Detection of noncovalent complexes in biological samples by intensity fading and high-mass detection MALDI-TOF mass spectrometry.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has not yet contributed widely to the study of intact noncovalent biomolecular complexes, because MALDI is known to cause dissociation of the interaction partners and induce formation of nonspecific aggregates. Here, we present a new strategy to circumvent this problem. It is based on intensity fading (in the low m/z range) and high-mass detection MALDI mass spectrometry (MS), using a cryodetector (in the high m/z range), with and without chemical cross-linking of the interaction partners. The study focuses on noncovalent interactions between the human enzyme carboxypeptidase A (hCPA) and three protease inhibitors (PCI, TCI, and LCI) present in heterogeneous mixtures of other nonbinding molecules derived from a biological source, an extract from leech (Hirudo medicinalis). Another example involves an extract of the sea anemone Stichodactyla helianthus, which is used without previous fractionation to detect the specific complex between the enzyme trypsin and the endogenous SphI-1 inhibitor. The results give insight into the mechanism of intensity fading MS and demonstrate that the specificity of binding is greatly favored when the overall concentrations of the analytes (nonbinding molecules, protease inhibitor and target enzyme) present in a biological sample of interest are kept at low concentrations, in the sub-micromolar range. Higher concentrations may lead to unspecific interactions and the formation of aggregates both during the MALDI process and during reaction with the cross-linking reagents. This strategy is expected to advance the field of high-throughput affinity-based approaches, by taking advantage of a new generation of high mass detectors for MALDI-TOF instruments.
Yanes
Oscar
O
Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
Nazabal
Alexis
A
Wenzel
Ryan
R
Zenobi
Renato
R
Aviles
Francesc X
FX
D000818:Animals; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry; D002457:Cell Extracts / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D015925:Cryopreservation; D047709:Hirudo medicinalis / Q000737:chemistry; D006801:Humans; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012615:Sea Anemones / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002457:Cell Extracts; D003432:Cross-Linking Reagents; D011480:Protease Inhibitors; D011506:Proteins; D002268:Carboxypeptidases