Identification of protein ligands in complex biological samples using intensity-fading MALDI-TOF mass spectrometry.
The easy detection of biomolecular interactions in complex mixtures using a minimum amount of material is of prime interest in molecular and cellular biology research. In this work, a mass spectrometry MALDI-TOF based approach, which we call intensity-fading (IF MALDI-TOFMS), and which was designed for just such a purpose, is reported. This methodology is based on the use of the MALDI ion intensities to detect quickly the formation of complexes between nonimmobilized biomolecules in which a protein is one of the partners (protein-protein, protein-peptide, protein-organic molecule, and protein-nucleic acid complexes). The complex is detected through the decrease (fading) of the molecular ion intensities of the partners as directly compared to the MALDI mass spectrum of the mixture (problem and control molecules) following the addition of the target molecule. The potential of the approach is examined in several examples of model interactions, mainly involving small nonprotein and protein inhibitors of proteases, at both the qualitative and semiquantitative levels. Using this method, different protein ligands of proteolytic enzymes in total extracts of invertebrate organisms have been identified in a simple way. The proposed procedure should be easily applied to the high-throughput screening of biomolecules, opening a new experimental strategy in functional proteomics.
Villanueva
Josep
J
Institut de Biotecnologia i de Biomedicina, and Departament de Bioquímica, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain.
Yanes
Oscar
O
Querol
Enrique
E
Serrano
Luis
L
Aviles
Francesc X
FX
D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry; D008024:Ligands; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry*; D012615:Sea Anemones; D012697:Serine Endopeptidases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008024:Ligands; D011506:Proteins; D002268:Carboxypeptidases; D012697:Serine Endopeptidases