Mass spectrometric characterization of a series of adenosylated peptides acting as bisubstrate analogs of protein kinases

Abstract

We are currently developing strategies to synthesize bisubstrate analogs as potential inhibitors of serine and tyrosine protein kinases; several such analogs have been synthesized. The initial target proteins were the cAMP dependent protein kinase (cAPK) and the Ca(+2)/calmodulin dependent protein kinase (CaM kiiase II). These bisubstrate analogs were based on either known peptide substrates such as kemptide, a seven amino acid peptide substrate of cAPK, or on inhibitory peptides such as a seventeen amino acid peptide encompassing the autoinhibitory domain of CaM kinase II. Peptides containing a single phosphoserine group were first synthesized and then adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), or adenosine 5'-triphosphate (ATP) was coupled through the serine phosphate with prior activation by 1,1-carbonyldiimidazole using either a solution or solid phase reaction scheme. In this current study, we report the characterization of the bisubstrate analogs by liquid secondary ionization mass spectrometry (LSIMS), matrix-assisted laser desorption mass spectrometry (MALDI), and tandem mass spectrometry (MS/MS).In the positive-ion mode, the LSIMS spectra of the bisubstrate analogs yielded a series of molecular ions containing mono-, di-, and trivalent cation adducts. Cation adducts were absent in the negative-ion mode where the dominant species were deprotonated molecular ions, [M - H](-), making this latter technique more useful for confirming product identity and assessing purity. Analysis of these compounds by MALDI in both the positive- and negative-ion modes yielded molecular ions which also contained metal ion adducts, although they were limited primarily to Fe(+2) adducts. Unlike LSIMS, the MALDI spectra showed no evidence for the elimination of the phosphoadenosine or other structural moieties. When these compounds were subjected to high energy collision-induced dissociation (CID), the dominant fragmentation pathways under positive-ion MS/MS conditions resulted from cleavage of the phosphate linkages to the adenosine moiety with charge retention on the peptide, although a major peak for 5'-deoxyadenosine was also seen at m/z 250. Charge retention in the negative-ion mode was most pronounced for ion fragments containing the highly acidic phosphate moieties and yielded phosphoadenosine related ions, for example, (AMP-H)(-), (AMP-H-H2O)(-), (ADP-H)(-), etc., as well as ions originating from the phosphate linker such as PO3 (-), H2PO4 (-), HP2O6 (-), H3P2O7 (-), and H2P3O9 (-). The largest phosphoadenosine ion in the negative-ion CID spectra for each bisubstrate analog, for example, m/z 426 (ADP-H)(-), m/z 506 (ATP-H)(-), or m/z 586 (AP4-H)(-), indicated that the desired covalent modification had been formed between the phosphoserine and APn moieties.

Characterization of AMPylation on Threonine, Serine, and Tyrosine Using Mass Spectrometry

“…Although fragmentation of the modification group itself was not commonly observed, the dissociation products of ADP and ATP were detected when kemptide-ADP and kemptide-ATP were subjected to CID experiments. In addition, the unique peak at m/z 250 shown in Figure 1b and d was also found as one of the dissociation products of kemptide-ADP and kemptide-ATP [18]. …”

“…Although the structure of kemptide-AMP was not studied, Gibson et al examined the tandem collision induced dissociation (CID) spectra of kemptide-ADP and kemptide-ATP, in addition to LSI-MS (liquid secondary ionization mass spectrometry) and MALDI-MS, to confirm that the AMPylated kemptides they synthesized had the desired structure [18]. They detected a series of fragment ions originating from the ADP and ATP groups, while the peptide sequence ions were insufficient to determine the position of the ADP/ATP modification site.…”

Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography–electrospray ionization tandem mass spectrometry

“…The ion which retained the phosphoadenosine group was observed at m/z 426 and corresponding −H 2 O ion at m/z 408 for each compound measured as described also in earlier studies [14]. The observed fragmentation is typical for the phosphoadenosine containing compounds [15]. The cleavage of phosphoadenosine was the major fragmentation pathway for all compounds producing ion 671.3 for acyl-CoA C17:0 in 0.5453 ± 0.0264 0.0603 ± 0.0349 0.9980 ± 0.0020…”