Optimized Method for Computing 18O/16O Ratios of Differentially Stable-Isotope Labeled Peptides in the Context of Postdigestion 18O Exchange/Labeling

摘要

Differential 18O/16O stable isotope labeling of peptidesnthat relies on enzyme-catalyzed oxygen exchange at theirncarboxyl termini in the presence of H2n18O has beennwidely used for relative quantitation of peptides/proteins.nThe role of tryptic proteolysis in bottom-up shotgunnproteomics and low reagent costs have made trypsincatalyzedn18O postdigestion exchange a convenient andnaffordable stable isotope labeling approach. However, itnis known that trypsin-catalyzed 18O exchange at thencarboxyl terminus is in many instances inhomogeneousincomplete. The extent of the 18Oexchange/incorporationnfluctuates from peptide to peptide mostly due to variablenenzyme-substrate affinity. Thus, accurate calculationnand interpretation of peptide ratios are analytically complicatednand in some regard deficient. Therefore, ancomputational approach capable of improved measurementnof actual 18O incorporation for each differentiallynlabeled peptide pair is needed. In this regard, we havendeveloped an algorithmic method that relies on thentrapezoidal rule to integrate peak intensities of all detectednisotopic species across a particular peptide ionnover the retention time, which fits the isotopic manifoldnto Poisson distributions. Optimal values for manifoldnfitting were calculated and then 18O/16O ratios derivednvia evolutionary programming. The algorithm is testednusing trypsin-catalyzed 18O postdigestion exchange tondifferentially label bovine serum albumin (BSA) at anpriori determined ratios. Both accuracy and precision arenimproved utilizing this rigorous mathematical approach.nWe further demonstrate the effectiveness of this methodnto accurately calculate 18O/16O ratios in a large scalenproteomic quantitation of detergent resistant membranenmicrodomains (DRMMs) isolated from cells expressingnwild-type HIV-1 Gag and its nonmyristylated mutant.