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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

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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.

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Acknowledgments

We acknowledge the contribution by the Verein zur Durchführung der wissenschaftlichen Forschung auf dem Gebiet der Neonatologie und Kinderintensivmedizin “Unser Kind”.

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The authors declare that they have no competing financial interests.

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Authors and Affiliations

  1. Department of Pediatrics, Medical University of Vienna, Währinger Gürtel 18, 1090, Vienna, Austria

    Wei-Qiang Chen & Gert Lubec

  2. Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, 20892, USA

    Elena Karnaukhova

Authors
  1. Wei-Qiang Chen
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  2. Elena Karnaukhova
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  3. Gert Lubec
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Corresponding author

Correspondence to Gert Lubec.

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Supplementary material 1 (TIFF 6138 kb)

Supplementary material 2 (TIFF 7317 kb)

Supplementary material 3 (DOC 128 kb)

Supplemental Figure 1 10 % SDS-PAGE (a) and 8 % native PAGE (b) separation of C1-1, C1-2, C1-3 and C1-4. C1-1 is C1INH in 10 mM phosphate buffer. C1-2 is C1INH after electro-elution. C1-3 is C1INH after PNGase F treatment. C1-4 is C1INH after asparaginase treatment.In order to show the effect of deamidation and partial deglycosylation, a 10 % SDS-PAGE was cast, all C1INH preparations were run at 50 V for 30 min, 100 V for 1 h 30 min and gels were stained using a Colloidal Coomassie blue staining kit. The band for C1-3 representing partial deglycosylation revealed a remarkable shift towards a lower apparent molecular weight.The band at the position of the boxes (solid line) in the native gel (b) was used for mass spectrometrical analyses. Electro-elution of proteins from an unstained gel run in parallel was carried out from the position as indicated by boxes (dashed). Several bands on the native gel may reflect splice variants, modifications including glycosylation or aggregates.

Supplementary Figure 2(a) The D-Tube has two membrane windows with specific molecular weight cut-off. Put gel band into D-Tube and add buffer. Electrophorese to elute the sample from gel. Put the D-tube into dialysis tank and change to appropriate buffer. (b) Put filter device into provided tube. Add sample into the filter device and cap. Centrifuge at 14,000×g for 10-30 min depending on volume. Immediately separate filter device and reversely put into another tube. 1,000×g for 2 min to spin down the concentrated solution.

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Chen, WQ., Karnaukhova, E. & Lubec, G. 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. Amino Acids 44, 1381–1389 (2013). https://doi.org/10.1007/s00726-013-1477-1

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  • DOI: https://doi.org/10.1007/s00726-013-1477-1

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