title: The use of native gels for the concomitant determination of protein sequences and modifications by mass spectrometry with subsequent conformational and functional analysis of native proteins following electro-elution.
abstract: The protocol consists of running a native gel with in-gel digestion by proteases, subsequent mass spectrometrical determination of protein sequence and modifications, followed by electro-elution and conformational analysis using melting point and circular dichroism. Finally, the eluted protein is tested for preserved function. Herein, C1 esterase inhibitor is applied on a native gel; in-gel digestion by proteases is carried out and peptides are identified by nano-LC-ESI-CID/ETD-MS/MS using an ion trap for generation of peptide sequences and protein modifications. Protein from replicate bands from the same gel is electro-eluted and used for determination of the melting point and used for circular dichroism analysis. Additional bands from the native gel are either in-gel digested with asparaginase to generate deamidation or PNGase F for deglycosylation, followed by mass spectrometry, conformational and functional studies. Preserved conformation and function of the C1 esterase inhibitor was shown. This protocol can be completed in 1 week.
authors.affiliation: Department of Pediatrics, Medical University of Vienna, Währinger Gürtel 18, 1090 Vienna, Austria. chenweiqiang@gmail.com
authors.forename: Wei-Qiang
authors.initials: WQ
authors.lastname: Chen
authors.forename: Elena
authors.initials: E
authors.lastname: Karnaukhova
authors.forename: Gert
authors.initials: G
authors.lastname: Lubec
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003174:Complement C1 Inactivator Proteins / Q000737:chemistry* / Q000302:isolation & purification; D050718:Complement C1 Inhibitor Protein; D006801:Humans; D008970:Molecular Weight; D063347:Native Polyacrylamide Gel Electrophoresis / Q000379:methods; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D020539:Sequence Analysis, Protein / Q000379:methods; D013050:Spectrometry, Fluorescence; D053719:Tandem Mass Spectrometry; D044366:Transition Temperature
chemical_list: D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D011506:Proteins; C469952:SERPING1 protein, human