Fred Regnier (Presenter)
Bio: During 40 years in the analytical division at Purdue my laboratory published 300 pier reviewed papers and has been granted 49 US patents on various aspects of protein/peptide/metabolite analysis; focusing on accelerating analyses through chromatography column development, stable isotope based quantification, immunological assay methods, cancer biomarker discovery, and what we call mass linked immune selection assays (MALISA). One recent example is the synthesis of immunobilized trypsin reactors that are temperature stable to 80 degrees; decreasing digestion times and increasing sensitivity in the bottom-up proteomics mode 50-100 fold. The subject of paper I wish to present will allow the preparation of 96 samples by Ab selection along with deposition on a MALDI plate in roughly 30 min.
Authorship: Fred E. Regnier
Professor of Chemistry, Department of Chemistry, Purdue University, West Lafayette, IN 47907.
A new form of antigen separation, mobile affinity sorbent chromatography (MASC) will be described that accelerates immune complex purification for analysis by MALDI-MS. This is achieved in 15 sec using 1-20 antibodies covalently linked to a soluble sorbent that subsequent to antigen binding produces a supramolecular complex in the range of 1000 kD. When introduced into a size exclusion column complexes are excluded from pore matrices, being washed continuously with fresh solvent during migration while lower affinity species dissociate and are resolved. Complexes thus resolved are continuously mixed with MALDI matrix and deposited on the MALDI plate.
INTRODUCTION. This paper will focus on accelerating sample preparation 10-20 fold through mobile affinity sorbent chromatography (MASC); a new separation method in which antigen:antibody (Ag:Ab) complexes are bound to a mobile affinity sorbent (AS) in the mobile phase of a size exclusion chromatography column. Immunosorbent affinity chromatography and magnetic bead methods are widely used in mass spectrometry (MS) based immunological assay methods; their great advantage being that they can purify a low abundance antigen a thousand fold or more in a single step within minutes. Limitations are that the captured antigens must be washed extensively to remove non-specifically bound species and there is the potential for analyte carryover in further assays. This causes the sample preparation component to be orders of magnitude slower than MS identification and quantification. MASC addresses these issues.
METHODS. MASC may be achieved in multiple ways. In one form (Case A) immune complexes (Ag:Ab-B) are formed in the sample solution through addition of biotinylated antibodies (Ab-B). Ag:Ab-B complexes thus form were then introduced into a size exclusion chromatography (SEC) column where the mobile phase contains an AS bearing covalently linked avidin (designated Av-AS). The speed with which biotin binds to avidin allowed Ag:Ab-B complexes to rapidly form of supramolecular complexes in the SEC column, generally exceeding 1000 kD. The general formula for these complexes is thought to be (Ag:Ab-B)n:(Av-As) where n can range from 0-10 on any particular Av-As. This complex is so large it is totally excluded from the SEC column pores, traveling through the column matrix non-retained in 15 sec or less. During migration the supramolecular complex was continuously washed as it encountered fresh solvent while lower affinity species that dissociated from the complex fell behind and were resolved. When the antigen bearing complexes emerged from the SEC column the remaining effluent was switch to waste while the complex was directed to the MALDI plate. Through column switching valves two SEC columns were operated 180o out of phase to increase throughput.
With an alternative form of MASC (Case B), antibodies are bound directly to the mobilize affinity sorbent; the construct being designated (Ab)n-AS where n has the same meaning as above and is controllable during synthesis. (Ab)n-AS was added to samples directly and subsequent to immune complex formation the (Ag:Ab)o-AS supramolecular complex was injected into the SEC column and the separation handled as above.
A third mode of operation (Case C) is to add the (Ab)n-AS complex to the mobile phase of the SEC column continuously. In this mode the sample is injected directly into the SEC column where the immune complex is form during the course of elution.
RESULTS. The presentation will being with examples of the three modes of MASC described above. This will be follow by antigen:antibody binding kinetics studies using these systems. The time required for transferrin to reach equilibrium at an antibody concentration of 1 ug/mL was examined using the systems described in Case A and B and will be presented as an example. As expected, the reaction time in Case C was so short that antigen capture was incomplete and the peaks were broad. Case C was not investigated further. Multiplexing was carried out using both Case A and B systems showing that at least 10 different antigens could be captured simultaneously. Complete validation of these assays is in progress. The Case B system was particularly useful in for studying cross-reactivity and the co-capture of proteoforms that differ in molecular weight due to post-translational modifications. It was observed in the case of Lewis-x targeting that large numbers of glycoproteins were captured as has been observed with solid immunosorbents. Relative quantification was achieved in the case of carbonylated proteins by derivatization with isotopically labeled reagents.
CONCLUSIONS. Preliminary studies indicate that MASC has the potential to substantially accelerate mass spectral based immunological assays by reducing sample preparation time.
References & Acknowledgements:
This work was supported in part by NIH grant number U24 CA 126480.
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