A facile and sensitive method for quantification of cyclic nucleotide monophosphates in mammalian organs: basal levels of eight cNMPs and identification of 2',3'-cIMP.

Emily Weinert,Fred Strobel,Xin Jia,Benjamin Fontaine

doi:10.3390/biom4041070

Abstract

A sensitive, versatile and economical method to extract and quantify cyclic nucleotide monophosphates (cNMPs) using LC-MS/MS, including both 3',5'-cNMPs and 2',3'-cNMPs, in mammalian tissues and cellular systems has been developed. Problems, such as matrix effects from complex biological samples, are addressed and have been optimized. This protocol allows for comparison of multiple cNMPs in the same system and was used to examine the relationship between tissue levels of cNMPs in a panel of rat organs. In addition, the study reports the first identification and quantification of 2',3'-cIMP. The developed method will allow for quantification of cNMPs levels in cells and tissues with varying disease states, which will provide insight into the role(s) and interplay of cNMP signalling pathways.

Highlights

Cyclic nucleotides adenosine 3',5'-cyclic monophosphate (3',5'-cAMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cGMP) are essential mammalian metabolites that function primarily within the cell as secondary messengers. 3',5'-cAMP and 3',5'-cGMP have been investigated for decades and haveBiomolecules 2014, 4 been found to perform crucial roles in regulating cellular metabolism and mediating actions of numerous mammalian hormones and neurotransmitters [1,2]
Development of the current method to extract and quantify cyclic nucleotide monophosphates (cNMPs) from mammalian tissues began with optimization of the separation and detection protocols by LC-MS/MS
Many existing protocols studying metabolomics use a combination of ultra-performance liquid chromatography (UPLC) coupled with a triple quadrupole

Summary

Introduction

Cyclic nucleotides adenosine 3',5'-cyclic monophosphate (3',5'-cAMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cGMP) are essential mammalian metabolites that function primarily within the cell as secondary messengers. 3',5'-cAMP and 3',5'-cGMP have been investigated for decades and haveBiomolecules 2014, 4 been found to perform crucial roles in regulating cellular metabolism and mediating actions of numerous mammalian hormones and neurotransmitters [1,2]. 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs), in mammalian tissues and cell lines has previously been reported [3,4,5] While their potential roles in tumorigenesis, cellular signal transduction and post-injury mechanisms have been suggested, their signalling pathways have yet to be elucidated [6,7,8]. CadD, an enzyme found in the pathogenic bacterium Leptospira interrogans, has been recently reported as a cAMP specific deaminase, resulting in the production of 3',5'-cIMP [7]. This conversion has suggested the possibility that cells sense 3',5'-cIMP levels [7,16]

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Discussion

Conclusion