Project description:The in-gel digestion of proteins for analysis by liquid chromatograph mass spectrometry has been used since the early 1990s. Although several improvements have contributed to increasing the quality of the data obtained, many recent publications still use sub-optimal approaches. We present an updated in-gel digestion protocol. We show that alternative reducing, alkylating agents and tryptic digestion buffers increase peptide and protein identification and reduce incubation times. Our results indicate that a simultaneous and short, high temperature reduction and alkylation reaction using Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and chloroacetamide (CAA) with a subsequent gel wash improve protein identification and sequence coverage, diminish peptide side reactions. Additionally, use of 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid buffer (HEPES) allows a significant reduction in the digestion time improving trypsin performance and increasing the peptide recovery. The updated in-gel digestion protocol described here is highly efficient and offers flexibility to be incorporated in any proteomic laboratory.
Project description:Development of an updated genome-scale metabolic model of Clostridium thermocellum and its application for integration of multi-omics datasets
Project description:<TO BE UPDATED> Chronic lymphocytic leukaemia (CLL) is a common, adult B-cell leukaemia that has challenges in prognosis and treatment. It is characterised by a heterogeneous clinical course with multiple distinct phenotypes currently defined genetically or with target-specific monoclonal antibodies. While many studies have examined specific protein targets or global mRNA expression in CLL, few have attempted to characterise expression across the whole proteome. To achieve a non-biased, global proteomics characterisation, 14 CLL samples representing the genetic mutant subgroups NOTCH1, SF3B1 and WT, were subjected to quantitative mass spectrometry and compared with normal B cells using two isobaric tag experiments (TMT 10-plex). 6150 proteins were fully quantitated revealing a strong correlation between the regulated proteins across the CLL samples, independent of subtype. >800 proteins demonstrated significant upregulation (p<0.05) across the CLL samples. In addition to several novel cell surface markers, overexpressed proteins were strongly indicative of dysregulation to mRNA processing, spliceosome activity, transcriptional control by RNA pol II and epigenetic mechanisms (all p<10-10). A strong enrichment was observed for proteins coded by chromosome 12, often observed with trisomy in CLL (p<0.001). Downregulated proteins included cell adhesion molecules such as integrins and suggested a reduced capacity for endothelial transmigration (both p<10-10). These findings confirm many previous observations of CLL-specific protein overexpression (eg. CD5, ROR1, matriptase) and identify several novel surface targets for investigation. They also suggest that strong patterns of protein expression exist across CLL subtypes. Together, these results demonstrate the potential of proteomics and advocate the characterisation of further cancer samples by such methods.
