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.