Soc. Ammonium formate is another volatile salt, but it is even less suitable for neutral pH buffering than ammonium acetate due to the pKa of formic acid/formate (3.75) . Chem. However, this solution is not buffered, and it will undergo significant pH changes upon addition of small amounts of H+ or OH−. The aqueous environment is an integral part of this interaction network. A more complete table of buffers can also be found on our eLearning site CHROMacademy > Buffer choice for HPLC separations. This issue deserves particular attention when probing enzymatic reactions that consume or produce pH-active compounds [46,47,48]. Ammonium acetate nonetheless remains a useful additive for native ESI-MS. The term “buffer” may also be used in different contexts. : Leveraging ultraviolet photodissociation and spectroscopy to investigate peptide and protein three-dimensional structure with mass spectrometry. The addition of organic acids is the most common approach for inducing protein unfolding in bulk solution. Various alkyl-ammonium acetate salts have been used for mechanistic investigations [ 81 , 94 ], but they also do not provide buffering at neutral pH based on the pK a values involved. −, and by transmembrane ion transporters [24, 25]. Conditions that are too gentle cause inadequate desolvation, whereas excess activation disrupts noncovalent contacts and causes gas-phase unfolding [39, 40]. 79, 5511–5520 (2007), Van Berkel, G.J., Asano, K.G., Schnier, P.D. Chem. Doubling the H+ concentration to 2 mM (e.g., as the result of water hydrolysis during ESI ) will lower the pH to 2.7. Chem. At any rate, the use of ammonium acetate solutions in native ESI-MS is preferable over pure water. This may be the main reasons why ammonium acetate has become the de facto standard for native ESI-MS. For a 100 mM solution the pH will only drop to 6.5 under otherwise identical conditions. 76, 3944–3950 (2004), Metwally, H., McAllister, R.G., Konermann, L.: Exploring the mechanism of salt-induced signal suppression in protein electrospray mass spectrometry using experiments and molecular dynamics simulations. Admittedly, the pH estimates obtained for the magenta (H+) profile of Figure 2a represent a worst-case scenario. Spectrom. 3, 454–461 (2017), Shelimov, K.B., Clemmer, D.E., Hudgins, R.R., Jarrold, M.F. This article takes a critical look at the properties of ammonium acetate in a native ESI-MS context. J. : Surface-induced protein unfolding in submicron electrospray emitters. Am. Chem. : Co-populated conformational ensembles of b2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry. Am. Chem. Mass Spectrom. Chem. 75, 4945–4955 (2003), Liang, Y., Du, F., Sanglier, S., Zhou, B.R., Xia, Y., Van Dorsselaer, A., Maechling, C., Kilhoffer, M.C., Haiech, J.: Unfolding of rabbit muscle creatine kinase induced by acid – a study using electrospray ionization mass spectrometry, isothermal titration calorimetry, and fluorescence spectroscopy. 22, 1187–1196 (2011), Scarff, C.A., Patel, V.J., Thalassinos, K., Scrivens, J.H. + is a weak acid. 18, 385–393 (2007), Peschke, M., Verkerk, U.H., Kebarle, P.: Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method. Soc. Conversely, experiments in negative ion mode likely result in final pH values around 9.25 ± 1 as a result of NH4 Correspondence to Ammonium acetate mitigates these effects by buffering the solution in the range of 4.75 ± 1. J. © 2020 Springer Nature Switzerland AG. This acidification is severe enough to cause the protonation of His (pKa ~7), but it will leave Asp and Glu carboxylates (pKa ~ 4) largely unaffected. Regions that lack buffering capacity are characterized by steep slopes. Again, MSA produces altered selectivity to TFA and there are reports that addition of MSA to TFA based eluent systems in HILIC mode can be used to ‘tune’ the selectivity in this separation mode . Soc. Am. 21, 286–289 (2010), Jecklin, M.C., Touboul, D., Bovet, C., Wortmann, A., Zenobi, R.: Which electrospray-based ionization method best reflects protein–ligand interactions found in solution? ESI-MS can readily deal with nonvolatile additives as long as on-line liquid chromatography or other sample clean-up steps are incorporated into the workflow. J. Soc. B 106, 521–533 (2002), Grdadolnik, J., Merzel, F., Avbelj, F.: Origin of hydrophobicity and enhanced water hydrogen bond strength near purely hydrophobic solutes. Acetate buffering will lessen this acidification by ensuring that the pH does not drop below 4.75 ± 1. Proc. J. Ammonium acetate has sparing solubility in acetonitrile and above 60% acetonitrile, vigilance is required to avoid the formation of colourless salt crystals within the eluent reservoir and inner surfaces of the HPLC equipment. Am. Anal. Natl. Examples of such events include thermal denaturation due to inadvertent heating [41, 42], as well as surface adsorption within the ESI capillary . As an added bonus, ammonium acetate can even suppress the formation of undesired salt adducts by competing with nonvolatile electrolytes for binding sites at the protein surface . When designing native ESI-MS experiments, one should nonetheless not dismiss the possibility that ESI droplets can undergo significant acidification. : Observation of the heme–globin complex in native myoglobin by electrospray-ionisation mass spectrometry. When adjusting the pH of the aqueous portion of the buffer to achieve a pH relative to a known or calculated analyte pKa (i.e. However, the increased electrolyte concentration will enhance the rate of electrochemical processes within the ESI source, such that the H+ production is expected to rise when more ammonium acetate is added . This area has experienced significant growth in recent years, fuelled by the development of instruments that are capable of performing both m/z analyses and ion mobility measurements [10,11,12,13,14,15].