Detection of Noncovalent Complexes in Biological Samples by Intensity Fading and High-Mass Detection MALDI-TOF Mass Spectrometry

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• 作者：Oscar Yanes ; Alexis Nazabal ; Ryan Wenzel ; Renato Zenobi ; Francesc X. Aviles
• 刊名：Journal of Proteome Research
• 出版年：2006
• 出版时间：October 2006
• 年：2006
• 卷：5
• 期：10
• 页码：2711 - 2719
• 全文大小：235K
• 年卷期：v.5,no.10(October 2006)
• ISSN：1535-3907

文摘

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has not yet contributed widelyto the study of intact noncovalent biomolecular complexes, because MALDI is known to causedissociation of the interaction partners and induce formation of nonspecific aggregates. Here, we presenta new strategy to circumvent this problem. It is based on intensity fading (in the low m/z range) andhigh-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 noncovalentinteractions 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 biologicalsource, an extract from leech (Hirudo medicinalis). Another example involves an extract of the seaanemone Stichodactyla helianthus, which is used without previous fractionation to detect the specificcomplex between the enzyme trypsin and the endogenous SphI-1 inhibitor. The results give insightinto the mechanism of intensity fading MS and demonstrate that the specificity of binding is greatlyfavored when the overall concentrations of the analytes (nonbinding molecules, protease inhibitor andtarget 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 ofaggregates both during the MALDI process and during reaction with the cross-linking reagents. Thisstrategy is expected to advance the field of high-throughput affinity-based approaches, by takingadvantage of a new generation of high mass detectors for MALDI-TOF instruments.