Evaluation of a surface-sampling probe electrospray mass spectrometry system for the analysis of surface-deposited and affinity-captured proteins.
A combined self-aspirating electrospray emitter/surfacing-sampling probe coupled with an ion trap mass spectrometer was used to sample and mass analyze proteins from surfaces. The sampling probe mass spectrometer system was used to sample and detect lysozyme that had been deposited onto a glass slide using a piezoelectric spotter or murine gamma-interferon affinity captured on a glass slide using surface-immobilized anti-gamma-interferon antibody. The detection level for surface-deposited lysozyme (spot size < or =200 microm) was approximately 1.0 fmol (approximately 100 fmol/mm2) as determined from the ability to measure accurately the protein molecular mass from the mass spectrum acquired by sampling the deposit. These detection limits may be sufficient for certain applications in which protein fractions from a separation method are collected onto a surface. Radiolabeled proteins were used to quantify the surface density of immobilized antibody and the efficiency of capture of the gamma-interferon on glass and higher surface area ceramic supports. The capture density of gamma-interferon at surface saturation ranged from about 23 to 50 fmol/mm2 depending on the capture surface. Nonetheless, mass spectrometric detection of affinity capture protein was successful in some cases, but the results were not reproducible. Thus, improvement of the sampling system, ionization efficiency and/or capture density will be necessary for practical sampling of affinity-captured proteins. The means to accomplish improved sampling system detection limits and to increase the absolute amounts of protein captured per unit area are discussed.
Van Berkel
Gary J
GJ
Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. vanberkelgj@ornl.gov
Ford
Michael J
MJ
Doktycz
Mitchel J
MJ
Kennel
Stephen J
SJ
D000345:Affinity Labels; D020099:Coated Materials, Biocompatible / Q000032:analysis*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008422:Materials Testing / Q000295:instrumentation* / Q000379:methods; D009113:Muramidase / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods; D013499:Surface Properties; D014159:Transducers
D000345:Affinity Labels; D020099:Coated Materials, Biocompatible; D009113:Muramidase