ammonium formate vs ammonium acetate
Figure 1: Pentafluoropropionic acid (PFPA, pKa 0.18) and Heptafluorobutyric acid (HFBA pKa 0.4). Am. J. 25, 347–379 (2006), Wortmann, A., Kistler-Momotova, A., Zenobi, R., Heine, M.C., Wilhelm, O., Pratsinis, S.E. 3, 454–461 (2017), Shelimov, K.B., Clemmer, D.E., Hudgins, R.R., Jarrold, M.F. 86, 1640–1647 (2014), Zhuang, X., Gavriilidou, A.F.M., Zenobi, R.: Influence of alkylammonium acetate buffers on protein–ligand noncovalent interactions using native mass spectrometry. W.H. Soc. Redox buffers stabilize the interfacial potential within the ESI source. It is hoped that the habit of referring to pH 7 solutions as ammonium acetate “buffer” will disappear from the literature. When 1 mM H+ is added to this buffer (corresponding to water oxidation under typical nanoESI conditions ), the buffer will respond with a slight acidification to pH 6.8. J. Soc. Chem. Mass Spectrom. 11, 961–966 (2000). Major interferences arise from the fact that cationic and anionic components (such as Na+, H2PO4 Anal. Soon afterwards it became apparent that even protein–ligand and protein–protein complexes can be studied using this approach [31,32,33]. This can be illustrated by considering a bulk solution that contains 1 mM H+, corresponding to pH 3. J. Biol. In addition to these gas-phase events, it is necessary to minimize structural perturbations that might take place while the protein is still surrounded by solvent. 92, 2426–2427 (1995), Hendricks, N.G., Julian, R.R. Soc. The acidity of these reagents should also be noted and a stationary phase with good low pH stability should be selected. J. ESI-induced pH artifacts are more apparent for proteins that start to unfold around pH 5–6. Proc. Also, the volatile nature of ammonium acetate makes it difficult to predict in how far elevated concentrations would help suppress pH changes within ESI droplets (Figure 2a). Table 2 lists several additional buffers that are sufficiently volatile for LC-MS use. Ammonium hydrogen carbonate is MS friendly and has a UV cut-off of 190nm. 36, 1125–1132 (2001), Shieh, I.-F., Lee, C.-Y., Shiea, J.: Eliminating the interferences from TRIS buffer and SDS in protein analysis by fused-droplet electrospray ionization mass spectrometry. 110, 7235–7239 (2013), Deng, L., Broom, A., Kitova, E.N., Richards, M.R., Zheng, R.B., Shoemaker, G.K., Meiering, E.M., Klassen, J.S. One potential pitfall is the occurrence of pH-induced changes that can affect the analyte while it is still surrounded by solvent. It is a stronger acid than TFA and, as such, will have sufficient capacity at lower concentrations than TFA (a 3mM solution of MSA gives a similar pH to 0.1% TFA). Readers interested in ESI-related redox chemistry can refer to recent reviews [49, 50]. Overall, the profiles of Figure 2a demonstrate that proteins within the ESI plume can experience significant pH-induced stress, with pH < 1 during the final stages of solvent evaporation (Figure 2b, c). : Native electrospray ionization and electron-capture dissociation for comparison of protein structure in solution and the gas phase. Soc. −, and by transmembrane ion transporters [24, 25]. Bear in mind the UV contribution of the additive when working at low UV wavelengths (<220nm) and be smart with UV detector settings to avoid sloping baselines [1,2]. Am. Under these circumstances, the ammonium ion is merely acting as an MS friendly counter ion in place of sodium or phosphorous ions. Soc. 233–258. 162, 173–181 (1997), Kitova, E.N., El-Hawiet, A., Schnier, P.D., Klassen, J.S. Retention time under reversed phase conditions will tend to increase with increasing ion pair chain length; however, care is required to add just enough ion pairing reagent for improved retention. Am. Sure they do. The addition of organic acids is the most common approach for inducing protein unfolding in bulk solution. Sci. Chem. 85, 2191–2199 (2013), Kaddis, C.S., Loo, J.A. Dashed lines indicate approximate radii of ubiquitin (Ubq, 8.6 kDa) and pyruvate kinase (PK, 230 kDa); (b) and (c) illustrate droplets where the entire Rayleigh charge is due to protons, representing intermediate stages of a protein CRM process. Unfortunately, ammonium bicarbonate tends to induce protein unfolding during ESI. -0.05 pH units per 10% acetonitrile, Low ionic strength solutions at near neutral pH – electrolytically produced protons are abundant at the droplet surface from which analyte ions are desorbed, In strongly basic ammonia solutions, gas phase protons transferred from ammonium ions can be the dominant charging mechanism, Discharge induced ionization also occurs with high ionic strength at neutral or high pH, 3. Although many native ESI-MS studies have employed ammonium acetate at concentrations around 10 mM [4, 12, 15, 16, 32, 82,83,84,85,86], higher concentrations can often be tolerated [1, 5, 7, 8, 11, 40, 86]. Biochem. This behavior is consistent with experimental observations, e.g., apomyoglobin in pure water produces high ESI charge states that indicate acid-induced unfolding. Analyst 136, 20–28 (2011), Kebarle, P., Verkerk, U.H. A proper buffer has its pKa within ± 1 unit of the desired pH . The initial solution volume was 10 mL for all titration curves. +, thereby generating a buffer around the pKa of ammonium (9.25). Solutions of sodium or ammonium acetate (for example) can be infused into the eluent flow post-column in order to promote adduct formation, which is often attendant with an increase in analyte signal. Chem. Chem. Anal. 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]. Compare this to the use of ammonium acetate or formate buffers at low pH where the buffering ranges of the ammonium species and the format or acetate are several pH units apart (see Table 1). Native ESI-MS involves the transfer of intact proteins and biomolecular complexes from solution into the gas phase. 19, 332–343 (2008), Wang, W., Kitova, E.N., Klassen, J.S. Ammonium formate is another volatile salt, but it is even less suitable for neutral pH buffering than ammonium acetate due to the pK a of formic acid/formate (3.75) . Chem. Mass Spectrom. 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. 85, 6469–6476 (2013), Susa, A.C., Mortenson, D.N., Williams, E.R. J. Rev. J. Phys. Both of the relevant pKa values (4.75 for acetic acid and 9.25 for ammonia) are 2.25 units away from the desired neutral pH. : Analyzing enzymatic ph activity profiles and protein titration curves using structure-based pKa calculations and titration curve fitting. This is especially useful in the analysis of peptides and proteins and typically 5mM of medronic acid can be added to buffered mobile phase (ammonium acetate for example) to provide highly-effective ‘deactivation’, resulting in improved peak shapes, detector sensitivity, and quantitative reproducibility. Charge-balancing redox processes within the ESI capillary affect the solution chemistry . 0 This change in terminology will help avoid false expectations regarding the acid/base environment of analytes under ESI conditions. 06513189, 2 Clarendon Road, Ashford, Middlesex TW15 2QE, United Kingdom, +44 (0)161 818 7434 email@example.com, Copyright © 1999 - 2019.
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