title: A secreted protein microarray platform for extracellular protein interaction discovery.
abstract: Characterization of the extracellular protein interactome has lagged far behind that of intracellular proteins, where mass spectrometry and yeast two-hybrid technologies have excelled. Improved methods for identifying receptor-ligand and extracellular matrix protein interactions will greatly accelerate biological discovery in cell signaling and cellular communication. These technologies must be able to identify low-affinity binding events that are often observed between membrane-bound coreceptor molecules during cell-cell or cell-extracellular matrix contact. Here we demonstrate that functional protein microarrays are particularly well-suited for high-throughput screening of extracellular protein interactions. To evaluate the performance of the platform, we screened a set of 89 immunoglobulin (Ig)-type receptors against a highly diverse extracellular protein microarray with 686 genes represented. To enhance detection of low-affinity interactions, we developed a rapid method to assemble bait Fc fusion proteins into multivalent complexes using protein A microbeads. Based on these screens, we developed a statistical methodology for hit calling and identification of nonspecific interactions on protein microarrays. We found that the Ig receptor interactions identified using our methodology are highly specific and display minimal off-target binding, resulting in a 70% true-positive to false-positive hit ratio. We anticipate that these methods will be useful for a wide variety of functional protein microarray users.
authors: Department of Protein Chemistry, Genentech, South San Francisco, CA 94080, USA.
authors: Sree R
authors: SR
authors: Ramani
authors: Irene
authors: I
authors: Tom
authors: Nicholas
authors: N
authors: Lewin-Koh
authors: Bernd
authors: B
authors: Wranik
authors: Laura
authors: L
authors: Depalatis
authors: Jianjun
authors: J
authors: Zhang
authors: Dan
authors: D
authors: Eaton
authors: Lino C
authors: LC
authors: Gonzalez
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005110:Extracellular Space / Q000378:metabolism*; D005189:False Positive Reactions; D015723:Gene Library; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D008863:Microspheres; D040081:Protein Array Analysis / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D055767:Immobilized Proteins; D011506:Proteins; D011971:Receptors, Immunologic