Devereux, S

Eagle, GL

Hillmen, P

Jenkins, R

Kitteringham, N

Oates, M

Park, K

Pettitt, A

Schuh, A

Zhuang, J

University of Liverpool Repository

Abstract Submission5. Chronic lymphocytic leukemia and related disorders - BiologyEHA-1293LINKING PROTEOME AND GENOME FOR BIOMARKER DISCOVERY IN CLL – ESTABLISHMENT OF A CLLPROTEIN DATABASEGina L. Eagle* 1, Jianguo  Zhuang1, Rosalind  Jenkins2, Neil  Kitteringham2, Melanie Oates1, Kevin Park2, Anna Schuh3,Stephen Devereux4, Peter Hillmen5, Andrew Pettitt11Department of Molecular & Clinical Cancer Medicine, 2MRC Centre for Drug Safety Science, Department of Molecularand Clinical Pharmacology, University of Liverpool, Liverpool, 3Department of Oncology, University of Oxford, Oxford, 4Department of Haematological Medicine, Kings College Hospital NHS Foundation Trust, London, 5Faculty of Medicineand Health, University of Leeds, Leeds, United KingdomBackground: Chronic Lymphocytic Leukaemia (CLL) is notable for its clinical variability, suggesting a role for precisionmedicine. Attempts at understanding the biological basis of this variability have mainly focussed on genomic alterationsand gene expression at the mRNA level. However, the molecular basis of CLL variability remains incompletelyunderstood. We speculate that this is because the clinical phenotype is ultimately determined by gene expression at theprotein level. We have therefore embarked on a global proteomic study of a large number of CLL trial samples for whichwhole genome sequencing data will be available through the Genomics England Ltd (GEL) CLL Pilot Project. Weanticipate that our study will bridge the gap in our understanding between genotype and clinical phenotype.Aims: Recently developed mass spectrometric approaches to highly parallel, label-free protein quantification provide anew opportunity to develop diagnostic, prognostic and predictive biomarkers. SWATH (Sequential Windowed Acquisitionof all THeoretical fragments) is a data-independent approach which generates a mass spectral library of fragment ionsfrom all detectable peptide precursors. The composite MS/MS spectra are deconvoluted by alignment with a high qualityand exhaustive, tissue specific database, whereupon patient samples may be stratified based on the quantitativeexpression profile of thousands of proteins. Here, we describe generation of a CLL-specific protein database that willprovide a permanent reference source for all future trial samples, as indicated in Figure 1.Methods: Protein extracts were prepared from CLL cells from 14 individual patients in various stages of the disease.Lysates were pooled and digested before being subjected to cation exchange chromatography. Forty fractions weredelivered into a TripleTOF 6600 mass spectrometer (SCIEX) via an Eksigent nanoLC 415 system (SCIEX). Datadependent acquisition was performed using 25 MS/MS per cycle to optimise for quality and 30 MS/MS per cycle tooptimise for coverage, and the combined data were searched using ProteinPilot 5.0 (SCIEX) against the SwissProtdatabase (Nov 2015, 20,193 human entries). Proteins lying within a 1% global false discovery rate were included in thedatabase.Results: The new CLL-specific database contains 7773 proteins. The database covers 50% of all humanUniProtKB/SwissProt entries which have evidence at the protein level. Pathway analysis of B cell receptor (BCR)signalling based on the GeneGo pathway maps in the MetaCore database (version 6.14, build 61508; Thomson Reuters)showed that this CLL database covers over 87% of the proteins involved in BCR signalling. The quality of the databaseentries is further underlined by high representation in the Human Genome database (20,814 entries) in aspect of GeneOntology biological processes and molecular functions, as determined by PANTHER over-representation tests.Image/Pictures:Summary/Conclusion: Our global protein expression database for CLL cells is thus the most complete database of itstype. By combining advanced computational biology to relate protein expression to whole genome sequencing data, thedatabase will provide a framework for elucidating the effect of genomic alterations on protein expression. Theconstruction of this comprehensive CLL database also provides the platform for applying SWATH/MS-based analysis ofindividual patient samples to identify novel drug targets and predictive biomarkers of disease progression and treatmentresponse.Keywords: Chronic lymphocytic leukemia, Proteomics

LINKING PROTEOME AND GENOME FOR BIOMARKER DISCOVERY IN CLL-ESTABLISHMENT OF A CLL PROTEIN DATABASE

LINKING PROTEOME AND GENOME FOR BIOMARKER DISCOVERY IN CLL-ESTABLISHMENT OF A CLL PROTEIN DATABASE

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