Petr Pompach (Presenter)
Institute of Microbiology
Authorship: Petr Pompach (1,2,3), Jana Nováková (1), Daniel Kavan (1,2), Oldřich Benada (1), Viktor Růžička (4), Michael Volný (2,3), Petr Novák (1,2,3)
(1) Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic, (2) Faculty of Science, Charles University in Prague, Prague, Czech Republic, (3) AffiPro, s.r.o., Mratin, Czech Republic, (4) BioVendor, a.s., Brno, Czech Republic
| Short Abstract Antibody-functionalized MALDI surfaces prepared by ambient ion landing allow efficient immuno-affinity enrichment of antigen from complex samples directly on the plate. The effectivity of antibody-functionalized MALDI surfaces to enrich protein antigen was demonstrated on determination of haptoglobin phenotype - an important biomarker in patient survival. MALDI plates were functionalized by polyclonal human anti-haptoglobin antibody using the lab-made apparatus. One microliter of serum was applied on the anti-haptoglobin spot and incubated for one hour. After several washing steps, samples were in-situ reduced and mixed with matrix. Automated sample analysis was used to acquire spectra of intact proteins. |
Long Abstract
Introduction
Combination of mass spectrometry with immuno-affinity techniques offers unambiguous detection of antigens in very little volumes of human body fluids. Antibody-functionalized MALDI surfaces, prepared by ambient ion landing at atmospheric pressure, allow efficient immuno-affinity enrichment of antigen from complex samples directly on the plate. The absence of any interlayer between conductive MALDI surface modified by ion landing and antibody affinity molecules reduces the non-specific interactions of other proteins in the sample and improves conductivity of modified surface. The effectivity of antibody-functionalized MALDI surfaces to enrich protein antigen was demonstrated on determination of haptoglobin phenotype - an important biomarker in patient survival.
Methods
MALDI plates were functionalized by ambient ion landing of polyclonal human anti-haptoglobin antibody using the lab-made apparatus consists of the syringe pump, nanoelectrospray emitter with applied high voltage and heated desolvation tube. The beam of charged species was deposited on the vertically mounted ITO glass slide that was kept on a high voltage of the opposite polarity with respect to the spray voltage. One microliter of serum was applied on the anti-haptoglobin spot on the functionalized MALDI plate and incubated for one hour. After several washing steps, samples were in-situ reduced and mixed with matrix. Automated sample analysis was used to acquire spectra of intact proteins. The haptoglobin phenotype was determined from the spectra by embedded software script.
Results
The MALDI slides were functionalized by anti-haptoglobin polyclonal antibody using the ambient ion landing procedure. The desolvation temperature was 45°C, which allowed efficient solvent removal while avoiding temperature denaturation of sprayed antibody. The arrays used in this study were composed of 16 spots with 9 mm raster and 2 mm immunoaffinity area. The deposited antibody on the ITO slides by ambient ion landing was first analyzed by scanning electron microscopy. To detect the alpha subunits by MALDI mass spectrometry, it was necessary to reduce the protein after the enrichment, which was achieved by applying the TCEP reagent directly on the spots. The DHAP matrix, which was directly mixed on the spot with the sample, was used for identification of alpha 1 or alpha 2 haptoglobin subunits. For the haptoglobin phenotype Hp 1-1, only an ion at m/z 9192 corresponding to molecular mass of alpha 1 subunit, was observed in the spectra. The spectrum of haptoglobin phenotype Hp 2-1 contained ions at m/z 9192 and at m/z 15945 (alpha 2 subunit). The spectrum of haptoglobin phenotype Hp 2-2 contained the dominant ion at m/z 15945 corresponding to the alpha 2 subunit. The method was tested using 116 human serum samples. The validation was performed by using SDS electrophoresis followed by western blot with chemiluminescent detection.
Conclusions
The analysis of the 116 patients cohort found fifty-two individuals (44%) with phenotype Hp 2-2, forty-seven (41%) with Hp 2-1 and seventeen (15%) with Hp 1-1, and the results obtained by Western blot were in 100% agreement.
References & Acknowledgements:
This work has been supported by the Institutional Research Concept of the Institute of Microbiology RVO61388971; grants from the Ministry of Education Youth and Sports of the Czech Republic and European Regional Development Funds (CZ.1.07/2.3.00/30.0003 and CZ.1.05/1.1.00/02.0109); and Charles University (project UNCE 204025/2012).
| Description | Y/N | Source |
| Grants | yes | Ministry of education, youth and sports |
| Salary | yes | Institute of Microbiology |
| Board Member | yes | Czech society for mass spectrometry |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |