title: Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry.
abstract: Phosphorylation events within cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth. Phosphopeptides derived from aberrantly phosphorylated proteins that are presented on tumors and not on normal tissues by human leukocyte antigen (HLA) class I molecules are promising candidates for future cancer immunotherapies, because they are tumor specific and have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry-compatible, iron(III)-immobilized metal affinity chromatography (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a peptide esterification step to increase phosphopeptide specificity from these low-input samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian cancer, leukemia and colorectal cancer have been identified using these highly selective phosphopeptide enrichment protocols in combination with a program called 'CAD Neutral Loss Finder' that identifies all spectra containing the characteristic neutral loss of phosphoric acid from phosphorylated serine and threonine residues. This methodology enables the identification of HLA-associated phosphopeptides presented by human tissue samples containing as little as nanograms of peptide material in 2 d.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Jennifer G
authors.initials: JG
authors.lastname: Abelin
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Paisley D
authors.initials: PD
authors.lastname: Trantham
authors.affiliation: Department of Clinical Immunology, University of Birmingham, Birmingham, UK.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Penny
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma, Oklahoma City, Oklahoma, USA.
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Patterson
authors.affiliation: Department of Clinical Immunology, University of Birmingham, Birmingham, UK.
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Ward
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma, Oklahoma City, Oklahoma, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Hildebrand
authors.affiliation: Department of Clinical Immunology, University of Birmingham, Birmingham, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Cobbold
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Dina L
authors.initials: DL
authors.lastname: Bai
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.affiliation: 1] Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA. [2] Department of Pathology, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D006649:Histocompatibility Antigens / Q000378:metabolism; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000032:analysis* / Q000378:metabolism
chemical_list: D006649:Histocompatibility Antigens; D010748:Phosphopeptides