title: An artifact in LC-MS/MS measurement of glutamine and glutamic acid: in-source cyclization to pyroglutamic acid.
abstract: 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 liquid chromatography (LC)-mass spectrometry (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.
authors.affiliation: Department of Bioinformatics and Computational Biology and ‡Proteomics and Metabolomics Facility, Department of Pathology, MD Anderson Cancer Center, University of Texas , Houston, Texas 77054, United States.
authors.forename: Preeti
authors.initials: P
authors.lastname: Purwaha
authors.forename: Leslie P
authors.initials: LP
authors.lastname: Silva
authors.forename: David H
authors.initials: DH
authors.lastname: Hawke
authors.forename: John N
authors.initials: JN
authors.lastname: Weinstein
authors.forename: Philip L
authors.initials: PL
authors.lastname: Lorenzi
mesh_terms: D016477:Artifacts; D002853:Chromatography, Liquid / Q000379:methods*; D003500:Cyclization; D017022:Flow Injection Analysis; D018698:Glutamic Acid / Q000032:analysis*; D005973:Glutamine / Q000032:analysis*; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D005973:Glutamine; D018698:Glutamic Acid; D011761:Pyrrolidonecarboxylic Acid