Determination of cyclic nucleotide-responsive protein kinase activity by quantitative fast atom bombardment mass spectrometry.

Abstract

Applicalion o f fast atom bombiirdnient mass s 1) 6: c t r omt! t r y 1 o c yc i i c nuc 1 e o t i d e siimplt!s, the utiliziilion of a n i . s . method involving rclativcly s o T 1 ionizalion, provides 11 means of gciier o t i ng m a s s spect. ra coritii i n i rig strong p rot ona ted molecular ions [ I . 2 ] , which previously had not been possihlc with lhtr m o r e wcll cslohlished, harsher. n 1 . s . rnelhods o f ionization. Collisionally induced ti i ssoc: i a t i 011 wi t h ina s s an a I y sed i on k i rie t i c ene rgy siicctrurn scann i nf: (c . i . d ./ni. i . k . e . s . ) constitutes a f'orni [ i f tandem m.s. generating valuable structural d a t a , and has been applied to the protonated moloculiir ions obtained by f .a.b.m.s. to identify cyclic nucleotidcs in lissue extracts [3-51, the product. and sidc-products o f cyclic nucleolidc b I (1 s y n t he s i s [ G ] , c e 1 1 p e rmea t i ng c yc 1 i c nuc 1 eo t i de derivatives [7] and derivarives necessary for cyclic nuclcotide radioimnunoassay [GI. Quantitative f.a.b./m.i.k.e.s. analysis has been successfully applied to studies of two cyclic nucIeotidc p h o s p h o d i e s t e r a s e s [ 9 , 1 0 1 b y c . i .d./m. i . k . e . s . scanning of the protonated molecular ions of the cyclic nucleotide substrate and inononucleotide product followed by determination of the pcak height of the diagnostic ions in the m.i.Ic.e. spectrum; the kinetic parameters obtained by quantilative f.a.b./ m.i.k.e.s. analysis showed close correlation with those obtained by the conventional radiometric phosphodiesterase assays. We have now dcveioped quantitative f.a.b.m.s. a s an alternative means of estimating protein kinase activities. By monitoring characteristic pcak heights in incubates containing active enzyme and controls with inactive enzyme the following parameters were calculated f o r a cyclic CMP-responsive protein kinase preparation:(a) cyclic CMP binding capacity (b) ability to bind other cyclic nucleotides (c) integral phosphodiesterase activity (d) integral phosphatase activity (e) protein kinase activity To calculate these parameters the levels of free. unbound o r unconsumed cyclic CMP. ATP, ADP, CMP, cyclic AMP and cyclic GMP were estimated by expressing the peak heights of the diagnostic peaks of each compound relative to matrix ion peaks:( f . o . I , . in. s . )