Surface Activity of Amines Provides Evidence for the Combined ESI Mechanism of Charge Reduction for Protein Complexes

Abstract

Charge reduction reactions are important for native mass spectrometry (nMS) because lower charge states help retain native-like conformations and preserve noncovalent interactions of protein complexes. While mechanisms of charge reduction reactions are not well understood, they are generally achieved through the addition of small molecules, such as polyamines, to traditional nMS buffers. Here, we present new evidence that surface-active, charge reducing reagents carry away excess charge from the droplet after being emitted due to Coulombic repulsion, thereby reducing the overall charge of the droplet. Furthermore, these processes are directly linked to two mechanisms for electrospray ionization, specifically the charge residue and ion evaporation models (CRM and IEM). Selected protein complexes were analyzed in solutions containing ammonium acetate and selected trialkylamines or diaminoalkanes of increasing alkyl chain lengths. Results show that amines with higher surface activity have increased propensities for promoting charge reduction of the protein ions. The electrospray ionization (ESI) emitter potential was also found to be a major contributing parameter to the prevalence of charge reduction; higher emitter potentials consistently coincided with lower average charge states among all protein complexes analyzed. These results offer experimental evidence for the mechanism of charge reduction in ESI and also provide insight into the final stages of the ESI and their impact on biological ions.

Probing Acid-Induced Compaction of Denatured Proteins by High-Pressure Electrospray Mass Spectrometry

“…At pH 4, myoglobin was only partially unfolded, and all detected ions were due to those of holomyoblobin with heme remaining intact (3a). This mass spectrum is similar to the typical “native mass spectrum” obtained using ammonium acetate as a “buffer,” although the role of ammonium salts in maintaining the native state of proteins is still controversial. − The abundance of the highly charged apomyoglobin (without heme) started to increase at pH 3.5 (3b) and reached its maximum at ∼ pH 3 (3c). Further decreasing the pH to 1 significantly increased the intensity for charge states +7 and +6, indicating the compaction of apomyoglobin (3d).…”

On the Chemistry of Aqueous Ammonium Acetate Droplets during Native Electrospray Ionization Mass Spectrometry

“…Protein mass spectra generated with NaCl show extensive peak tailing due to the formation of heterogeneous [M + z H + n (Na – H) + m (Cl + H)] z+ adducts, illustrated in Figure A for lysozyme. Figure A also shows abundant chemical noise in the form of salt clusters. ,, In contrast, ESI of lysozyme in 100 mM aqueous NH 4 Ac yielded clean [M + z H] z+ signals with greatly reduced background noise (Figure B), highlighting the favorable properties of this volatile salt. ,,,− …”

“…Typical native ESI experiments start with bulk solutions with an initial pH of 7, containing C 0 = 10 to 100 mM NH 4 Ac. ,,,− Thus, we will focus on these two C 0 values. Nascent droplets formed at the tip of the Taylor cone have not undergone extensive evaporation yet, such their NH 4 Ac concentrations will be close to C 0 , albeit with some Ac – → HAc conversion, see point ( v ).…”

“…Most native ESI experiments employ aqueous solutions containing ammonium acetate (NH 4 Ac). ,,,− The popularity of this additive stems from two attributes: (1) NH 4 Ac decomposes into NH 3 and acetic acid (HAc), both of which are volatile such that the resulting gaseous analyte ions are free of nonspecific adducts, although some adduction can persist for certain systems. , (2) NH 4 Ac yields neutral pH when dissolved in water . The second point has led to the misconception that NH 4 Ac is a neutral pH buffer. , A buffer is defined as a “ solution containing appreciable amounts of both a weak acid and its conjugate weak base .” NH 4 Ac is not a buffer at pH 7 because NH 4 + and Ac – are not a conjugate acid/base pair .…”

Ion parking in native mass spectrometry

“…Many involve altering solution conditions, such as the use of additives, to shift charge state distributions to lower values. 19,20 The exposure of electrospray droplets to acids or bases has been shown to be effective in reducing charge states [21][22][23] and the use of gasphase ion/molecule reactions for charge state reduction has been described. [24][25][26] The most robust means for charge reduction in the gas phase is via ion/ion reactions either prior to sampling ions into a mass spectrometer [27][28][29] or within the mass spectrometer.…”