title: Assessment of microwave-assisted enzymatic digestion by measuring glycated hemoglobin A1c by mass spectrometry.
abstract: Enzymatic digestion of proteins and analysis of the resulting peptides by mass spectrometry is an established approach in proteomics and in clinical and environmental chemistry. The long digestion times of several hours prevent the fast turnover of samples and results. Qualitative applications showed that microwave radiation profoundly shortens enzymatic digestion. However, its usefulness for quantitative applications had not been assessed. In this study, the microwave-assisted enzymatic digestion of hemoglobin at different temperatures, buffer concentrations, and digestion times was assessed and compared with conventional digestion for the proteolytic enzymes trypsin and Glu-C. A microwave-assisted enzymatic digestion method optimized for digestion time and temperature was applied for the analysis of glycated hemoglobin HbA1c and compared with a reference method. Using trypsin, complete digestion was obtained at 50 degrees C within 20 min. Under these conditions, the digestion efficiency was 20% higher than with conventional trypsin digestion. These effects were not observed with Glu-C as enzyme, probably because of the decreased stability of Glu-C at elevated temperatures in comparison with the trypsin used. The comparison of the optimized microwave-assisted digestion method using trypsin with the reference method for HbA1c using Glu-C gave a close correlation in the results (R2: 0.996). A significant bias of 0.33% HbA1c was observed, with higher values obtained with the microwave-assisted tryptic digest; this finding might have resulted from the use of a different enzyme. This study showed that microwave-assisted enzymatic digestion can substantially reduce digestion times to minutes and can be used in qualitative as well as quantitative applications.
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Hwy, NE (MS F-25), USA. HVesper@cdc.gov
authors.forename: Hubert W
authors.initials: HW
authors.lastname: Vesper
authors.forename: Luchuan
authors.initials: L
authors.lastname: Mi
authors.forename: Archibold
authors.initials: A
authors.lastname: Enada
authors.forename: Gary L
authors.initials: GL
authors.lastname: Myers
mesh_terms: D002384:Catalysis; D004789:Enzyme Activation / Q000187:drug effects; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006442:Glycated Hemoglobin / Q000032:analysis* / Q000737:chemistry* / Q000528:radiation effects; D013058:Mass Spectrometry; D008872:Microwaves; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000528:radiation effects; D014357:Trypsin / Q000737:chemistry* / Q000528:radiation effects
chemical_list: D006442:Glycated Hemoglobin; D012697:Serine Endopeptidases; D014357:Trypsin; C028344:glutamyl endopeptidase