Detection of intact hemoglobin from aqueous solution with laser desorption mass spectrometry

Wattenberg, Andreas; Sobott, Frank; Brutschy, Bernhard

doi:10.1002/(SICI)1097-0231(20000530)14:10<859::AID-RCM956>3.3.CO;2-D

Title

Detection of intact hemoglobin from aqueous solution with laser desorption mass spectrometry

Author

Wattenberg, Andreas

Sobott, Frank

Brutschy, Bernhard

Abstract

Laser induced liquid beam ionization/desorption mass spectrometry (LILBID-MS) is a new desorption method recently developed in our laboratory. This method allows ions to be desorbed directly from the liquid phase into the high-vacuum region of a mass spectrometer. This method has now been applied to the detection of noncovalent protein-protein complexes. The example given in this paper is the quartenary complex of human hemoglobin. For the first time, the intact hemoglobin could be detected by laser desorption mass spectrometry. Furthermore, evidence for the specificity of the complex is given. Copyright © 2000 John Wiley & Sons, Ltd. Studying noncovalent interactions of proteins with mass spectrometry is a very difficult field of research. The difficulties arise from the fragility of these complexes in the gas phase. In solution they are held together only by weak forces, mainly hydrophobic and dipole-dipole interactions. First of all the sample preparation on the target must not destroy this delicate equilibrium. Upon desorption of the ions into the gas phase, the stabilizing forces change with polar interactions, such as salt bridges and H-bonds, being responsible for the stability of the noncovalent complex. As a result, the gas-phase structure of the complexes generally differs from that in solution. This fact alone makes it difficult to detect noncovalent complexes by mass spectrometry and to correlate mass spectra with solution behavior. Moreover, these complexes tend to dissociate in the gas phase, due to thermal excitation during the desorption process and due to their internal energy. This dissociation rate must be slow enough to detect the complexes on the time-scale of the experiment, i.e. in the microsecond regime (for a time-of-flight analyzer). As a result, great care must be taken not to destroy the complexes and not to produce artefacts by the detection process. Once these problems have been solved, a broader application of mass spectrometry in biological sciences can be anticipated. Electrospray ionization mass spectrometry (ESI-MS) has been mainly used for the detection of noncovalent interactions,16, although Matrix-assisted laser desorption/ionization (MALDI) has recently been applied to a lesser extent.79. MALDI suffers from two main shortcomings: first the sample preparation (crystallization of the analyte/matrix mixture) removes the aqueous environment and, secondly, the desorption process itself seems to hinder the detection of specific noncovalent interactions in most cases. A benchmark system that challenges the potential of mass spectrometry for the detection of molecular aggregation is the hemoglobin complex. Hemoglobin is an important part of mammalian blood and transports oxygen to the cells. The intact human protein consists of four polypeptide chains, two α-chains and two β-chains, and four heme groups nested therein. These subunits are held together by noncovalent interactions, mainly salt bridges and hydrophobic interactions. The structure is sensitive to the pH-value of the solvent. Only at a neutral pH does the complex remain stable. While the intact (αβ)2-complex has been observed using ESI-MS,1012 this was not the case with MALDI-MS, even when using conditions that allowed the detection of other noncovalent complexes.13 In our group we have developed a new laser desorption method that uses a beam of the solution as matrix. This circumvents the first problem associated with MALDI. The ions preformed in the liquid are directly desorbed by means of an infrared laser beam.14 Recently, this method has also been adapted to the study of aqueous solutions. This is of prime interest, as most biochemical reactions take place in an aqueous environment. Most proteins and especially their noncovalent complexes are only stable in water, which may be considered as nature's solvent. It has recently been shown that the interaction of a peptide with a metal cation in water can be monitored using this method and that the mass spectra can be correlated with the solution behavior.15 In this paper we show the first results studying noncovalent protein-protein complexes, which are much more fragile than the previously studied system.

Language

English

Source (journal)

Rapid communications in mass spectrometry. - London

Publication

London : 2000

ISSN

0951-4198

DOI

10.1002/(SICI)1097-0231(20000530)14:10<859::AID-RCM956>3.0.CO;2-M

10.1002/(SICI)1097-0231(20000530)14:10<859::AID-RCM956>3.3.CO;2-D

Volume/pages