title: Optimization of immunoaffinity enrichment and detection: toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry.
abstract: Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.
authors.affiliation: Department of Physical and Analytical Chemistry, Uppsala University, SE-751 24 Uppsala, Sweden. konstantin.artemenko@kemi.uu.se
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Artemenko
authors.forename: Sara
authors.initials: S
authors.lastname: Bergström Lind
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Elfineh
authors.forename: Corina
authors.initials: C
authors.lastname: Mayrhofer
authors.forename: Roman A
authors.initials: RA
authors.lastname: Zubarev
authors.forename: Jonas
authors.initials: J
authors.lastname: Bergquist
authors.forename: Ulf
authors.initials: U
authors.lastname: Pettersson
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation; D020014:K562 Cells; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis*; D020543:Proteome / Q000032:analysis*
chemical_list: D000911:Antibodies, Monoclonal; D010455:Peptides; D020543:Proteome; D019000:Phosphotyrosine