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Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra.

ABSTRACT: It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated for all the OIEs sharing this OIP. The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated. The final individual abundance of the OIP for each OIE is the individual ideal experimental abundance multiplied by 1?+?RD. Initial studies were performed using higher-energy collisional dissociation tandem mass spectra on myoglobin (with direct infusion) and the intact E. coli proteome (with liquid chromatographic separation). Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved. The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available.

SUBMITTER: Xiao K

PROVIDER: S-EPMC4593959 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra.

Xiao Kaijie K Yu Fan F Fang Houqin H Xue Bingbing B Liu Yan Y Tian Zhixin Z

Scientific reports 20151006

It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated ...[more]

PMID: 26439836

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Project description:BackgroundMethods used to quantify protein from biological samples are often inaccurate with significant variability that requires care to minimize. The errors result from losses during protein preparation and purification and false detection of interfering compounds or elements. Amino acid analysis (AAA) involves a series of chromatographic techniques that can be used to measure protein levels, avoiding some difficulties and providing specific compositional information. However, unstable derivatives, that are toxic and can be costly, incomplete reactions, inadequate chromatographic separations, and the lack of a single hydrolysis method with sufficient recovery of all amino acids hinder precise protein quantitation using AAA.ResultsIn this study, a hydrophilic interaction chromatography based method was used to separate all proteinogenic amino acids, including isobaric compounds leucine and isoleucine, prior to detection by multiple reaction monitoring with LC-MS/MS. Through inclusion of commercially available isotopically labeled (13C, 15N) amino acids as internal standards we adapted an isotopic dilution strategy for amino acid-based quantification of proteins. Three hydrolysis methods were tested with ubiquitin, bovine serum albumin, (BSA), and a soy protein biological reference material (SRM 3234; NIST) resulting in protein estimates that were 86-103%, 82-94%, and 90-99% accurate for the three protein samples respectively. The methane sulfonic acid hydrolysis approach provided the best recovery of labile amino acids including: cysteine, methionine and tryptophan that are challenging to accurately quantify.ConclusionsAccurate determination of protein quantity and amino acid composition in heterogeneous biological samples is non-trivial. Recent advances in chromatographic phases and LC-MS/MS based methods, along with the availability of isotopic standards can minimize difficulties in analysis and improve protein quantitation. A robust method is described for high-throughput protein quantification and amino acid compositional analysis. Since accurate measurement of protein quality and quantity are a requirement for many biological studies that relate to crop improvement or more generally, our understanding of metabolism in living systems, we envision this method will have broad applicability.

Dataset Information

Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra.

Publications

Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra.

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