mpc

Thursday, June 27th & Friday, June 28th

 Hands-On Workshop

9.00-12.30h / 13.30-16.00h

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Ansgar Poetsch & Andreas Schrötter & Fouzi El Magraoui

 

Preparation and label free and label-based Quantification of Membrane Proteins

One of the most important methods in order to identify changes in cellular signaling pathways and disease-specific biomarkers is the relative and absolute quantitation of protein abundances. Many diseases such as Morbus Alzheimer are membrane protein dependent disorders and quantifying abundances of membrane proteins is a fundamental step in understanding the underlying pathomechanisms.

In the first part of the workshop, we demonstrate a label-free quantification approach based on 1D-gel sample separation followed by LC-MS/MS analysis and relative protein quantification (spectral counting). Furthermore the participants will improve their skills regarding the isolation of membrane proteins. Therefore we will focus on two different subclasses of common used detergents, the anionic surfactant SDS and the non-ionic glycoside digitonin. While SDS triggered membrane preparation results in a high output of free membrane proteins, the presence of the mild non ionic detergent digitonin allows the preparation of native membrane protein complexes.

In the second part of the workshop, strengths and weaknesses of absolute quantification strategies for membrane proteins will be discussed based on self-conducted experiments. For this purpose, an integral membrane protein will be quantified with AQUA as well as the PSAQ technique on a triple-quad instrument (TSQ Vantage). Apart from the different quantification approaches, important sample preparation aspects (solubilization efficiency, protein/peptide recovery,..) for absolute membrane protein quantification will be explained in an accompanying seminar. At the end of the workshop, participants should have acquired enough knowledge to devise a sample preparation protocol for their own membrane protein(s) of interest and to choose an appropriate quantification technique.

A maximum number of 5 participants can participate in this workshop.

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