on 06-05-201405:22 AM - edited on 10-15-202107:23 AM by AnalyteGuru
Purwaha P, Silva LP, Hawke DH, Weinstein JN, Lorenzi PL. Anal Chem. 2014 Jun 3. Advances in metabolomics, particularly for research on cancer, have increased the demand for accurate, highly sensitive methods for measuring glutamine (Gln) and glutamic acid (Glu) in cell cultures and other biological samples. N-terminal Gln and Glu residues in proteins or peptides have been reported to cyclize to pyroglutamic acid (pGlu) during LC-MS analysis, but cyclization of free Gln 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 Gln, Glu, and pGlu, we found that free Gln 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 µM indicated that a minimum of 33% and maximum of almost 100% of Gln 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 Gln, 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.