates of analyte and standard digestion by trypsin. We have expressed, purified, and characterized a set of seven 15N-labeled QconCATs that cover seven tryptic peptides from human clusterin with a length of natural flanking sequences ranging from none (+0) to six amino acid residues (+6) for each tryptic peptide. Individual QconCATs were mixed with recombinant human clusterin at a 1:1 molar ratio, digested, and actual ratios for each combination of peptide/flanking sequence was measured with a multiple reaction monitoring assay. Data analysis suggested that natural flanking sequences shorter than +6 residues can cause a quantitative error since the random appearance of other amino acid residues in close proximity to trypsin cleavage sites have unpredictable consequences for the digestion rates of QconCATs.

Citation

Analytical Chemistry

Volume

87

Issue

2

Pub Type

Journals

Download Paper

DOI Link

Keywords

Targeted proteomics, Internal standard, QconCAT, natural flanking sequence

Citation

Cheung, S. , Anderson, K. , Wang, M. and Turko, I. (2015), Natural Flanking Sequences for Peptides Included in Quantification Concatamer Internal Standard, Analytical Chemistry, [online], https://doi.org/10.1021/ac503697j (Accessed March 13, 2025)