NDLI: 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

Content Provider	Springer Nature Link
Author	Chen, Wei Qiang Karnaukhova, Elena Lubec, Gert
Copyright Year	2013
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.
Starting Page	1381
Ending Page	1389
Page Count	9
File Format	PDF
ISSN	09394451
Journal	Amino Acids
Volume Number	44
Issue Number	5
e-ISSN	14382199
Language	English
Publisher	Springer Vienna
Publisher Date	2013-03-20
Publisher Place	Vienna
Access Restriction	Subscribed
Subject Keyword	Conformation CD Protein structure Native gel Biochemistry Analytical Chemistry Biochemical Engineering Life Sciences Proteomics Neurobiology
Content Type	Text
Resource Type	Article
Subject	Organic Chemistry Biochemistry Clinical Biochemistry

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