Characterization of High Molecular Weight Multimeric States of Human Haptoglobin and Hemoglobin-Based Oxygen Carriers by High-Mass MALDI MS



Tatiana Pimenova1, Claudia P. Pereira2, Dominik J. Schaer2, and Renato Zenobi1


  1. Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland
  2. Internal Medicine Research Unit, University of Zurich, Zurich, Switzerland


High-mass MALDI-TOF mass spectrometry (MS) is a novel analytical approach to study large biomolecules and their interactions. It is a powerful alternative method to gel electrophoresis (GE) and size exclusion chromatography (SEC) for obtaining information on the molecular weights of macromolecules and for determining protein complexes. The precision of mass measurements (mass accuracy), high sensitivity, speed of the analysis, and tolerance toward sample heterogeneity are the major features of this MS-based approach. Remarkably, MS provides direct stoichiometric information of macromolecular protein complexes, when noncovalent interactions are stabilized during desorption/ionization by use of chemical cross-linking reagents. In this study, high-mass MALDI-TOF MS was applied to characterize the multimeric state of the human plasma protein haptoglobin (Hp), which is in the mass range of 150–300 kDa. Also, higher order structures of hemoglobin-based oxygen carriers (HBOCs) and their interactions with human haptoglobin were analyzed. These investigations are of clinical importance and contribute to the overall understanding of specific toxicity and clearance of HBOCs.

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Complex Tracker


Human Hp 1-1 and Hp 2-2 were obtained in powder form and dissolved in 50 mM potassium phosphate buffer (pH 7.4) to a concentration of 1 mg/ml. The proteins were stabilized by adding 1 μL of 2 mg/ml DSS (disuccinimidyl suberate) in dimethylformamide to 10 μl of protein solution (2-5 μM) that was in 10 mM triethylammonium bicarbonate buffer (pH 9.2). The samples were incubated at room temperature for 2 hours before equal amounts were mixed with the sinapic acid matrix (10 mg/mL in 50% ACN/0.1% TFA) and 1 μL of each final mixture was spotted on a MALDI target and dried at room temperature. The target was analyzed using a MALDI-TOF mass spectrometer that had been modified with a CovalX HM1 detection system. The data was interpreted using the CovalX Complex Tracker software.

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