Purwaha Preeti, Silva Leslie P, Hawke David H, Weinstein John N, Lorenzi Philip L
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.
Anal Chem. 2014 Jun 17;86(12):5633-7. doi: 10.1021/ac501451v. Epub 2014 Jun 5.
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.
代谢组学的进展，尤其是在癌症研究方面，使得对在细胞培养物和其他生物样品中测量谷氨酰胺（Gln）和谷氨酸（Glu）的准确、高灵敏度方法的需求增加。据报道，在液相色谱（LC）-质谱（MS）分析过程中，蛋白质或肽中的N端Gln和Glu残基会环化形成焦谷氨酸（pGlu），但在LC-MS分析过程中游离Gln和Glu环化形成游离pGlu的情况尚未得到充分表征。使用我们开发的用于分离Gln、Glu和pGlu的LC-MS/MS方案，我们发现游离Gln和Glu在电喷雾电离源中环化形成pGlu，揭示了代谢组学研究中一种以前未被表征的假象。对浓度范围为0.39至200μM的Gln标准品进行分析表明，在电离源中，至少33%且最多近100%的Gln转化为pGlu，转化程度取决于碎裂电压。我们得出结论，通过使用（i）能充分分离这三种代谢物的色谱条件，（ii）同位素内标以校正源内pGlu的形成，以及（iii）用户优化的碎裂电压来采集MS谱图，基于电喷雾电离的质谱对Gln、Glu和pGlu定量的灵敏度和准确性可得到显著提高。这些发现对使用LC-MS技术的代谢组学和代谢研究有直接影响。
