Detection of noncovalent complexes in biological samples by intensity fading and high-mass detection MALDI-TOF mass spectrometry

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Author(s) Yanes, Oscar, Nazabal, Alexis, Wenzel, Ryan, Zenobi, Renato, Aviles, Francesc X.
Publication Type Journal Items, Publication Status: Published
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Title Detection of noncovalent complexes in biological samples by intensity fading and high-mass detection MALDI-TOF mass spectrometry
Author(s) Yanes, Oscar
Nazabal, Alexis
Wenzel, Ryan
Zenobi, Renato
Aviles, Francesc X.
Journal or Series Title Journal of proteome research
Volume Number 5
Issue Number 10
Start Page 2711
End Page 2719
ISSN 1535-3893
1535-3907
Publisher American Chemical Society
Publication Place Washington, DC
Publication Date 2006
Keyword(s) mass spectrometry
MALDI-TOF
high-mass detection
cryodetection
microchannel plates
intensity fading
noncovalent complexes
cross-linking
Abstract 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 Sphl-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.
DOI 10.1021/pr060202f
Additional Notes Received 1 May 2006, Published online 30 August 2006
Document Type Article
Publication Status Published
Language English
Assigned Organisational Unit(s) 03430
Organisational Unit(s)
NEBIS System Number 004296166
Source Database ID PP-25378
WOS-000241053100023
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@article{Yns2006,
  author = "Yanes, Oscar and Nazabal, Alexis and Wenzel, Ryan and Zenobi, Renato and Aviles, Francesc X.",
  title = "{D}etection of noncovalent complexes in biological samples by intensity fading and high-mass detection {M}{A}{L}{D}{I}-{T}{O}{F} mass spectrometry",
  journal = "Journal of proteome research",
  year = 2006,
  volume = "5",
  number = "10",
  pages = "2711--2719",
}


E-Citations record created: Thu, 01 Apr 2010, 16:30:13 CET