Mass spectrometric identification of proteins from Western blots

Eppendorf tubes with protein
Alphalyse
May 2. 2016

Question:

Is there a protocol for preparing PVDF membrane for mass spectrometry?

We have an unknown protein recognized in Western blot by autoantibodies that we need to identify.

Is it possible to send in a piece of a PVDF western blotting membrane for mass spectrometric protein identification? Do you have any special requirements or protocols that we must follow?

Answer:

It is not possible to analyze proteins detected by antibodies in Western Blots directly with mass spectrometric peptide mapping. The main reason is that the Western Blot protocol adds other proteins to the PVDF membrane for detection. E.g., proteins for blocking the membrane, typically milk protein powder or bovine serum albumin (BSA), and primary and secondary antibodies for staining [1, 2].

Instead, you can run a parallel 1D or 2D gel to identify the unknown protein. First, stain the gel with a sensitive silver stain optimized for compatibility with mass spec analysis, then cut out the protein spot of interest. Finally, analyze the spot using trypsin digestion and nano-LC MS/MS or MALDI MS/MS peptide analysis for protein identification [1, 3, 4].

Also, the antibodies used for detection can be very specific and identity minute amounts of a particular protein even though other proteins may be present at the same position in the 1D or 2D gel. The mass spectrometric analysis requires that the protein of interest is relatively pure at the gel position [2, 4].

Learn more about our protein identification services here:

www.alphalyse.com/protein-identification

References:

[1]          Shevchenko et al.Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels” Analytical Chemistry1996

[2]          Jonsson et al.Recovery of gel-separated proteins for in-solution digestion and mass spectrometry,” Analytical Chemistry, 2001

[3]          Liang et al.: “Characterization of SDS–PAGE-separated proteins by matrix-assisted laser desorption/ionization mass spectrometry,” Analytical Chemistry1996

[4]         Jørgensen et al.Efficacy and compatibility with mass spectrometry of methods for elution of proteins from sodium dodecyl sulfate-polyacrylamide gels and polyvinyl difluoride membranes,” Analytical Chemistry2004

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Alphalyse aims to deliver high quality data and customer service to all of our clients in a fast and convenient way.

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