Petr Pompach (Presenter)
Institute of Microbiology
Bio: Petr Pompach is scientist in charge in the Institute of Microbiology of the Czech Academy of Sciences and research assistant at Department of Biochemistry, Charles University in Prague. He is a member of the executive board of the Czech Society for Mass Spectrometry. Petr is a co-developer of the technology for surface modification by reactive ion landing under atmospheric pressure. This technology was successfully used for enrichment of clinically important antigens from human serum followed by MALDI mass spectrometric detection. The other applications of this technology include enrichment of glycans/glycopeptides by lectin chips, phosphopeptides by TiO2 chips and digestion of proteins by different types of protease chips. Petr Pompach is author of more than 39 articles and co-author of national, European and US patents.
Authorship: Petr Pompach (1,2); Petra Darebna (1,2); Viktor Ruzicka (3); Michael Volny (4); Petr Novak (1,2)
(1) Institute of Microbiology, v.v.i., CAS, Prumyslova 595, Vestec, Czech Republic, (2) Faculty of Science, Charles University in Prague, Albertov 6, Prague, Czech Republic, (3) Biovendor, Karasek 1767, Brno, Czech Republic, (4) AffiPro, s.r.o., Zloninska 256, Mratin, Czech Republic
The bioanalytical prospect of MALDI-compatible protein chips prepared by ambient ion soft landing is demonstrated on in-situ enrichment, detection and quantification of human transferrin and its carbohydrate deficient forms. The assay is based on the interaction between the immobilized antibody and the sampled analyte directly on the chip and subsequent analysis by MALDI mass spectrometry. 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 maintains the original conductivity of the MALDI plate, which provides efficient ionization.
Modern biochemical tests, used in medicine, veterinary, research and industry, are often based on the interaction of the protein with its biological partner. In case the protein is an antibody and the analyte an appropriate antigen, the test refers to the immunoassay. The antigen can be visualized by several different techniques like chemiluminescence, fluorescence and radiation. Since these techniques relays on the specificity of used antibody a novel detection procedure exploring desorption mass spectrometry has been introduced.
The presented technology allows preparation of MALDI compatible protein chips by ambient ion landing (1). The electrosprayed proteins are immobilized on various conductive surfaces such as metal, stainless steel or indium tin oxide glass. The soft ion landing procedure performed under atmospheric pressure keeps landed proteins in their native forms. The automated ion landing apparatus enables manufacturing of protein affinity chips with predefined array of sample positions, or any other geometry of choice. The protein chips prepared by this technique are stable enough to be used for in-situ enrichment of antigens and their direct MALDI mass spectrometric detection. Compare to other materials, non-reactive surfaces suffer minimal nonspecific interactions with chemical species in the investigated sample and are thus an ideal substrate for selective protein chips (2, 3).
A novel immunoMALDI workflow involves anti-transferrin antibody functionalized surfaces for enrichment of clinically important protein transferrin from human serum and its follow up detection by MALDI mass spectrometry.
A solution of polyclonal anti-transferrin antibody in 20 mM ammonium bicarbonate buffer, pH 7.8 was used as an immunoaffinity modifier. The solution was delivered by the syringe pump. The 20 µm capillary was connected with nanoelectrospray emitter with 20 µm diameter. The positive voltage (+1.5kV) generated by a high voltage power supply was applied on the liquid junction. Pre-heated nitrogen gas was used as a nebulizer gas. The charged aerosol was dried by passing through the desolvation tube that was kept on ground potential and externally heated to 45°C. The dried aerosol consisted of multiply charged antibody ions presumably at different stages of desolvation. This 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 (-1.5kV). The apparatus for ambient ion landing was equipped with remotely controlled stepper electromotors.
One microliter of serum was applied on the spot and incubated for 1 hour at room temperature. After the incubation, the whole chip was washed three times in PBS buffer. The 2,5-dihydroxyacetophenone MALDI matrix was premixed directly on the spot with the sample and let dried. The samples were analyzed by Ultraflex III MALDI-TOF/TOF operating in a linear positive mode.
Our results show the successful in-situ enrichment of transferrin and its carbohydrate deficient forms (CDT) by functionalized chips. The disialotransferrin was observed at m/z 38700 as a doubly charged ion, which correspond to the loss of one complex glycan (molecular weight 2200 Da). In case of heavy alcoholic consumers, the asialotransferrin at m/z 37600, corresponding to the loss of two glycans, was observed. The 2,5-dihydroxyacetophenone matrix improved the performance of protein detection by formation of up to quadruply charged ions with resolution allowing separation of transferrin glycoforms. This was not achieved with other matrixes. The percentage of CDT was calculated as a ratio of the sum of asialo- and disialo-transferrin and the transferrin. A cohort of 80 individuals was analyzed by immunoMALDI workflow and the results were compared with capillary electrophoresis method from SEBIA (4).
Conclusions & Discussion
From its discovery, carbohydrate deficient transferrin is routinely used for clinical screen of excessive alcohol abuse. A need exists for a reliable method for high-throughput clinical applications. In principle, mass spectrometry enables fast detection and quantification of transferrin isoforms since they have different molecular masses. Our results show the successful in-situ enrichment of CDT by functionalized chips and their follow up detection and relative quantification by MALDI. This opens the door to immunoMALDI assays that allow sensitive determination of antigens in serum and leads to fast clinical assays that can be easily automated for high-throughput screening.
References & Acknowledgements:
1) Volny M. et al., Anal Chem. 2005 Aug 1;77(15):4890-6
2) Pompach P. et al., Clin Chem. 2016 Jan;62(1):270-8.
3) Pompach P. et al., Anal Chem. 2016 Sep 6;88(17):8526-34.
4) Schellenberg F. et al., Clinica chimica acta 2007 382(1-2):48-53.
This work has been supported by grants from the Ministry of Education, Youth and Sports of the Czech Republic (LH15010, LD15089), the Czech Science Foundation (16-24309S), Grant Agency of Charles University (932316) and European Regional Development Funds (CZ.1.05/1.1.00/02.0109).
|Grants||yes||Ministry of Education, Youth and Sports of the Czech Republic|
|Salary||yes||Institute of Microbiology|
|Board Member||yes||Czech Society for Mass Spectrometry|
IP Royalty: no
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