Observation of intact (heme‐bound) myoglobin by electrospray ionization on a double‐focusing mass spectrometer

Abstract

A noncovalently bound complex, the heme-apomyoglobin protein system (M, 17 568), was detected using electrospray ionization on a double-focusing mass spectrometer with an array detector. The kilovolt energy conditions used for ion transmission and focusing did not cause significant amounts of fragmentation of the weaklybound complex. With a high-performance array detector, ferntomolar sensitivity was achieved for myoglobin in water.Noncovalent interactions play many significant roles in biochemistry. Proteins are folded into specific molecular conformations maintained by intramolecular noncovalent interactions. Protein activity is highly dependent on this 'secondary structure.' Many types of superstrate/substrate binding are examples of intermolecular noncovalent interactions, including antibodylantigen, protein/cofactor, receptor/ligand, and enzyme/substrate pairings. Intermolecular noncovalent interactions are also responsible for aggregation of proteins into multimers (quaternary structure). These weak interactions can be disrupted by a variety of conditions, such as pH, temperature, solvent composition, and the presence of denaturing agents such as guanidine hydrochloride or urea.Since the first reports of electrospray (ES) ionization mass spectra of biomolecules by Fenn et ale,] ES has become an almost indispensable method in the determination of peptide and protein primary ~tructure.~" In electrospray, highly charged liquid droplets containing the analyte are formed at atmospheric pressure. Ionization takes place by attachment (or detachment) of charge-carrying species (typically protons) to the molecular entity. By virtue of its ability to. form stable gas-phase ions with a paucity of fragmentation, even from nonvolatile polar molecules such as polypeptides and proteins, ES has been classified as an 'ultrasoft' ionization method. However the typical solution conditions used in electrospray ionization (i.e., water + methanol or water + acetonitrile with 1-5% (v) acetic acid for positive ionization) are rather harsh from the standpoint of maintaining native conformations and noncovalent interactions necessary for biological activity. Thus, there is usually little information about secondary structure or noncovalently bound complexes in the electrospray mass spectrum.The denaturing effect of pH$' organic solvent^'.^ and ternperat~re'.~ on the ES mass spectra of proteins has been reported for a few systems. Recently, several groups have shown that ES mass spectrometry has the capabiliti18 of examining noncovalently bound systems, such as myogl~bin,~~-'~ by adjustment of solution and pH conditions. Author to whom correspondence should be addressed.Because noncovalent bond energies are about an order of magnitude lower than typical covalent bond strengths, the multiply charged molecular entities generated by ES from such weakly bound biological systems are very susceptible to collisionally activated dissociation (CAD).*' Previous ES work on noncovalently bound systems has been performed on quadrupole mass spe...

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry

“…Currently, we have no definitive explanation for this charge-shifting behavior. Denaturation has been shown to lead to higher charging (Katta & Chait, 1991aLeBlanc et al, 1991;Loo et al, 1991Loo et al, , 1993a. However, we consider the possibility of further denaturation to be unlikely under these conditions because the myoglobin should already be completely denatured in a 4% acetic acid/50% acetonitrile solution and this same effect is also observed with peptides as small as 6 residues, which are unlikely to have higher-order structure (data not shown).…”

“…The dominant ion in the spectrum obtained without surfactant corresponds to the myoglobin-heme complex (MI 17,568) (Katta & Chait, 1991b;Loo et al, 1993a), whereas the surfactant spectrum shows primarily apo-myoglobin (MI 16,951) in higher charge states; only a small amount of the binary myoglobin- …”

“…The dominant ion in the spectrum obtained without surfactant corresponds to the myoglobin-heme complex (MI 17,568) (Katta & Chait, 1991b;Loo et al, 1993a), whereas the surfactant spectrum shows primarily apo-myoglobin (MI 16,951) in higher charge states; only a small amount of the binary myoglobin- A similar experiment compared spectra of bovine ubiquitin in H 2 0 with and without 0.01% CTAB; addition of CTAB eliminated the ubiquitin dimer ion contribution, albeit at a serious loss in signal intensity overall. Ubiquitin dimer was stable to less denaturing detergents such as n-dodecyl glucoside.…”

“…ESI-MS, in particular, is increasingly being used to examine noncovalent complexes between protein subunits or proteins and their ligands. ESI-MS conditions can be mild enough that proteins retain their native conformations and enzyme-substrate complexes or subunit interactions can be observed (Ganem et al, 1991a(Ganem et al, , 1991bKatta & Chait, 1991b;Baca & Kent, 1992;Ganguly et al, 1992;Drummond et al, 1993;Goodlett et al, 1993;Light-Wahl et al, 1993aLoo et al, 1993aLoo et al, , 1993bOgorzalek Loo et al, 1993). A number of investigators have taken advantage of the dramatic increase in charge state observed when some proteins are denatured to probe protein structure (Katta & Chait, 1991aLeBlanc et al, 1991;Loo et al, 1991Loo et al, , 1993b Mirzaet al, 1993).…”

Activation of Peptide Ions by Blackbody Radiation: Factors That Lead to Dissociation Kinetics in the Rapid Energy Exchange Limit

“…Five additional frequencies (23,125,159,267, and 945 cm −1 ) were systematically varied to reproduce REX limit Arrhenius pre-exponential factors of 10 9.9 , 10 12.4 , 10 14.5 , and 10 16.8 s −1 .…”