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Quantification of isotopically overlapping deamidated and 18o-labeled peptides using isotopic envelope mixture modeling.

ABSTRACT: A robust peptide quantification method was developed where overlapping peptide isotopic distributions were fit with predicted peptide isotopic envelope mixture models (IEMMs). Application to two difficult quantitative problems was demonstrated. The first was the quantification of deamidation, where masses of isotopic peaks differ by 1 Da, and the second was (18)O labeling, where the isotopic peaks are shifted 2 and 4 Da. In both cases, peptide quantification cannot be performed by simple integration of extracted ion chromatograms, because the isotopic envelopes of mass-shifted peptides are normally not resolved. To test the methodology for quantification of deamidation, several synthetic peptides and their corresponding deamidated forms were mixed at various ratios (1:0, 1:2, 2:1, 4:1, 10:1, and 20:1) and analyzed using the IEMM method, resulting in a high correlation (R(2) = 0.96) between measured and known percentages of deamidation. The IEMM method was then incorporated into a workflow for deamidation quantification in a large-scale proteomics experiment. A series of normal (3 day, 2 year, 35 year, and 70 year) and cataractous (93 year) human lenses were analyzed using two-dimensional liquid chromatography tandem mass spectrometry, and deamidation quantities of several gammaS-crystallin peptides ([N14-Q16], N53, [Q63-Q70], and N143) were determined. Two peptides (N53 and [Q63-Q70]) had more extensive deamidation in the water-insoluble portions of normal lens samples, and deamidation at N143 was more extensive in the 93 year water-insoluble cataractous sample. The utility of the technique for analysis of (18)O-labeled peptides was examined using mixtures of labeled BSA peptides in known (16)O/(18)O ratios (10:1, 4:1, 1:1, 1:4, and 1:10). The methodology allowed for accurate measurements of ratios of (16)O/(18)O peptides over the wide range of relative abundances.

SUBMITTER: Dasari S

PROVIDER: S-EPMC2693468 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Quantification of isotopically overlapping deamidated and 18o-labeled peptides using isotopic envelope mixture modeling.

Dasari Surendra S Wilmarth Phillip A PA Reddy Ashok P AP Robertson Lucinda J G LJ Nagalla Srinivasa R SR David Larry L LL

Journal of proteome research 20090301 3

A robust peptide quantification method was developed where overlapping peptide isotopic distributions were fit with predicted peptide isotopic envelope mixture models (IEMMs). Application to two difficult quantitative problems was demonstrated. The first was the quantification of deamidation, where masses of isotopic peaks differ by 1 Da, and the second was (18)O labeling, where the isotopic peaks are shifted 2 and 4 Da. In both cases, peptide quantification cannot be performed by simple integra ...[more]

PMID: 19173613

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Project description:BackgroundQuantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-beta treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable.ResultsThe presented method assesses, in complete cell lysates, the degree of phosphorylation of specific peptide residues from MS spectra using 18O/16O labeling. The abundance of each observed phospho-peptide from two cell states was estimated from three overlapping isotope contours. The influence of peptide-specific labeling efficiency was removed by performing a label swapped experiment and assuming that the labeling efficiency was unchanged upon label swapping. Different degrees of phosphorylation were reported using the fold change measure which was extended with a confidence interval found to reflect the quality of the underlying spectra. Furthermore a new way of method assessment using simulated data is presented. Using simulated data generated in a manner mimicking real data it was possible to show the method's robustness both with increasing noise levels and with decreasing labeling efficiency.ConclusionThe fold change error assessable on simulated data was on average 0.16 (median 0.10) with an error-to-signal ratio and labeling efficiency distributions similar to the ones found in the experimentally observed spectra. Applied to experimentally observed spectra a very good match was found to the model (<10% error for 85% of spectra) with a high degree of robustness, as assessed by data removal. This new method can thus be used for quantitative signal cascade analysis of total cell extracts in a high throughput mode.

Project description:Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-?-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard.

Project description:Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) coupled with stable isotope standards (SIS) has been used to quantify native peptides. This peptide quantification by MALDI-TOF approach has difficulties quantifying samples containing peptides with ion currents in overlapping spectra. In these overlapping spectra the currents sum together, which modify the peak heights and make normal SIS estimation problematic. An approach using Gaussian mixtures based on known physical constants to model the isotopic cluster of a known compound is proposed here. The characteristics of this approach are examined for single and overlapping compounds. The approach is compared to two commonly used SIS quantification methods for single compound, namely Peak Intensity method and Riemann sum area under the curve (AUC) method. For studying the characteristics of the Gaussian mixture method, Angiotensin II, Angiotensin-2-10, and Angiotenisn-1-9 and their associated SIS peptides were used. The findings suggest, Gaussian mixture method has similar characteristics as the two methods compared for estimating the quantity of isolated isotopic clusters for single compounds. All three methods were tested using MALDI-TOF mass spectra collected for peptides of the renin-angiotensin system. The Gaussian mixture method accurately estimated the native to labeled ratio of several isolated angiotensin peptides (5.2% error in ratio estimation) with similar estimation errors to those calculated using peak intensity and Riemann sum AUC methods (5.9% and 7.7%, respectively). For overlapping angiotensin peptides, (where the other two methods are not applicable) the estimation error of the Gaussian mixture was 6.8%, which is within the acceptable range. In summary, for single compounds the Gaussian mixture method is equivalent or marginally superior compared to the existing methods of peptide quantification and is capable of quantifying overlapping (convolved) peptides within the acceptable margin of error.

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Quantification of isotopically overlapping deamidated and 18o-labeled peptides using isotopic envelope mixture modeling.

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Quantification of isotopically overlapping deamidated and 18o-labeled peptides using isotopic envelope mixture modeling.

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