Ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact hemoglobin complex from whole human blood.
Ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOFMS) was applied to the analysis of intact human hemoglobin complex directly from whole human blood. The less acidic matrix substance 2,6-dihydroxyacetophenone provided sufficient insensitivity to the impurities present in this crude biological matrix to make any sample pretreatment except dilution dispensable. This matrix facilitated exact molecular mass determination of the non-assembled hemoglobin alpha- and beta-chain (SD < or = +/-0.28 Da and Deltam < or =6.4 ppm for n = 10) if trifluoroacetic acid was used as an additive. Replacement of the denaturing additive trifluoroacetic acid by the neutral salt ammonium acetate allowed the detection of the intact hemoglobin alpha(2)beta(2)H(n)-assembly (n = 0-4) and the alphabeta-subassembly. A prerequisite for the detection of the noncovalent complex was the application of a very soft sample preparation procedure. Crystal morphology, sample concentration and laser pulse energy were also found to be important parameters for the analysis of the intact protein complexes. However, comparison with published electrospray ionization (ESI)-MS results on mammalian hemoglobin molecules shows that, even under the applied gentle conditions, MALDI does not provide a completely reliable picture of the solution-phase equilibrium. In contrast to ESI, especially extensive loss of the heme b (H) groups was noticed. The disruption of the rather stable heme b-globin interaction is assumed to be induced by photo-excitation during the desorption/ionization process.
Zehl
Martin
M
Institute of Chemical Technologies and Analysis, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria.
Allmaier
Günter
G
D006454:Hemoglobins / Q000032:analysis*; D006801:Humans; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D006454:Hemoglobins