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Anal Chem. 2010 Jul 1;82(13):5878-86. doi: 10.1021/ac101284c.

Optimized method for computing (18)O/(16)O ratios of differentially stable-isotope labeled peptides in the context of postdigestion (18)O exchange/labeling.

Author information

1
Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Maryland 21702, USA.

Abstract

Differential (18)O/(16)O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H(2)(18)O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs have made trypsin-catalyzed (18)O postdigestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed (18)O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the (18)O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme-substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual (18)O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then (18)O/(16)O ratios derived via evolutionary programming. The algorithm is tested using trypsin-catalyzed (18)O postdigestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both accuracy and precision are improved utilizing this rigorous mathematical approach. We further demonstrate the effectiveness of this method to accurately calculate (18)O/(16)O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its nonmyristylated mutant.

PMID:
20540505
PMCID:
PMC3479679
DOI:
10.1021/ac101284c
[Indexed for MEDLINE]
Free PMC Article

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