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Characterizing the connectivity of poly-ubiquitin chains by selected reaction monitoring mass spectrometry.

ABSTRACT: Protein ubiquitination is an essential post-translational modification (PTM) involved in the regulation of a variety of cellular functions, including transcription and protein degradation. Proteins can be both mono- or poly-ubiquitinated. Poly-ubiquitin chains vary in the manner by which the ubiquitin proteins are linked and their total length. Different poly-ubiquitin structures are thought to specify different fates for the target protein but the correlation between poly-ubiquitin structures and their specific cellular function(s) is not well understood. We have developed a set of specific and quantitative targeted mass spectrometry assays to determine the frequency of different types of inter-ubiquitin linkages in poly-ubiquitin chains relative to the total ubiquitin concentration. We chemically synthesized heavy isotope labeled reference peptides that represent the products generated by tryptic digestion of the known forms of inter-ubiquitin links for the yeast Saccharomyces cerevisiae and human, in addition to all peptides from tryptic digestion of a single ubiquitin molecule for these two species. We used these peptides to develop optimized Selected Reaction Monitoring (SRM) assays for their unambiguous detection in biological samples. We used these assays to profile the frequency of the different types of inter-ubiquitin linkages in a mixture of in vitro assembled human poly-ubiquitin chains and 15 isolated poly-ubiquitinated proteins from S. cerevisiae. We then applied the method to detect toxin induced changes in the poly-ubiquitination profile in complex and enriched protein samples.

SUBMITTER: Mirzaei H

PROVIDER: S-EPMC3057100 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Characterizing the connectivity of poly-ubiquitin chains by selected reaction monitoring mass spectrometry.

Mirzaei Hamid H Rogers Richard S RS Grimes Barbara B Eng Jimmy J Aderem Alan A Aebersold Ruedi R

Molecular bioSystems 20100806 10

Protein ubiquitination is an essential post-translational modification (PTM) involved in the regulation of a variety of cellular functions, including transcription and protein degradation. Proteins can be both mono- or poly-ubiquitinated. Poly-ubiquitin chains vary in the manner by which the ubiquitin proteins are linked and their total length. Different poly-ubiquitin structures are thought to specify different fates for the target protein but the correlation between poly-ubiquitin structures a ...[more]

PMID: 20694217

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Project description:For many research questions in modern molecular and systems biology, information about absolute protein quantities is imperative. This information includes, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes, or quantitative comparisons of different proteins within one sample or across samples. To date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers of samples. Here we describe and demonstrate the utility of a targeting MS technique for the estimation of absolute protein abundance in unlabeled and nonfractionated cell lysates. The method is based on selected reaction monitoring (SRM) mass spectrometry and the "best flyer" hypothesis, which assumes that the specific MS signal intensity of the most intense tryptic peptides per protein is approximately constant throughout a whole proteome. SRM-targeted best flyer peptides were selected for each protein from the peptide precursor ion signal intensities from directed MS data. The most intense transitions per peptide were selected from full MS/MS scans of crude synthetic analogs. We used Monte Carlo cross-validation to systematically investigate the accuracy of the technique as a function of the number of measured best flyer peptides and the number of SRM transitions per peptide. We found that a linear model based on the two most intense transitions of the three best flying peptides per proteins (TopPep3/TopTra2) generated optimal results with a cross-correlated mean fold error of 1.8 and a squared Pearson coefficient R(2) of 0.88. Applying the optimized model to lysates of the microbe Leptospira interrogans, we detected significant protein abundance changes of 39 target proteins upon antibiotic treatment, which correlate well with literature values. The described method is generally applicable and exploits the inherent performance advantages of SRM, such as high sensitivity, selectivity, reproducibility, and dynamic range, and estimates absolute protein concentrations of selected proteins at minimized costs.

Dataset Information

Characterizing the connectivity of poly-ubiquitin chains by selected reaction monitoring mass spectrometry.

Publications

Characterizing the connectivity of poly-ubiquitin chains by selected reaction monitoring mass spectrometry.

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