Investigation of calmodulin-peptide interactions using matrix-assisted laser desorption/ionization mass spectrometry

Abstract

In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium-calmodulin as well as the calmodulin-peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin. Relative binding affinities of different peptides towards the protein could also be estimated using IF-MALDI-MS. This study may extend the application of IF-MALDI-MS in the analysis of noncovalent complexes and offer a perspective into the utility of MALDI-MS as an alternative approach to study the peptides binding to calmodulin.

Screening calmodulin‐binding ligands using intensity‐fading matrix‐assisted laser desorption/ionization mass spectrometry

“…Drug–CaM interactions can also be studied through measuring CaM‐enzyme activity inhibited by the ligand . However, most of these methods require large quantities of samples . Mass spectrometry has become one of the important techniques because of its low sample consumption and fast analysis time, particularly after the development of soft ionization techniques .…”

“…Much of the study of noncovalent interactions has been performed with electrospray ionization mass spectrometry (ESI‐MS) as the intact complexes can be maintained in the gas phase. However, ESI has a low tolerance toward high ionic strength sample solutions . Relatively few cases have been published showing the capacity of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to detect specific intact complexes successfully .…”

“…Relatively few cases have been published showing the capacity of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to detect specific intact complexes successfully . One of the problems when observing complexes by MALDI‐MS is that the method generally causes dissociation of the interaction partners, while another difficulty is the presence of nonspecific aggregates (false positives) . However, MALDI‐MS is still a suitable analytical technique for protein agent screening due to its high sensitivity, salt tolerance and easy automation .…”

“…This method was based on a selective decrease (fading) of the ion abundance of the specific ligands after the addition of the target protein. It has been used to detect the formation of protein‐organic compounds and protein‐nucleic acids, as well as discovery ligands in biological extracts …”

Distinct Mechanisms of Calmodulin Binding and Regulation of Adenylyl Cyclases 1 and 8

“…This is explained by the auxiliary cation binding sites that CaM possesses in addition to the four high-affinity sites, 28 as shown by other ESI experiments. 21,22,29,30 When the mass spectra were recorded with AC8-C2b and 1 mM Ca 2+ , there were two signals corresponding to free CaM and peptide-bound CaM with four to eight Ca 2+ ions (Figure 3H). In contrast, there was no signal for free CaM when AC1-C1b or AC8-Nt was used (Figure 3D,F), demonstrating that all CaM molecules in the solution formed a complex with these two peptides in the presence of Ca 2+ .…”