Exploring the "Intensity Fading" Phenomenon in the Study of Noncovalent Interactions by MALDI-TOF Mass Spectrometry

Oscar Yanes, Francesc X. Aviles, Peter Roepstorff, Thomas J.D. Jørgensen

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19 Citations (Scopus)

Abstract

The difficulties to detect intact noncovalent complexes involving proteins and peptides by MALDI-TOF mass spectrometry have hindered a widespread use of this approach. Recently, "intensity fading MS" has been presented as an alternative strategy to detect noncovalent interactions in solution, in which a reduction in the relative signal intensity of low molecular mass binding partners (i.e., protease inhibitors) can be observed when their target protein (i.e., protease) is added to the sample. Here we have performed a systematic study to explore how various experimental conditions affect the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs when the binding assay is carried out in the sub-μM range and the interacting partners are present in complex mixtures of nonbinding compounds. Thus, at these experimental conditions, the specific inhibitor-protease interaction causes a selective reduction in the relative abundance of the inhibitor. Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower high-mass sensitivity of MCP detectors. © 2007 American Society for Mass Spectrometry.
Original languageEnglish
Pages (from-to)359-367
JournalJournal of the American Society for Mass Spectrometry
Volume18
Issue number2
DOIs
Publication statusPublished - 1 Feb 2007

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