Journal
ANALYTICAL CHEMISTRY
Volume 86, Issue 12, Pages 5633-5637Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac501451v
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Funding
- U.S. National Cancer Institute (NCI) [CA143883, CA083639]
- Cancer Prevention Research Institute of Texas (CPRIT) [RP130397]
- NIH [1S10OD012304-01]
- Chapman Foundation
- Michael and Susan Dell Foundation
- MD Anderson's NCI Cancer Center [CA016672]
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Advances in metabolomics, particularly for research on cancer, have increased the demand for accurate, highly sensitive methods for measuring glutamine (Gin) and glutamic acid (Glu) in cell cultures and other biological samples. N-terminal Gin and Glu residues in proteins or peptides have been reported to cyclize to pyroglutamic acid (pG1u) during liquid chromatography (LC)-mass spectrometry (MS) analysis, but cyclization of free Gin and Glu to free pGlu during LC-MS analysis has not been well-characterized. Using an LC-MS/MS protocol that we developed to separate Gin, Glu, and pG1u, we found that free Gin and Glu cyclize to pGlu in the electrospray ionization source, revealing a previously uncharacterized artifact in metabolomic studies. Analysis of Gln standards over a concentration range from 0.39 to 200 mu M indicated that a minimum of 33% and maximum of almost 100% of Gin was converted to pGlu in the ionization source, with the extent of conversion dependent on fragmentor voltage. We conclude that the sensitivity and accuracy of Gin, Glu, and pGlu quantitation by electrospray ionization-based mass spectrometry can be improved dramatically by using (i) chromatographic conditions that adequately separate the three metabolites, (ii) isotopic internal standards to correct for in-source pGlu formation, and (iii) user-optimized fragmentor voltage for acquisition of the MS spectra. These findings have immediate impact on metabolomics and metabolism research using LC-MS technologies.
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