Detection and identification of plasma proteins that bind GlialCAM using ProteinChip arrays, SELDI-TOF MS, and nano-LC MS/MS.
In order to fully understand biological processes it is essential to identify interactions in protein complexes. There are several techniques available to study this type of interactions, such as yeast two-hybrid screens, affinity chromatography, and coimmunoprecipitation. We propose a novel strategy to identify protein-protein interactions, comprised of first detecting the interactions using ProteinChips and SELDI-TOF MS, followed by the isolation of the interacting proteins through affinity beads and RP-HPLC and finally identifying the proteins using nano-LC MS/MS. The advantages of this new strategy are that the primary high-throughput screening of samples can be performed with small amounts of sample, no specific antibody is needed and the proteins represented on the SELDI-TOF MS spectra can be identified with high confidence. Furthermore, the method is faster and less labor-intensive than other current approaches. Using this novel method, we isolated and identified the interactions of two mouse plasma proteins, mannose binding lectin C and properdin, with GlialCAM, a type 1 transmembrane glycoprotein that belongs to the Ig superfamily.
Favre-Kontula
Linda
L
Merck Serono International, Geneva Research Center, 9 Chemin des Mines, Geneva, Switzerland.
Sattonnet-Roche
Pascale
P
Magnenat
Edith
E
Proudfoot
Amanda E I
AE
Boschert
Ursula
U
Xenarios
Ioannis
I
Vilbois
Francis
F
Antonsson
Bruno
B
D000818:Animals; D019062:Cell Adhesion Molecules, Neuron-Glia / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D020382:Interleukin-18 / Q000378:metabolism; D037601:Mannose-Binding Lectin / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D036103:Nanotechnology / Q000379:methods; D011414:Properdin / Q000032:analysis / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D019062:Cell Adhesion Molecules, Neuron-Glia; D018797:Cell Cycle Proteins; C487620:HEPACAM protein, human; D036341:Intercellular Signaling Peptides and Proteins; D020382:Interleukin-18; D037601:Mannose-Binding Lectin; C445639:Mbl2 protein, mouse; D011506:Proteins; C118370:interleukin-18 binding protein; D011414:Properdin