title: Optimised two-dimensional electrophoresis procedures for the protein characterisation of structural tissues.
abstract: 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.
authors.affiliation: University of Nottingham Division of Ophthalmology and Visual Sciences, EENT Centre, UK. andy.hopkinson@nottingham.ac.uk
authors.forename: Andrew
authors.initials: A
authors.lastname: Hopkinson
authors.forename: Richard S
authors.initials: RS
authors.lastname: McIntosh
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
authors.forename: John
authors.initials: J
authors.lastname: Keyte
authors.forename: Harminder S
authors.initials: HS
authors.lastname: Dua
authors.forename: Paddy J
authors.initials: PJ
authors.lastname: Tighe
mesh_terms: D000650:Amnion / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D009928:Organ Specificity; D040081:Protein Array Analysis / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D013440:Sulfides
chemical_list: D011506:Proteins; D013440:Sulfides; D003545:Cysteine