Anal. Chem. 1995, 67, 4462-4465
Matrix-Assisted Laser Desorption/lonization of Noncovalently Bound Compounds A. S. Woods,+ J. C. Buchsbaum,* T. A. Worrall,* J.
M. Berg,* and R. J.
cotter*^*^@
Department of Oncology, Department of Biophysics and Biophysical Chemistry, and Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205
Matrix-assisted laser desorptiodionhtion (MALDI) mass spectrometry was used to obtain spectra of peptide-metal ion complexes formed by a zinc fhger peptide of the transcription factor IIIA (Cysz-Hisz) type and zinc and cobalt ions, as well as peptide-enzyme complexes. Peptides and proteins analyzed by mass spectrometry are generally dissolved in 0.1%aqueous TFA (pH < 2.0). At this pH, peptides and proteins denature. We therefore reasoned that MALDI mass spectrometry might be able to detect noncovalently bound compounds if conditions were used to prepare the samples that allowed macromolecular assemblies to retain tertiq structure. Samples were dissolved in 1 M ammonium bicarbonate, and a saturated matrix solution was prepared using ethanolammonium bicarbonate (1 :1) solution or ethanol-ammonium citrate (1: 1) solution. All preparations of zinc finger peptides were done in a glovebox under nitrogen to prevent oxidation of the metal binding cysteine residues. Using this approach, we have been able to demonstrate that MALDI mass spectrometry can be used to study both noncovalent metal binding complexes and noncovalent peptide-enzyme complexes. Proteins are biopolymers made of amino acid subunits joined head to tail by covalent peptide bonds between the carboxylic acid group of one amino acid and the amino group of the next. The primary amino acid sequence folds into the tertiary structure necessary for biological activity. Tertiary structure is maintained by much weaker, noncovalent bonds. These weak bonds usually form between different parts of the same protein and between different molecules. The work of Christian Anfinsen has provided much of the insight into the relationship between the amino acid sequence of a protein and its conformation.' As the environment of a folded protein is altered by changing the pH, temperature, or pressure, its structure and biological activity do not change gradually. Instead, the protein exhibits little change until a denaturation point is reached, at which there is a sudden loss of tertiary structure and biological function. Denaturation leads to disruption of noncovalent bonds, resulting in the loss of tertiary structure and protein function. Ganem et al.2.3were the f i s t to demonstrate that noncovalently bound compounds could be studied by mass spectrometry (MS) using ion spray (electrospray ionization, ESI) MS and dissolving Department of Oncology. * Department of Biophysics and Biophysical Chemistry. + Department of Pharmacology and Molecular Sciences. (1) Anfinsen C. B. Science 1973,181,223-230. (2) Ganem. B.; Li Y.; Henion, J. J. Am. Chem. SOC.1991,113,6294-6296. (3) Ganem, B., Li Y.; Henion, J. J. Am. Chem. SOC. 1991,113, 7818-7819. +
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Analytical Chemisfry, Vol. 67, No. 24, December 15, 7995
the receptor-ligand complex in 10 mM NH40Ac buffer (PH 7.5). Hutchens and Allen observed the specific binding of one Zn or Cu ion to each of two C y s ~ C y zinc s ~ finger regions in the DNAbinding domain of the estrogen receptor protein (ERDBD). Complexation of the protein with Cu ions leads to a conformational state distinctly different from that of the fully reduced ERDBD apoprotein or the ERDBD-Zn h~lopeptide.~JFenselau et al. utilized ESI MS to observe coordination of seven Zn or Cd ions with the 20 cysteines in a metallothionein.6 The observation of coordinatecovalent and noncovalent binding has been far more elusive for matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry. In this type of analysis, peptide and protein samples are usually dissolved in an aqueous solution of 0.1%TFA, 20%or more CH&N (PH ~ 2 . 0 ) .The matrix of choice is saturated 3,5dimethoxy-4-hydroxycinnamicacid in 1:l CH&N or CzHsOH-HzO @H
