title: A proteomic approach to identify phosphoproteins encoded by cDNA libraries.
abstract: We report a method for large-scale rapid analysis of phosphoproteins in tissues or cells by combining immobilized metal affinity chromatography (IMAC) with phage display cDNA library screening. We expressed a testis cDNA library as fusion proteins on phage and, using IMAC, enriched for sequences encoding phosphoproteins. Selected clones were polymerase chain reaction amplified and sequenced. The majority of the clones sequenced (80%) encoded known proteins previously identified as phosphoproteins. Immunoblotting with phosphotyrosine antibodies confirmed that some of the selected sequences encoded tyrosine phosphorylated proteins when expressed on phage. An advantage of this method is the rapid identification of phosphoproteins encoded by a cDNA library, which can identify proteins that are potentially phosphorylated in vivo. When this method is combined with limited enzymatic digestion and tandem mass spectrometric techniques, the specific phosphorylation site in a protein can be identified. This technique can be used in proteomics studies to effectively detect phosphorylated proteins and avoid time-consuming and expensive peptide sequencing.
authors.affiliation: Department of Animal Sciences, University of Illinois, 1207 West Gregory Drive, Urbana, IL 61801, USA.
authors.forename: Xudong
authors.initials: X
authors.lastname: Shi
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Belton
authors.forename: Heather R
authors.initials: HR
authors.lastname: Burkin
authors.forename: Ana P
authors.initials: AP
authors.lastname: Vieira
authors.forename: David J
authors.initials: DJ
authors.lastname: Miller
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D015723:Gene Library; D007501:Iron / Q000378:metabolism; D019151:Peptide Library; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D019151:Peptide Library; D010750:Phosphoproteins; D007501:Iron