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Optimised two‐dimensional electrophoresis procedures for the protein characterisation of structural tissues
Author(s) -
Hopkinson Andrew,
McIntosh Richard S.,
Layfield Robert,
Keyte John,
Dua Harminder S.,
Tighe Paddy J.
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401073
Subject(s) - chemistry , chromatography , dithiothreitol , protein purification , extraction (chemistry) , sample preparation , membrane , membrane protein , solubility , urea , thiourea , gel electrophoresis , biochemistry , organic chemistry , enzyme
The protein analysis of structural tissues is typically highly problematic. Amniotic membrane displays unique wound healing and anti‐scarring properties; however, little is known concerning its active protein content. The structural nature of amniotic membrane necessitated development and extensive optimisation of the entire two‐dimensional (2‐D) workflow. Proteins were extracted using powerful solubilisation buffers and analysis carried out using 2‐D electrophoresis followed by mass spectrometry (MS) identification. Preservation and processing resulted in prefractionation of soluble from structural and membrane‐associated proteins. Enhanced protein solubility was achieved by cysteine blocking using both N,N ‐dimethylacrylamide (DMA) alkylation and bis(2‐hydroxyethyl) disulphide (HED); an alternative procedure for the effective application of HED is demonstrated. The benefits of precipitation and cup‐loading versus in‐gel rehydration were also assessed, with procedures for the employment of HED with the latter described. Following optimisation, a representative sample 21 proteins were identified from amniotic membrane using MS verify procedures were MS‐compatible. Our results demonstrate that techniques for the reproducible separation of proteins from a proteinaceous structural tissue have been optimised. Briefly, proteins are extracted using a thiourea/urea extraction buffer containing carrier ampholytes, dithiothreitol (DTT), and 3‐(cyclohexylamino)‐1‐propanesulfonic acid (CHAPS). After DMA alkylation, proteins were precipitated (using the 2‐D clean‐up kit from Amersham Biosciences) and resolubilised in extraction buffer containing a lower concentration of DTT. Samples were either cup‐loaded onto rehydrated HED‐containing strips or rebuffered into HED‐containing buffer followed by in‐gel rehydration.