Surface-plasmon-resonance-based chemical proteomics: efficient specific extraction and semiquantitative identification of cyclic nucleotide-binding proteins from cellular lysates by using a combination of surface plasmon resonance, sequential elution and liquid chromatography-tandem mass spectrometry.
Chemical proteomics is a powerful methodology for identifying the cellular targets of small molecules, however, it is biased towards abundant proteins. Therefore, quantitative strategies are needed to distinguish between specific and nonspecific interactions. Here, we explore the potential of the combination of surface plasmon resonance (SPR) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) as an alternative approach in chemical proteomics. We coupled cGMP molecules to the SPR chip, and monitored the binding and dissociation of proteins from a human lysate by using sequential elution steps and SPR. The eluted proteins were subsequently identified by LC-MS/MS. Our approach enabled the efficient and selective extraction of low-abundant cyclic-nucleotide-binding proteins such as cGMP-dependent protein kinase, and a quantitative assessment of the less- and nonspecific competitive binding proteins. The data show that SPR-based chemical proteomics is a promising alternative for the efficient specific extraction and quantitative identification of small-molecule-binding proteins from complex mixtures.
Visser
Natasja F C
NF
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Scholten
Arjen
A
van den Heuvel
Robert H H
RH
Heck
Albert J R
AJ
D002460:Cell Line; D002853:Chromatography, Liquid; D017869:Cyclic GMP-Dependent Protein Kinases / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D009712:Nucleotides, Cyclic / Q000737:chemistry; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D020349:Surface Plasmon Resonance
D009712:Nucleotides, Cyclic; D017869:Cyclic GMP-Dependent Protein Kinases