Proteins

Buffer systems are generally neutral or mildly acidic/basic aqueous solutions; appropriate concentrations of buffer (> 10 mM) are generally employed to avoid fluctuations in pH during the desolvation process; aqueous solutions at pH 6-8 with 10-100 mM buffer are typical [6, 7].

In many cases, organic solvent is co-mixed with the solution to aid gas-phase desolvation. Typical organic solvents such as methanol can alter the solution conformation of proteins dramatically [8]. The general rule of thumb for organic solvents with proteins is that a little (5-10%) can significantly improve desolvation, and therefore increase sensitivity, while a lot (>10%) will produce high signal-to-noise spectra of denatured proteins to which complexation (if any) has no biological significance.

These concepts are illustrated in Fig. 10.1 which shows the difference in appearance between myoglobin electrosprayed from denaturing and nondenaturing solutions. Myoglobin is comprised of a monomeric protein (MW = 16951) which, under physiological conditions, binds a single heme cofactor (MW = 615.190). The spectrum in Fig. 10.1a was acquired from a solution containing 2 mM myoglobin in 50% MeOH, 49% H2O, and 1% HOAc. It is comprised of a wide distribution of charge states representing apomyoglobin with charge states ranging from (M+23H)23+ to (M+10H)10+. Also clearly evident in the spectrum is a peak at m/z 616.192 corresponding to the unbound, protonated heme group. There are no peaks detected which correspond to the intact holomyoglobin species (MW = 17 566). The spectrum in Fig. 10.1b was acquired under the identical interface and source conditions (see figure caption) and the same myoglobin concentration. The only difference between the conditions used to acquire the two spectra in Fig. 10.1a, b is the composition of the buffer solution. The spectrum in Fig. 10.1b was acquired from a solution containing 2 mM myoglobin in 10 mM NH4OAc (pH 7). In this spectrum only two primary charge states are observed, corresponding to the (M+9H)9+ and (M+8H)8+ charge states of the holo-myoglobin species. Note also the absence of the free heme moiety at m/z 616.192. Finally, it is worth making note of the disparate signal-to-noise ratios of the two spectra in Fig. 10.1a, b. While it may not be obvious in the normalized y-axis of the spectra in Fig. 10.1, the two primary peaks corresponding to the holomyo-globin in Fig. 10.1b are nearly 3-fold less intense than the peaks corresponding to the most abundant peaks, the (M+17H)17+ and (M+18H)18+ charge states of the apomyoglobin, in Fig. 10.1a. The more acidic environment and the presence of MeOH increases the overall ionization efficiency of the myoglobin in Fig. 10.1a.

Fig. 10.1 Effect of buffer composition on the ESI-FTICR-MS spectrum of myoglobin. The spectrum in (a) was acquired from a solution containing 2 mM myoglobin in 50% MeOH, 49% H2O, and 1% HOAc. Peaks corresponding to apomyoglobin and free heme dominate the spectrum. The spectrum in (b) was acquired from a solution containing 2 mM myoglobin in 10 mM NH4OAc (pH 7) under otherwise identical instrument conditions. In this spectrum only two primary charge states are observed, corresponding to the (M+9H)9+ and (M+8H)8+ charges states of the holomyoglobin species.

Fig. 10.1 Effect of buffer composition on the ESI-FTICR-MS spectrum of myoglobin. The spectrum in (a) was acquired from a solution containing 2 mM myoglobin in 50% MeOH, 49% H2O, and 1% HOAc. Peaks corresponding to apomyoglobin and free heme dominate the spectrum. The spectrum in (b) was acquired from a solution containing 2 mM myoglobin in 10 mM NH4OAc (pH 7) under otherwise identical instrument conditions. In this spectrum only two primary charge states are observed, corresponding to the (M+9H)9+ and (M+8H)8+ charges states of the holomyoglobin species.

The MeOH serves not only to denature the protein, exposing more charge-carrying residues to the solvent, it also aids desolvation by reducing the surface tension of the microdroplets. Thus, the addition of organic solvents for the analysis of proteins is a double-edged sword - the enhanced sensitivity is achieved by denaturing the protein to the extent that it can no longer bind the heme moiety. In many cases (including the myoglobin system) 5-10% MeOH does not induce a significant extent of denaturation but can enhance the ionization efficiency and produce a more stable electrospray plume.

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