Dataset Information

An 18O-labeling assisted LC/MS method for assignment of aspartyl/isoaspartyl products from Asn deamidation and Asp isomerization in proteins.

ABSTRACT: An (18)O-labeling assisted LC/MS method was designed for unambiguous assignment of aspartyl/isoaspartyl products produced by Asn deamidation and Asp isomerization. By preparing the acid- and base-catalyzed deamidation standards in H2(18)O, isomer-specific mass tags were introduced to aspartyl- and isoaspartyl-containing peptides, which could be easily distinguished by mass spectrometry (MS). In contrast to the traditional ways of assigning the isomers on the basis of their elution order in reverse phase HPLC, the new method is more reliable and universal. Furthermore, the new method can be applied to the entire protein digest, and is therefore more time- and cost-effective compared with existing methods that use synthetic aspartyl- and isoaspartyl-containing peptide standards. Finally, since the identification of isomers in the new method only relies on LC/MS analysis, it can be easily implemented using the most basic and inexpensive MS instrumentation, thus providing an attractive alternative to tandem MS based approaches. The feasibility of this new method is demonstrated using a model peptide as well as the entire digest of human serum transferrin.

SUBMITTER: Wang S

PROVIDER: S-EPMC3702055 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An 18O-labeling assisted LC/MS method for assignment of aspartyl/isoaspartyl products from Asn deamidation and Asp isomerization in proteins.

Wang Shunhai S Kaltashov Igor A IA

Analytical chemistry 20130613 13

An (18)O-labeling assisted LC/MS method was designed for unambiguous assignment of aspartyl/isoaspartyl products produced by Asn deamidation and Asp isomerization. By preparing the acid- and base-catalyzed deamidation standards in H2(18)O, isomer-specific mass tags were introduced to aspartyl- and isoaspartyl-containing peptides, which could be easily distinguished by mass spectrometry (MS). In contrast to the traditional ways of assigning the isomers on the basis of their elution order in rever ...[more]

PMID: 23713887

Similar Datasets

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.

Dataset Information

An 18O-labeling assisted LC/MS method for assignment of aspartyl/isoaspartyl products from Asn deamidation and Asp isomerization in proteins.

Publications

An 18O-labeling assisted LC/MS method for assignment of aspartyl/isoaspartyl products from Asn deamidation and Asp isomerization in proteins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets