On probe affinity capture (OPAC) of proteins for analysis by MALDI-mass spectrometry

Analysis of low concentrations of active biomolecules found in biological media is a constant challenge of bioanalytical chemistry. Thus the coupling of separation/purification methods with MALDI-MS represents a significant goal for future applications of MALDI-MS. This project focuses on the development of On Probe Affinity Capture (OPAC) methods for the analysis of targeted biomolecules in biological mixtures by MALDI mass spectrometry.; OPAC MALDI-MS probes are developed by using RF plasma polymer deposition to create probe surfaces for use as is, or following subsequent chemical modification. Using this approach a variety of OPAC MALDI probes are developed for various applications. Controlled mixtures of peptide or proteins are used to test the efficacy of the specific functional MALDI probes for mixture fractionation. For example, biotin and avidin MALDI probes are used to selectively capture avidin and biotin labeled biomolecules; carboxylic acid functional MALDI probe are used to separate biomolecules according to their pI, leading to a reduction in the influence of ion suppression effect; allyl alcohol modified MALDI probes are used to separate biomolecues according to their hydrophobicities; and thermo-responsive MALDI probe are used for protein purification. All of these surfaces are shown to be very effective for the selective capture of targeted biomolecules in mixtures.; In addition, because of the detrimental influence of the ion suppression effect in MALDI analysis of mixtures, a method to separate peptides on the basis of pI is developed using chemically modified commercial PMMA. Introduction of carboxylic acid and amine functional groups into this material allows peptides to be separated on the basis of electrostatic interaction with the surface and results in improved detection of the spectrum of components in the peptide mixture.