Preservation of non‐covalent associations in electrospray ionization mass spectrometry: Multiply charged polypeptide and protein dimers

Abstract

The 'softness' of the electrospray ionization (ESI) method provides a direct link between solution chemistry and the inherent gas-phase environment of mass spectrometry. Available results related to the preservation of non-covalent associations into the gas phase after ESI are reviewed. These associations include the possible retention of elements of higher order protein structure, noncovalent polypeptide-heme associations and enzyme complexes. Experimental results are presented showing that non-covalently bound polypeptide and protein dimer ions are relatively common as low level contributions to ESI mass spectra. It is argued that these dimers are reflective of multimeric species in solution since Coulombic barriers preclude dimerization after ESI, although uncertainty remains regarding whether they exist prior to the formation of highly charged droplets. The dissociation of dimers is facile, and for proteins can yield monomers having a broad distribution of charge states. The detection of non-covalently associated dimers requires gentle ESI mass spectrometer interface conditions, yielding relatively low levels of internal excitation. Under such conditions, incomplete molecular ion desolvation can result in experimental artifacts for tandem mass spectrometric experiments. ESI mass spectrometry may have broad potential for the study of noncovalent liquid phase associations.

Investigation of the Gas-Phase Structure of Electrosprayed Proteins Using Ion-Molecule Reactions

“…Comparisons in reactivity were also made with DEA and OMA; no differences in reactivities were noted for additions over the ranges 3 x 10-5 t03 X 10-4 g/sec(DEA) and 2 X 10-5 to 3 X 10-4 glsec (OMA) for ions generated from ESI of different solution conformations. A small contribution from ubiquitin dimer, (2M + 9H)9+, appears in the H 2 0 spectrum when water is used as a sheath liquid, Its presence under mild ESI conditions is not unusual [12,15,16] but it is interesting to note that the dimer 9 + charge state disappears upon addition of 6 X 10-5 g/sec of TMA (Figure 7b). Such an observation suggests that either the proton transfer reactions are sufficiently exothermic to dissociate noncovalently bound complexes, or that the noncovalent dimers might be more reactive to proton transfer than monomers with half as many charges.…”

“…Measurement methods reflecting primary structure of biopolymers (e.g., the sequence for proteins) have been rapidly developed, with molecular weight determinations, peptide mapping, and tandem mass spectrometry (MS/MS) finding immediate applications in structure confirmation [5]. Questions regarding preservation of noncovalent associations in the gas phase, while more difficult to answer than those regarding primary structure, are proving to be experimentally tractable because they also rely upon molecular weight measurements [6][7][8][9][10][11][12][13][14][15][16][17]. Examining the higher order gas-phase structure of proteins by mass spectrometry is particularly difficult [18][19][20] because in most cases the relevant structural differences do not lead to differences in molecular weight, although in some cases arguments can be posed based on the observation of buried solvent molecules in protein crystals and their presence or absence in ESI mass spectra [9].…”

Coexisting stable conformations of gaseous protein ions.

“…Dramatic new ionization methods for mass spectrometry (MS) have made possible the formation of protein ions in the gas phase to measure molecular weight and primary sequence information (2-4), even on fmol samples (5, 6). Recent studies indicate that protein conformations in solution can affect the resulting charge distribution of the gaseous multiply charged ions formed by electrospray ionization (ESI) (7-9) and that even noncovalent complexes can survive ESI to form gaseous multiply charged ions (10)(11)(12)(13)(14)(15).…”

“…Dramatic new ionization methods for mass spectrometry (MS) have made possible the formation of protein ions in the gas phase to measure molecular weight and primary sequence information (2-4), even on fmol samples (5, 6). Recent studies indicate that protein conformations in solution can affect the resulting charge distribution of the gaseous multiply charged ions formed by electrospray ionization (ESI) (7-9) and that even noncovalent complexes can survive ESI to form gaseous multiply charged ions (10)(11)(12)(13)(14)(15).Critical information concerning solvent effects on the conformation and dynamic properties of proteins has come from NMR (16) and from isotope-exchange experiments with 2H20 (17), including those before and during ESI/MS (18,19). With an activation energy of 17-20 kcal/mol (1 cal = 4.184 J) (17), the H/2H exchange rate depends on the pH (17), electrostatic effects (20), proximity of the solvent-accessible surface (21), and conformational flexibility with hydrogen bond cleavage and formation during local unfolding and folding (22).…”

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics

“…Native MS analysis of purified protein complexes has been reported since the early 1990's (25,26). This was followed by gas-phase ejection of subunits (27), with the fragmentation of these by MS/MS and their identification being a more recent advance (23). The development of a separation technique termed native gel-eluted liquid fraction entrapment electrophoresis (native GELFrEE), allowed its linked use with native MS (23) to fully characterize intact complexes from endogenous systems (28).…”