Optimizing the surface plasmon resonance/mass spectrometry interface for functional proteomics applications: how to avoid and utilize nonspecific adsorption.
A great challenge in functional or interaction proteomics is to map protein networks and establish a functional relationship between expressed proteins and their effects on cellular processes. These cellular processes can be studied by characterizing binding partners to a "bait" protein against a complex background of other molecules present in cells, tissues, or biological fluids. This so-called ligand fishing process can be performed by combining surface plasmon resonance biosensors with MS. This combination generates a unique and automated method to quantify and characterize biomolecular interactions, and identify the interaction partners. A general problem in chip-based affinity separation systems is the large surface-to-volume ratio of the fluidic system. Extreme care, therefore, is required to avoid nonspecific adsorption, resulting in losses of the target protein and carry-over during the affinity purification process, which may lead to unwanted signals in the final MS analysis and a reduction in sensitivity. In this study, carry-over of protein and low-molecular weight substances has been investigated systematically and cleaning strategies are presented. Furthermore, it is demonstrated that by the introduction of colloidal particles as a capturing and transporting agent, the recovery yield of the affinity-purified ligand could be improved nearly twofold.
Department of Chemical Engineering, Mälardalen University, Eskilstuna, Sweden.
Helén
H
Larsericsdotter
Osten
O
Jansson
Andrei
A
Zhukov
Daphne
D
Areskoug
Sven
S
Oscarsson
Jos
J
Buijs
D000327:Adsorption; D000818:Animals; D015374:Biosensing Techniques; D002417:Cattle; D008024:Ligands; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*; D040081:Protein Array Analysis; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000295:instrumentation* / Q000379:methods*
D008024:Ligands; D012709:Serum Albumin