title: Mass spectrometry after capture and small-volume elution of analyte from a surface plasmon resonance biosensor.
abstract: The identification of binding partners of proteins by mass spectrometry following specific capture on a biosensor surface is a promising tool for proteomics research and the identification and characterization of protein-protein interactions. Previous approaches include the direct ionization of analyte from the biosensor chip on a matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOFMS) apparatus and the on-chip digestion followed by elution, chromatographic concentration of the fragments, and electrospray mass spectrometry. In the present paper, using the small-volume microfluidic sample manipulation technique with oscillatory flow reported recently (Abrantes et al. Anal. Chem. 2001, 73, 2828-2835), analyte is shown to be eluted from the sensor surface into a small volume of buffer that promotes dissociation from the capture surface and delivery to the mass spectrometer. Both the incubation of the sensor surface with the sample and the recovery of analyte can be achieved with a few microliters and conducted until steady-state is attained. Because the procedure is non-destructive for the sensor surface, multiple cycles of capture and elution allow the transfer and concentration of analyte into the elution buffer. The eluted analyte can be studied directly by MALDI-TOFMS, or subjected to proteolytic digestion for protein identification. Transfer into the elution buffer and MALDI-TOFMS detection was achieved from 5 microL of starting samples containing <50 fmol of analyte. Examples are presented for the specific detection and recovery of a protein from a complex mixture of cytosolic proteins.
authors.affiliation: Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Gilligan
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Yergey
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D008832:Microchemistry / Q000379:methods; D009113:Muramidase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D011506:Proteins; D009113:Muramidase