title: Effects of ammonium bicarbonate on the electrospray mass spectra of proteins: evidence for bubble-induced unfolding.
abstract: Many protein investigations by electrospray ionization (ESI) mass spectrometry (MS) strive to ensure a "native" solvent environment, i.e., nondenaturing conditions up to the point of gas-phase ion formation. Ideally, these studies would employ a volatile pH buffer to mitigate changes in H(+) concentration that can occur during ESI. Ammonium acetate is a commonly used additive, despite its low buffering capacity at pH 7. Ammonium bicarbonate provides greatly improved pH stabilization, thus offering an interesting alternative. Surprisingly, protein analyses in bicarbonate at pH 7 tend to result in the formation of very high charge states, similar to those obtained when electrospraying unfolded proteins in a denaturing solvent. This effect has been reported previously (Sterling, H. J.; Cassou, C. A.; Susa, A. C.; Williams, E. R. Anal. Chem. 2012, 84, 3795), but its exact mechanistic origin remains unclear. ESI-mediated unfolding does not take place in acetate under otherwise identical conditions. We demonstrate that heating of protein-containing bicarbonate solutions results in extensive foaming, caused by CO2 outgassing. In contrast, acetate solutions do not generate foam. Protein denaturation caused by gas bubbles is a well-known phenomenon. Adsorption to the gas/liquid interface is accompanied by major conformational changes that allow the protein to act as a surfactant. The foaming of beer is a manifestation of this effect. Bubble formation in bicarbonate during ESI is facilitated by collisional and blackbody droplet heating. Our data imply that heat and bubbles act synergistically to cause unfolding during the electrospray process, while proteins reside in ESI droplets. Because of this effect we advise against the use of ammonium bicarbonate for native ESI-MS. Ammonium acetate represents a gentler droplet environment, despite its low buffering capacity.
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
authors.forename: Jason B
authors.initials: JB
authors.lastname: Hedges
authors.forename: Siavash
authors.initials: S
authors.lastname: Vahidi
authors.forename: Xuanfeng
authors.initials: X
authors.lastname: Yue
authors.forename: Lars
authors.initials: L
authors.lastname: Konermann
mesh_terms: D000818:Animals; D001639:Bicarbonates / Q000737:chemistry* / Q000633:toxicity; D005740:Gases / Q000737:chemistry* / Q000633:toxicity; D006736:Horses; D009211:Myoglobin / Q000032:analysis* / Q000737:chemistry; D058767:Protein Unfolding / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D001639:Bicarbonates; D005740:Gases; D009211:Myoglobin; C027043:ammonium bicarbonate