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Desalting protein ions in native mass spectrometry using supercharging reagents.

ABSTRACT: Effects of the supercharging reagents m-NBA and sulfolane on sodium ion adduction to protein ions formed using native mass spectrometry were investigated. There is extensive sodium adduction on protein ions formed by electrospray ionization from aqueous solutions containing millimolar concentrations of NaCl, which can lower sensitivity by distributing the signal of a given charge state over multiple adducted ions and can reduce mass measuring accuracy for large proteins and non-covalent complexes for which individual adducts cannot be resolved. The average number of sodium ions adducted to the most abundant ion formed from ten small (8.6-29 kDa) proteins for which adducts can be resolved is reduced by 58% or 80% on average, respectively, when 1.5% m-NBA or 2.5% sulfolane are added to aqueous solutions containing sodium compared to without the supercharging reagent. Sulfolane is more effective than m-NBA at reducing sodium ion adduction and at preserving non-covalent protein-ligand and protein-protein interactions. Desalting with 2.5% sulfolane enables detection of several glycosylated forms of 79.7 kDa holo-transferrin and NADH bound to the 146 kDa homotetramer LDH, which are otherwise unresolved due to peak broadening from extensive sodium adduction. Although sulfolane is more effective than m-NBA at protein ion desalting, m-NBA reduces salt clusters at high m/z and can increase the signal-to-noise ratios of protein ions by reducing chemical noise. Desalting is likely a result of these supercharging reagents binding sodium ions in solution, thereby reducing the sodium available to adduct to protein ions.

SUBMITTER: Cassou CA

PROVIDER: S-EPMC4245020 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Desalting protein ions in native mass spectrometry using supercharging reagents.

Cassou Catherine A CA Williams Evan R ER

The Analyst 20141001 19

Effects of the supercharging reagents m-NBA and sulfolane on sodium ion adduction to protein ions formed using native mass spectrometry were investigated. There is extensive sodium adduction on protein ions formed by electrospray ionization from aqueous solutions containing millimolar concentrations of NaCl, which can lower sensitivity by distributing the signal of a given charge state over multiple adducted ions and can reduce mass measuring accuracy for large proteins and non-covalent complexe ...[more]

PMID: 25133273

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Project description:RationaleThe performance of mass spectrometry (MS) analysis is often affected by the presence of salt ions. To achieve optimal MS detection results, desalting is necessary for samples with high salt concentrations. We report a rapid, low-cost and flexible online desalting method using Nafion-coated sponge. This method is easy to perform and can be implemented to a wide range of customized fluidic systems.MethodsNafion-coated melamine sponge was fabricated by soaking a glass tube containing a melamine sponge in Nafion solution and then drying overnight. The online desalting workflow is comprised of three major parts: (1) Syringe pump, which provides a continuous flow for the online fluid system; (2) Nafion sponge in a glass tube, where the online desalting of sample solution happens; (3) Capillary Vibrating Sharp-Edge Spray Ionization (cVSSI), which is an ionization technique to ionize the desalted analytes.ResultsEffective online desalting of a 10 mM NaCl solution was demonstrated for a wide range of molecules including small molecules, peptides, DNAs, and proteins using a flow rate of 10 μL/min. By incorporating multiple pieces of the Nafion-coated sponge, effective desalting for ubiquitin and cytochrome c (Cyt-c) from physiological buffers, including phosphate-buffered saline (PBS) and tris-buffered saline (TBS), were also achieved. For molecules that are sensitive to low pH conditions after desalting, a R-SO3 NH4 -type Nafion-coated sponge was fabricated. Desalting of ubiquitin, oligosaccharide, and DNA oligomers from 10 mM NaCl or 10 mM KCl solutions was demonstrated.ConclusionsFlexible, low-cost, and efficient online desalting was achieved by the Nafion-coated sponge. A variety of molecules ranging from small molecules, peptides, proteins to oligosaccharides and DNAs can be desalted for MS analysis. The desalting by Nafion sponge has great potential for desalting applications that require customized fluidic design and rapid analysis.

Dataset Information

Desalting protein ions in native mass spectrometry using supercharging reagents.

Publications

Desalting protein ions in native mass spectrometry using supercharging reagents.

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