Abstract
The effect of salts, detergents and chaotropic agents on mass spectrometric analysis are relatively well understood, mainly due to their actions decreasing the performance of ESI interface in mass spectrometric analysis. However, there are few studies in the literature characterizing the effect of protein stabilization by glycerol, followed in some circumstances by the suppression of protein signal when ESI interface is used. The aim of the present research was to investigate in details the mass spectrometric behavior of some proteins in presence of high levels of glycerol during ESI–MS analysis. Thus, horse heart myoglobin and chicken ovalbumin were used as standard proteins. It was demonstrated that the presence of 1% (v/v) glycerol suppressed the signal of these proteins during the ESI–MS analysis, even when the sample nozzle potential was scanned from 28 to 80 V. However, when the glycerol concentration was decreased to 0.5% (v/v) and the sample cone voltage adjusted to 50 V, a perfect envelope of peaks was observed, allowing the spectrum deconvolution and the molecular mass determination with mass accuracy lower than 0.01% in each situation. A molecular explanation for this suppressive effect and for the analytical overcoming of this difficult is proposed.
Highlights
Electrospray ionization (ESI) mass spectrometry (MS) is a rapid and precise method for determining masses of proteins and can be used to validate protein sequences [1]; in addition to this it may be used as an important technique to evaluate the protein purity/homogeneity
When 50 pmoles of the fresh protein preparation was diluted in acetonitrile to produce a concentration of 50% (v/v) of the solvent, in presence of 1% (v/v) glycerol, no ESI–MS signal was observed (Fig. 1c)
No setting up of the instrument was enough to permit the visualization of the envelope of peaks characteristic of proteins during the ESI–MS analysis under this specific experimental conditions
Summary
Electrospray ionization (ESI) mass spectrometry (MS) is a rapid and precise method for determining masses of proteins and can be used to validate protein sequences [1]; in addition to this it may be used as an important technique to evaluate the protein purity/homogeneity. The mass accuracy of ESI–MS is generally within the limit from 0.01% to 0.05% of the calculated masses [1,2] and has been used to characterize many recombinant proteins [3,4]. ESI–MS may be used to characterize unusual posttranslational modifications [5], and to identify errors in cDNA sequences [6]. Many commercial recombinant proteins used as molecular biology tools, and even some of those academically-made preparations are maintained in presence of high glycerol concentrations after purification to keep stable the biological activity
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