Abstract
Phosphorylation is one of the most important forms of posttranslational modification. Dysregulation of phosphorylation is implicated in tumorigenesis, with cancerous signaling pathways activated by kinases. For immunotherapy with neoantigen-based peptides, phosphopeptides derived from aberrantly phosphorylated proteins presented by major histocompatibility complex (MHC) are promising candidates due to their specificity to elicit cytotoxic T-cell responses. Unlike other MHC peptides, phosphorylated MHC peptides cannot be predicted from DNA sequences, and their identification relies on the direct detection of phosphopeptides using mass spectrometry (MS). For MS detection, it is extremely important to enrich phosphorylated peptides from the complex repertoire of MHC peptides. Herein, we describe the combined use of immobilized metal affinity chromatography and titanium dioxide nanoparticles for phosphopeptides enrichment from immunopeptidome.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Hunter T (2000) Signaling—2000 and beyond. Cell 100:113–127
Ubersax JA, Ferrell JE Jr (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Bio 8:530–541
Brognard J, Hunter T (2011) Protein kinase signaling networks in cancer. Curr Opin Genet Dev 21:4–11
Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA et al (2010) A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143:1174–1189
Francavilla C, Lupia M, Tsafou K, Villa A, Kowalczyk K, Jersie-Christensen RR et al (2017) Phosphoproteomics of primary cells reveals druggable kinase signatures in ovarian cancer. Cell Rep 18:3242–3256
Mohammed F, Cobbold M, Zarling AL, Salim M, Barrett-Wilt GA, Shabanowitz J et al (2008) Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self. Nat Immunol 9:1236–1243
Locard-Paulet M, Lim L, Veluscek G, McMahon K, Sinclair J, Van Weverwijk A et al (2016) Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration. Sci Signal 9:ra15-ra15
Yi T, Zhai B, Yu Y, Kiyotsugu Y, Raschle T, Etzkorn M et al (2014) Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells. Proc Natl Acad Sci U S A 111:E2182–E2190
K-L H, Li S, Mertins P, Cao S, Gunawardena HP, Ruggles KV et al (2017) Proteogenomic integration reveals therapeutic targets in breast cancer xenografts. Nat Commun 8:14864
Dazert E, Colombi M, Boldanova T, Moes S, Adametz D, Quagliata L et al (2016) Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient. Proc Natl Acad Sci U S A 113:1381–1386
Zarling AL, Polefrone JM, Evans AM, Mikesh LM, Shabanowitz J, Lewis ST et al (2016) Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy. Proc Natl Acad Sci U S A 103:14889–14894
Bassani-Sternberg M, Bräunlein E, Klar R, Engleitner T, Sinitcyn P, Audehm S et al (2016) Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry. Nat Commun 7:13404
Laumont CM, Daouda T, Laverdure J-P, Bonneil É, Caron-Lizotte O, Hardy M-P et al (2016) Global proteogenomic analysis of human MHC class I-associated peptides derived from non-canonical reading frames. Nat Commun 7:10238
Olsen JV, Mann M (2013) Status of large-scale analysis of post-translational modifications by mass spectrometry. Mol Cell Proteomics 12:3444–3452
Andersson L, Porath J (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem 154:250–254
Pinkse MW, Uitto PM, Hilhorst MJ, Ooms B, Heck AJ (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76:3935–3943
Zhou H, Low TY, Hennrich ML, van der Toorn H, Schwend T, Zou H et al (2011) Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment. Mol Cell Proteomics 10:M110.006452
Potel CM, Lin M-H, Heck AJ, Lemeer S (2018) Defeating major contaminants in Fe3+-IMAC phosphopeptide enrichment. Mol Cell Proteomics 17:1028–1034
Abelin JG, Trantham PD, Penny SA, Patterson AM, Ward ST, Hildebrand WH et al (2015) Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry. Nat Protoc 10:1308–1318
Chen R, Fauteux F, Foote S, Stupak J, Tremblay T-L, Gurnani K et al (2018) Chemical derivatization strategy for extending the identification of MHC class I immunopeptides. Anal Chem 90:11409–11416
Chambers MC, Maclean B, Burke R, Amodei D, Ruderman DL, Neumann S et al (2012) A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol 30:918–920
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Chen, R., Li, J. (2019). Enrichment of Phosphorylated MHC Peptides with Immobilized Metal Affinity Chromatography and Titanium Dioxide Particles. In: Fulton, K., Twine, S. (eds) Immunoproteomics. Methods in Molecular Biology, vol 2024. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9597-4_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9597-4_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9596-7
Online ISBN: 978-1-4939-9597-4
eBook Packages: Springer Protocols