One of the great challenges of biology is to understand how organisms respond to food, exercise or disease. This requires analysis of parameters that closely reflect the dynamic behaviour of the system. These parameters must also reflect the integrated output of all system components including the genome, the transcriptome, the proteome, and its modifications.
We focus on protein phosphorylation because every biological process is regulated by altered protein phosphoryaltion. We’ve developed technology to measure this in cells and tissues both comprehensively and quantitively. We’re now using this technology to see how systems respond to changes in exercise, diet or drugs. By combining this technology with new mathematical approaches, we intend to gain insights into the architecture and behaviour of complex systems.
We have a variety of methods to globally enrich and site-specifically quantify phosphorylation depending on the study requirements. This includes enrichment using various metal-ion affinity chromatography methods and quantification with either stable isotope labelling (dimethylation, SILAC, TMT, iTRAQ, synthetic standards) or label free approaches. This is coupled with either fractionation or single-shot analysis and a variety of acquisition strategies including DDA, DIA, PRM or SRM. We are also further interested in high-throughput analysis of phosphorylation using EasyPhos.
The attachment of glycans to protein substrates produces enormous diversity. We are particularly interested in N-linked and O-linked glycosylation and have coupled stable isotope labelling with a variety of glycopeptide enrichment approaches such as hydrophilic interaction liquid chromatography (HILIC) to globally quantify the glycoproteome. We use various tandem mass spectrometry fragmentation methods (HCD/ETD/CID) to site-specifically quantify intact glycopeptides and investigate changes in heterogeneity.
Figure 1. Glycopeptide Quantification
Figure 2. Analysis of glycopeptides using tandem mass spectrometry and complementary fragmentation approaches