Characterisation of pancreatic ductal adenocarcinoma subtypes by global phosphotyrosine profiling
Here we use high-resolution mass spectrometry (MS) to characterise the magnitude and complexity of global tyrosine phosphorylation PDAC. We profiled the endogenous tyrosine phosphorylation-based signaling in 36 cell lines from two cell line populations, quantifying around 2,000 tyrosine phosphorylation sites. This approach facilitated consistent segregation of PDAC cell lines into three subtypes with distinct signalling profiles, using both their global tyrosine phosphorylation patterns and a Random Forest derived 8 phosphorylation site signature. We extracted segregation-driving phosphosites, and subsequent pathway and network analysis to characterise the identified subtypes.
Sample Processing Protocol
Two cohorts of pancreatic cancer cell lines were used. Cohort 1 cell lines (termed the ATCC Cohort) were purchased from and authenticated by the American Type Culture Collection (AsPC-1, BxPC-3, CFPAC-1, Capan-1, Capan-2, HPAC, HPAF-II, Hs700T, Hs766T, Panc 02.03, Panc 03.27, Panc 04.03, Panc05.04, Panc 08.13, Panc 10.05, Panc-1, PL45, MiaPaca-2, SU.86.86, SW1990) and were used and cultured according to ATCC protocols (ATCC, VA, USA). Cohort 2 cell lines (termed the TKCC Cohort) were isolated from primary human pancreatic ductal adenocarcinoma xenografts in house (TKCC 2.1, TKCC 04, TKCC 05, TKCC 06, TKCC 07, TKCC 09, TKCC 10, TKCC 12, TKCC 14, TKCC 15, TKCC 16, TKCC 17, TKCC 18, TKCC 19, TKCC 22, TKCC 26, TKCC 27). Preparation of lysates for mass spectrometry analysis. 30mg of cell line lysate in 1mM urea was digested overnight (RT) with TPCK treated trypsin (Worthington, ratio of 1:100). Phosphopeptide immunoprecipitation. Purified peptides were lyophilized overnight and dissolved in immunoaffinity purification (IAP) buffer (50mM MOPS with 10mM sodium phosphate dibasic, 130mM sodium chloride and 0.5% NP40) and sepharose 4B beads pre-conjugated to 100ug of PY100 antibody (Cell Signalling Technology) in a 1:4 ratio were added. Peptides were incubated with antibody overnight at 4C with nutation. Non-specific binding peptides were removed by three washes with IAP buffer and three washes with dH2O. pY peptides were eluted with 0.15% trifluoroacetic acid (TFA) in 40% acetonitrile.LC –MS/MS, Identification and Quantitation. Eluted peptides were resuspended in MS loading buffer (2% MeCN, 0.3% trifluoroacetic acid (TFA)) and were separated on a 12 cm 75 μM ID analytical column pulled to an internal diameter of 5 μM by a P-2000 laser puller (Sutter Instruments Co) and packed with C18 Magic reverse phase material using a Dionex Ultimate 3000 LC system. Peptides were separated over a gradient of 60 minutes at a ﬂow rate of 200 nL/min and electrosprayed directly into the MS using a spray voltage of 1.8-2.1 kV. Mass spectrometry was performed using either a Thermo Fisher Scientiﬁc (San Jose, CA) Orbitrap Velos in positive ion mode for ATCC Cohort samples, or a Q-Exactive for TKCC Cohort samples.
Data Processing Protocol
Mass spectrometry data were analysed using MaxQuant software version 1.1.25. Database searching was performed using the Andromeda search engine integrated into the MaxQuant environment against the human Uniprot database release 2010_10, concatenated with known contaminants as well as the reversed sequences of all entries. A stepwise approach was developed to identify stable clustering topology. Firstly, hierarchical clustering was performed multiple times with an incremental inclusion of phosphorylation sites from top 10 variable to top 412 variable phosphorylation sites and the most frequent clustering topology identified.KOBAS was used to perform pathway enrichment analysis. The hypergeometric test was selected to test statistical enrichment of KEGG and Reactome pathways, and the p-values were corrected for multiple comparisons. The protein-protein interactions among proteins of interest were retrieved from the Protein Interaction Network Analysis platform, and substrate-kinase relationships were downloaded from the PhosphoSitePlus database. The networks were generated and visualized using a PINA Cytoscape plugin.
Emily Humphrey, Max Planck Institute of Biochemistry
Professor Roger Daly, Head, Department of Biochemistry and Molecular Biology, Monash University Head, Signalling Network Laboratory, Monash University Head, Biomedicine Discovery Institute Cancer Program, Monash University ( lab head )
Humphrey ES, Su SP, Nagrial AM, Hochgräfe F, Pajic M, Lehrbach GM, Parton RG, Yap AS, Horvath LG, Chang DK, Biankin AV, Wu J, Daly RJ. Resolution of novel pancreatic ductal adenocarcinoma subtypes by global phosphotyrosine profiling. Mol Cell Proteomics. 2016 Jun 3. pii: mcp.M116.058313 PubMed: 27259358