| Autor: |
Kirsch ZJ; Department of Chemistry, University of Massachusetts Amherst; zkirsch@umass.edu., Arden BG; Department of Chemistry, University of Massachusetts Amherst; barden@umass.edu., Vachet RW; Department of Chemistry, University of Massachusetts Amherst; Molecular and Cellular Biology Program, University of Massachusetts Amherst; rwvachet@chem.umass.edu., Limpikirati P; Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University; patanachai.l@pharm.chula.ac.th. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of visualized experiments : JoVE [J Vis Exp] 2021 Jun 15 (172). Date of Electronic Publication: 2021 Jun 15. |
| DOI: |
10.3791/61983 |
| Abstrakt: |
Characterizing a protein's higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein's structure by MS, specific chemical reactions are performed in solution that encode a protein's structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as β2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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