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Integration of online digestion and electrolytic reduction with mass spectrometry for rapid disulfide-containing protein structural analysis.

ABSTRACT: Bottom-up structural analysis of disulfide-bond containing proteins usually involves time-consuming offline enzymatic digestion, chemical reduction and thiol protection prior to mass spectrometric detection, which takes many hours. This paper presents an expedited bottom-up approach, employing desorption electrospray ionization-mass spectrometry (DESI-MS) coupled with online pepsin digestion and online electrochemical reduction of disulfide bonds. Peptides are generated in high digestion yield as its precursor protein in acidic aqueous solution flows through a pepsin column, which can undergo direct electrolysis. The electrolytic behaviors of peptides, as online monitored by DESI-MS, suggest the presence or absence of disulfide bonds in the peptides, and also provide information to relate disulfide bond-containing peptide precursors to their corresponding reduced products. Furthermore, selective electrolysis simply using different reduction potentials can be adopted to generate either partially or fully reduced peptides to assist disulfide bond mapping. In addition, it turns out that DESI is suitable for ionizing peptides in water without organic solvent additives (organic solvent additives would not be compatible with the use of pepsin column). The feasibility of this method was demonstrated using insulin, a protein carrying three pairs of disulfide-bonds as an example, in which all disulfide bond linkages and most of the protein sequence were successfully determined. Strikingly, this method shortens the sample digestion, reduction and MS detection from hours to less than 7 min, which could be of high value in high-throughput proteomics research.

SUBMITTER: Zheng Q

PROVIDER: S-EPMC4240030 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Integration of online digestion and electrolytic reduction with mass spectrometry for rapid disulfide-containing protein structural analysis.

Zheng Qiuling Q Zhang Hao H Chen Hao H

International journal of mass spectrometry 20131101

Bottom-up structural analysis of disulfide-bond containing proteins usually involves time-consuming offline enzymatic digestion, chemical reduction and thiol protection prior to mass spectrometric detection, which takes many hours. This paper presents an expedited bottom-up approach, employing desorption electrospray ionization-mass spectrometry (DESI-MS) coupled with online pepsin digestion and online electrochemical reduction of disulfide bonds. Peptides are generated in high digestion yield a ...[more]

PMID: 25419170

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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

Integration of online digestion and electrolytic reduction with mass spectrometry for rapid disulfide-containing protein structural analysis.

Publications

Integration of online digestion and electrolytic reduction with mass spectrometry for rapid disulfide-containing protein structural analysis.

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