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Rapid identification of plasmalogen molecular species using targeted multiplexed selected reaction monitoring mass spectrometry.

ABSTRACT: Plasmalogens (Pls) levels are reported to be altered in several neurological and metabolic diseases. Identification of sn-1 fatty alcohols and sn-2 fatty acids of different Pls species is necessary to determine the roles and mechanisms of action of Pls in different diseases. Previously, full-scan tandem mass spectrometry (MS/MS) was used for this purpose but is not effective for low-abundance Pls species. Recently, multiplexed selected reaction monitoring MS (SRM/MS) was found to be more selective and sensitive than conventional full-scan MS/MS for the identification of low-abundance compounds. In the present study, we developed a liquid chromatography (LC)-targeted multiplexed SRM/MS system for the identification and quantification of different Pls choline (Pls-PC) and Pls ethanolamine (Pls-PE) species. We determined five precursor-product ion transitions to identify sn-1 and sn-2 fragments of each Pls species. Consequently, sn-1 and sn-2 fatty acyl chains of 22 Pls-PC and 55 Pls-PE species were identified in mouse brain samples. Among them, some species had C20:0 and C20:1 fatty alcohols at the sn-1 position. For quantification of Pls species in mouse brain samples, a single SRM transition was employed. Thus, our results suggest that the LC-targeted multiplexed SRM/MS system is very sensitive for the identification and quantification of low-abundance lipids such as Pls, and is thus expected to make a significant contribution to basic and clinical research in this field in the future.

SUBMITTER: Azad AK

PROVIDER: S-EPMC8662339 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Rapid identification of plasmalogen molecular species using targeted multiplexed selected reaction monitoring mass spectrometry.

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