Protein extraction and 2-DE of water- and lipid-soluble proteins from bovine pericardium, a low-cellularity tissue.
Bovine pericardium (BP) is an important biomaterial used in the production of glutaraldehyde-fixed heart valves and tissue-engineering applications. The ability to perform proteomic analysis on BP is useful for a range of studies, including investigation of immune rejection after implantation. However, proteomic analysis of fibrous tissues such as BP is challenging due to their relative low-cellularity and abundance of extracellular matrix. A variety of methods for tissue treatment, protein extraction, and fractionation were investigated with the aim of producing high-quality 2-DE gels for both water- and lipid-soluble BP proteins. Extraction of water-soluble proteins with 3-(benzyldimethylammonio)-propanesulfonate followed by n-dodecyl beta-D-maltoside extraction and ethanol precipitation for lipid-soluble proteins provided the best combination of yield, spot number, and resolution on 2-DE gels (Protocol E2). ESI-quadrupole/ion trap or MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular prefractionation of resolved proteins. Twenty-five unique, predominantly cytoplasmic bovine proteins were identified from the water-soluble fraction. Thirty-two unique, predominantly membrane bovine proteins were identified from the lipid-soluble fraction. These results demonstrated that the final protocol produced high-quality proteomic data from this important tissue for both cytoplasmic and membrane proteins.
Griffiths
Leigh G
LG
Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA. lggriffiths@ucdavis.edu
Choe
Leila
L
Lee
Kelvin H
KH
Reardon
Kenneth F
KF
Orton
E Christopher
EC
D017738:Alkanesulfonic Acids / Q000737:chemistry; D000818:Animals; D001593:Benzyl Compounds / Q000737:chemistry; D002417:Cattle; D005591:Chemical Fractionation / Q000379:methods*; D003593:Cytoplasm / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D000431:Ethanol / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D010496:Pericardium / Q000737:chemistry*; D055585:Physical Phenomena; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D054457:Tissue Scaffolds
C520493:3-(benzyldimethylammonio)propanesulfonate monohydrate; D017738:Alkanesulfonic Acids; D001593:Benzyl Compounds; D005960:Glucosides; D008565:Membrane Proteins; D011506:Proteins; D000431:Ethanol; C040358:dodecyl maltoside