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Quantification of peptide m/z distributions from 13C-labeled cultures with high resolution mass spectrometry
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With the introduction of orbital trap mass spectrometers molecular masses can be determined with great precision and accuracy. In addition, orbital trap spectrometers (Orbitraps) are sensitive and possess a linear dynamic range of multiple orders of magnitude. These qualities make the Orbitrap well-suited for both identification and quantification of compounds, and Orbitraps have become an instrument of choice for many proteomics applications. Orbitrap methods frequently include isotope labeling; therefore these instruments are potentially amenable to 13C metabolic flux analysis (MFA). Peptides synthesized from isotopically-labeled amino acids in metabolism, contain spatial and temporal information associated with the origin of the peptide, consequently, proteins that are expressed in a distinct tissue or during a specific developmental stage enable targeted metabolic studies. We investigated the suitability of Orbitrap measurements for isotope labeling quantification. Orbitrap spectra were carefully quantified; missing values were gap-filled; and standard deviations were estimated experimentally and compared to the expected multinomial sampling variance. Observed m/z distributions of labeled soy and E. coli peptides indicated no significant differences compared to simulated distributions. Gas chromatography mass spectrometry methods to quantify isotopic labeling in amino acids were extended and used with hydrolyzed protein from the same labeling experiments. The amino acids measured from GC-MS were mathematically convolved and compared with those obtained directly from the Orbitrap resulting in differences due to temporal labeling of proteins in metabolism. We conclude that Orbitrap measurements faithfully represent mass distributions, are suitable for quantification of isotope labeling-based studies, and are capable of assessing temporal differences in metabolism whereas other approaches cannot.
Doug K. Allen
James K. Gierse
Igor G.L. Libourel
Analytical Chemistry 2014 1 3 v.86
Journal Articles, USDA Authors, Peer-Reviewed
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