Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues.
Because of their sensitivity to solubilizing detergents, membrane protein assemblies are difficult to study. We describe a protocol that covalently conserves protein interactions through time-controlled transcardiac perfusion cross-linking (tcTPC) before disruption of tissue integrity. To validate tcTPC for identifying protein-protein interactions, we established that tcTPC allowed stringent immunoaffinity purification of the gamma-secretase complex in high salt concentrations and detergents and was compatible with mass spectrometric identification of cross-linked aph-1, presenilin-1 and nicastrin. We then applied tcTPC to identify more than 20 proteins residing in the vicinity of the cellular prion protein (PrPC), suggesting that PrP is embedded in specialized membrane regions with a subset of molecules that, like PrP, use a glycosylphosphatidylinositol anchor for membrane attachment. Many of these proteins have been implicated in cell adhesion/neuritic outgrowth, and harbor immunoglobulin C2 and fibronectin type III-like motifs.
Schmitt-Ulms
Gerold
G
Institute for Neurodegenerative Disease, San Francisco, California 94143, USA. g.schmittulms@utoronto.ca
Hansen
Kirk
K
Liu
Jialing
J
Cowdrey
Cynthia
C
Yang
Jian
J
DeArmond
Stephen J
SJ
Cohen
Fred E
FE
Prusiner
Stanley B
SB
Baldwin
Michael A
MA
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D006348:Cardiac Surgical Procedures; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D005557:Formaldehyde / Q000737:chemistry; D017261:Glycosylphosphatidylinositols; D007163:Immunosorbent Techniques; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D019006:Neural Cell Adhesion Molecules / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010477:Perfusion / Q000379:methods*; D018621:PrPC Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D053764:Presenilin-1; D025941:Protein Interaction Mapping / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019006:Neural Cell Adhesion Molecules; D018621:PrPC Proteins; D053764:Presenilin-1; C413604:nicastrin protein; D005557:Formaldehyde; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D014357:Trypsin; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse