Proteomics, Genomics & Microarrays


Case study: Mapping signalling networks in colorectal tumours with SureQuant-based phosphoproteomics


Low-level cell signalling pathway modifi cations, such as tyrosine phosphorylation (pTyr) on proteins, play a key role in cell signalling, and have been found to be commonly dysregulated in cancer cells. Profi ling the tumour pTyr proteome may, therefore, reveal insights central to the development of therapeutic oncology treatments. However, traditional MS-based methods do not offer adequate sensitivity, reproducibility and depth of coverage given that pTyr proteins account for less than 1% of cellular phosphorylation.


To overcome these challenges and enable research into phosphoproteomics, Professor Forest White’s research group at the Massachusetts Institute of Technology (MIT), implemented SureQuant methodology, achieving reproducible and sensitive targeted quantitation as a result. White used this technique to leverage isotopically-labelled trigger peptides and reliably quantify several hundred commonly dysregulated pTyr targets in human colorectal tumour samples.


“SureQuant is uniquely positioned for phosphotyrosine tumour studies,” said White, Professor of Biological Engineering at MIT. “Targeted approaches typically allow observation of dozens of targets at one time, but the SureQuant setups available instead allow thousands - and the number of pTyr events within the cell likely number in the same range, with at least 2,000 sites. We aim to expand our SureQuant work to develop a larger-scale model that can cover all functionally important pTyr sites, so we can monitor the phosphotyrosine signalling network.”


Figure 5 SureQuant


Up to 500 pairs of H/L targets/30 min


Value Prop


*Highest targeting efficiency


*Complete quan profile every analysis


*Maintain sensitivity AND target scale


Quantification


Performance (Precision/ Accuracy)


Sensitivity (LLOQ)


Scale


(# Targets) Efficiency


(Productive Scans) Load-and-play


(Minimal adjustment) PRM/SRM


Up to 100 pairs of H/L targets in 30 min (60K – 116ms)


*Favouring sensitivity PRM/SRM


Up to 500 pairs of H/L targets in 30 min (7.5K – 10ms)


*Favouring target number


Summary of PRM/SRM and SureQuant capabilities. Traditional targeted approaches require tradeoffs between high-density targeted panels or high-sensitivity measurements, while SureQuant acquisition offers both large-scale profi ling while still maintaining the highest quantifi cation performance.


Summary of PRM/SRM and SureQuant capabilities. Traditional targeted approaches require tradeoffs between high-density targeted panels or high-sensitivity measurements, while SureQuant acquisition offers both large-scale profiling while still maintaining the highest quantification performance.


Conclusion


Quantitative proteomics methodologies are essential tools in the drug discovery toolkit. Discovery-based and targeted methods of quantitative proteomics are perceived as having opposing aims, with one type positioned to take a broader view and one a more detailed view. However, novel and refi ned methods of IS-PRM are capable of achieving large-scale target profi ling with superior quantitative performance, fi lling a critical gap in protein quantifi cation workfl ows. As well as enabling a deeper understanding of the molecular mechanisms behind cancers, such technology has great potential beyond oncological proteomics. The SureQuant method promises to play a crucial role in advancing our understanding of a wide range of complex diseases and signalling pathways, unlocking a wealth of knowledge about cell behaviour and health - and signposting potential routes to timely, effective treatments.


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Automated Cell Counter Wins Best New Life Science Product Award


DeNovix Inc, a developer and manufacturer of instrumentation and assays for the life sciences has been awarded the prestigious Best New Life Science Product of the Year Award for the CellDrop Automated Cell Counter which enables scientists to Count Cells Without Slides™.


The Scientists’ Choice Awards recognise the innovations which have had the greatest impact on global scientific advancement - as nominated by scientists around the world. With a drive towards sustainability and reducing the environmental impact of the laboratory, the DeNovix CellDrop Cell Counter, with its unique DirectPipette™ Technology, incorporates a novel sample loading method that eliminates the need for disposable plastic slides typically required for routine cell counting.


The CellDrop features both dual fluorescence and brightfield optics and a variable height chamber, enabling accurate measurement across a wide range of cell types, densities and sizes. Additionally, its easy-to-use software and 7″ touchscreen interface and network integration, enable users to capture, review and export data easily and securely.


“Since introducing CellDrop to the market in April 2019 the reviews and feedback from scientists around the world has been excellent and we are delighted it has been recognised with this prestigious award,” said Fred Kielhorn, CEO of DeNovix. “Researchers are actively seeking out ways to improve the environmental footprint of their labs and CellDrop enables this while decreasing consumable costs and bringing new functionality.”


More information online: ilmt.co/PL/vgzm 52288pr@reply-direct.com


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