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Proteomics-based strategies to identify proteins relevant to chronic lymphocytic leukemia

Significance statement

Chronic lymphocytic leukemia (CLL) is a malignant disease of B lymphocytes and characterized by variable clinical outcomes. The main cause of Chronic lymphocytic leukemia remains unclear although some factors have been shown to increase the risk of developing the disease. Different molecular prognostic markers were reported in Chronic lymphocytic leukemia and some have been widely used. However, improvement in the accuracy of Chronic lymphocytic leukemia prognosis is still required. Proteomics is an attractive approach to identify disease-related proteins in order to deepen our understanding of Chronic lymphocytic leukemia molecular biology and to discover useful prognostic markers. We therefore performed qualitative and quantitative proteomics analyses on primary Chronic lymphocytic leukemia samples from 12 patients and reported the largest and most conserved proteomics data set in Chronic lymphocytic leukemia up to date; 728 proteins with false discovery rate (FDR) equal to 0%. Then we applied two novel strategies and two previously described methods on our reported Chronic lymphocytic leukemia proteome to identify Chronic lymphocytic leukemia -related proteins. Collectively the four strategies detected 63 proteins as CLL-related proteins, of which 10 proteins have been previously linked to different types of cancer including Chronic lymphocytic leukemia. Additional investigations on 4 of the 63 proteins in an additional Chronic lymphocytic leukemia cohort showed that thyroid hormone receptor-associated protein 3 (TR150), S100A8 and T-cell leukaemia/lymphoma protein 1A (TCL-1) were associated with high-risk CLL and early need for treatment. In contrast, myosin-9 exhibited reduced expression in high-risk Chronic lymphocytic leukemia. The above reported results fit well with the biological roles of TR150, S100A8, TCL-1 and myosin-9. For example, TR150 is required for the expression of Cyclin D1, which is an important factor in the proliferation of Chronic lymphocytic leukemia cells. In addition, high expression of S100A8 leads to greater activity of nuclear factor kappa B (NF-κB), which is an important transcription factor in Chronic lymphocytic leukemia cells. Furthermore, TCL-1 interacts with ATM, enhances the activation of NF-κB and leads to CLL- like disease when overexpressed in transgenic mice. Finally, reduced expression of mysosin-9 leads to prolonged cell attachment with high-endothelial venules (HEVs) and enhances cell retention in lymphnodes which promote survival and proliferation in Chronic lymphocytic leukemia cells. This study does not only help to improve our understanding about Chronic lymphocytic leukemia molecular biology but also report potentially good prognostic markers in Chronic lymphocytic leukemia. In addition, it represents a rich source of Chronic lymphocytic leukemia proteome and CLL-related proteins that can be used in future studies to identify prognostic markers and/or therapeutic targets in Chronic lymphocytic leukemia.

Figure LegendExample of tandem mass spectrometry analysis of the digested proteins extracted from primary CLL samples. Base peak chromatography (BPC) shows an example of MS scan of peptides spotted on a MALDI plate, where in almost all eluted spots (1530 spots) there were detected peptides reflecting the quality of the 2D nano-LC separation (A). Following the assessment of peptide masses, precursor fragmentation occurred to conduct peptide sequencing. This figure shows MS/MS spectra of a peptide (ISGLIYEETR) that was assigned to histone H4; one of the CLL-related proteins reported in this study (B). iTRAQ relative quantification of this peptide showed that high-risk CLL sample labelled with iTRAQ reagent 115 had reduced expression of histone H4 compared with low-risk CLL samples labelled with iTRAQ reagents 116 and 117 (C). The sample that was labelled with iTRAQ reagent 114 was used as a reference sample.

Proteomics-Based Strategies To Identify Proteins Relevant to Chronic Lymphocytic Leukemia. Global Medical Discovery













Journal Reference

Alsagaby SA, Khanna S, Hart KW, Pratt G, Fegan C, Pepper C, Brewis IA, Brennan P. J Proteome Res. 2014 ;13(11):5051-62.

Institute of Cancer & Genetics, School of Medicine, Cardiff University , Heath Park, Cardiff, CF14 4XN, United Kingdom.


Chronic lymphocytic leukemia (CLL), a malignant B-cell disorder, is characterized by a heterogeneous clinical course. Two-dimensional nano liquid chromatography (2D-nano-LC) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) (LC-MALDI) was used to perform qualitative and quantitative analysis on cellular extracts from 12 primary CLL samples. We identified 728 proteins and quantified 655 proteins using isobaric tag-labeled extracts. Four strategies were used to identify disease-related proteins. First, we integrated our CLL proteome with published gene expression data of normal B-cells and CLL cells to highlight proteins with preferential expression in the transcriptome of CLL. Second, as CLL’s outcome is heterogeneous, our quantitative proteomic data were used to indicate heterogeneously expressed proteins. Third, we used the quantitative data to identify proteins with differential abundance in poor prognosis CLL samples. Fourth, hierarchical cluster analysis was applied to identify hidden patterns of protein expression. These strategies identified 63 proteins, and 4 were investigated in a CLL cohort (39 patients). Thyroid hormone receptor-associated protein 3, T-cell leukemia/lymphoma protein 1A, and S100A8 were associated with high-risk CLL. Myosin-9 exhibited reduced expression in CLL samples from high-risk patients. This study shows the usefulness of proteomic approaches, combined with transcriptomics, to identify disease-related proteins.

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