Identification of plasma protease derived metabolites of glucagon and their formation under typical laboratory sample handling conditions
File(s)
Accepted version
Author(s)
Howard, JW
Kay, RG
Tan, T
Minnion, J
Creaser, CS
Type
Journal Article
Abstract
RATIONALE
Glucagon modulates glucose production, and it is also a biomarker for several pathologies. It is known to be unstable in human plasma, and consequently stabilisers are often added to samples, although these are not particularly effective. Despite this, there have not been any studies to identify in vitro plasma protease derived metabolites; such a study is described here. Knowledge of metabolism should allow the development of more effective sample stabilisation strategies.
METHODS
Several novel metabolites resulting from the incubation of glucagon in human plasma were identified using high-resolution mass spectrometry with positive electrospray ionisation. Tandem mass spectrometric (MS/MS) scans were acquired for additional confirmation using a QTRAP. Separation was performed using reversed-phase ultra-high-performance liquid chromatography. The formation of these metabolites was investigated during a time-course experiment and under specific stress conditions representative of typical laboratory handling conditions. Clinical samples were also screened for metabolites.
RESULTS
Glucagon3-29 and [pGlu]3glucagon3-29 were the major metabolites detected, both of which were also present in clinical samples. We also identified two oxidised forms of [pGlu]3glucagon3-29 as well as glucagon19-29, or 'miniglucagon', along with the novel metabolites glucagon20-29 and glucagon21-29. The relative levels of these metabolites varied throughout the time-course experiment, and under the application of the different sample handling conditions. Aprotinin stabilisation of samples had negligible effect on metabolite formation.
CONCLUSIONS
Novel plasma protease metabolites of glucagon have been confirmed, and their formation characterised over a time-course experiment and under typical laboratory handling conditions. These metabolites could be monitored to assess the effectiveness of new sample stabilisation strategies, and further investigations into their formation could suggest specific enzyme inhibitors to use to increase sample stability. In addition the potential of the metabolites to affect immunochemistry-based assays as a result of cross-reactivity could be investigated.
Glucagon modulates glucose production, and it is also a biomarker for several pathologies. It is known to be unstable in human plasma, and consequently stabilisers are often added to samples, although these are not particularly effective. Despite this, there have not been any studies to identify in vitro plasma protease derived metabolites; such a study is described here. Knowledge of metabolism should allow the development of more effective sample stabilisation strategies.
METHODS
Several novel metabolites resulting from the incubation of glucagon in human plasma were identified using high-resolution mass spectrometry with positive electrospray ionisation. Tandem mass spectrometric (MS/MS) scans were acquired for additional confirmation using a QTRAP. Separation was performed using reversed-phase ultra-high-performance liquid chromatography. The formation of these metabolites was investigated during a time-course experiment and under specific stress conditions representative of typical laboratory handling conditions. Clinical samples were also screened for metabolites.
RESULTS
Glucagon3-29 and [pGlu]3glucagon3-29 were the major metabolites detected, both of which were also present in clinical samples. We also identified two oxidised forms of [pGlu]3glucagon3-29 as well as glucagon19-29, or 'miniglucagon', along with the novel metabolites glucagon20-29 and glucagon21-29. The relative levels of these metabolites varied throughout the time-course experiment, and under the application of the different sample handling conditions. Aprotinin stabilisation of samples had negligible effect on metabolite formation.
CONCLUSIONS
Novel plasma protease metabolites of glucagon have been confirmed, and their formation characterised over a time-course experiment and under typical laboratory handling conditions. These metabolites could be monitored to assess the effectiveness of new sample stabilisation strategies, and further investigations into their formation could suggest specific enzyme inhibitors to use to increase sample stability. In addition the potential of the metabolites to affect immunochemistry-based assays as a result of cross-reactivity could be investigated.
Date Issued
2014-12-15
Date Acceptance
2014-10-31
Citation
Rapid Communications in Mass Spectrometry, 2014, 29 (2), pp.171-181
ISSN
1097-0231
Publisher
Wiley
Start Page
171
End Page
181
Journal / Book Title
Rapid Communications in Mass Spectrometry
Volume
29
Issue
2
Copyright Statement
This is the peer reviewed version of the following article: Howard J. W., Kay R. G., Tan T., Minnion J., and Creaser C. S. (2014) Identification of plasma protease derived metabolites of glucagon and their formation under typical laboratory sample handling conditions, Rapid Commun. Mass Spectrom., 29, pages 171–181, doi: 10.1002/rcm.7090, which has been published in final form at https://dx.doi.org/10.1002/rcm.7090. This article may be used for non-commercial purposes in accordance With Wiley Terms and Conditions for self-archiving.
Subjects
Science & Technology
Life Sciences & Biomedicine
Physical Sciences
Technology
Biochemical Research Methods
Chemistry, Analytical
Spectroscopy
Biochemistry & Molecular Biology
Chemistry
METHIONINE SULFOXIDE REDUCTASE
DIABETIC SUBJECTS
MINIGLUCAGON
ENDOPEPTIDASE
QUANTITATION
SENSITIVITY
MEMBRANES
ANALOGS
Publication Status
Published