Planar Functionalized Surfaces for Direct Immunoaffinity Desorption/Ionization Mass Spectrometry.
BACKGROUND
Recent studies show that the haptoglobin phenotype in individuals with diabetes mellitus is an important factor for predicting the risk of myocardial infarction, cardiovascular death, and stroke. Current methods for haptoglobin phenotyping include PCR and gel electrophoresis. A need exists for a reliable method for high-throughput clinical applications. Mass spectrometry (MS) can in principle provide fast phenotyping because haptoglobin α 1 and α 2, which define the phenotype, have different molecular masses. Because of the complexity of the serum matrix, an efficient and fast enrichment technique is necessary for an MS-based assay.


METHODS
MALDI plates were functionalized by ambient ion landing of electrosprayed antihaptoglobin antibody. The array was deposited on standard indium tin oxide slides. Fast immunoaffinity enrichment was performed in situ on the plate, which was further analyzed by MALDI-TOF MS. The haptoglobin phenotype was determined from the spectra by embedded software script.


RESULTS
The MALDI mass spectra showed ion signals of haptoglobin α subunits at m/z 9192 and at m/z 15 945. A cohort of 116 sera was analyzed and the reliability of the method was confirmed by analyzing the identical samples by Western blot. One hundred percent overlap of results between the direct immunoaffinity desorption/ionization MS and Western Blot analysis was found.


CONCLUSIONS
MALDI plates modified by antihaptoglobin antibody using ambient ion landing achieve low nonspecific interactions and efficient MALDI ionization and are usable for quick haptoglobin phenotyping.
Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic; Faculty of Science, Charles University in Prague, Prague, Czech Republic; AffiPro, s.r.o., Mratin, Czech Republic;
Petr
P
Pompach
Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic;
Jana
J
Nováková
Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic; Faculty of Science, Charles University in Prague, Prague, Czech Republic;
Daniel
D
Kavan
Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic;
Oldřich
O
Benada
BioVendor, a.s., Brno, Czech Republic.
Viktor
V
Růžička
Faculty of Science, Charles University in Prague, Prague, Czech Republic; AffiPro, s.r.o., Mratin, Czech Republic;
Michael
M
Volný
Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic; Faculty of Science, Charles University in Prague, Prague, Czech Republic; AffiPro, s.r.o., Mratin, Czech Republic; pnovak@biomed.cas.cz.
Petr
P
Novák
D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D006242:Haptoglobins / Q000032:analysis* / Q000276:immunology*; D006801:Humans; D010641:Phenotype; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013499:Surface Properties
D000906:Antibodies; D006242:Haptoglobins