While we cannot guarantee to see any specific protein, pathway or phosphorylation site, with a coverage of approximately 130,000 unique peptides and 41,000 unique phosphopeptides that map to approximately 15,000 unique phosphorylation sites, we have a very good chance of finding relevant biological processes in your study. We can also devise methods with include-lists to ensure the mass spectrometer triggers to analyze peptides of interest if they are present in the sample. Finally, all our SysQuant® customers are offered an additional bioinformatics and data interpretation consultancy service which is tailor-made to each client to focus on their specific pathway/protein(s)/phosphorylation site(s) of interest.
We use metal affinity chromatography as it has the best overall performance for enrichment of phosphorylated peptides relative to the sample requirements. We use both TiO2 & IMAC resins to maximize coverage of singly and multiply phosphorylated peptides.
SysQuant® is designed for the analysis of tissues & cell lysates from discovery, pre-clinical & clinical studies across all therapeutic areas.
For each sample the minimum protein requirement is 1mg total protein with a maximum requirement of 2mg. This is approximately 40mg wet tissue, ½ a core needle biopsy or 106 – 107 cells. Protein recoveries are sample dependent and our team will discuss requirements for specific samples types during planning phase.
No, pre-treat of cells is not required for SysQuant®. Best practice protocols for preparation of cells for a SysQuant® study can be provided on request.
SysQuant® outputs are typically in the range 8,000 protein groups, 130,000 unique peptides & 15,000 unique phosphorylation sites with confidence score greater than pRS = 75%. Approximately 85% of all peptide spectral matches (PSM) have quantitation in all ten samples within each TMT® 10plex across a SysQuant® study.
There is no definition of a phosphoproteome in terms of numbers. We identify approximately 41,000 unique phosphopeptides in a typical human tissue study that map to approximately 15,000 unique phosphorylation site (with pRS score >=75% or high confident phospho-sites).
We are often asked what is the sensitivity of the SysQuant® workflow, however it is impossible to give definitive values; with greater than 8,000 proteins covered this will include many low abundance features.
Using data from an in-house CNS SysQuant® study of disease vs control samples we reliably can detect a significant fold change of 20% or more. For phosphorylation sites a change of approximately 25% was considered reliable.
Using data from an in-house CNS SysQuant® study of disease vs control samples a median biological CV (n=3) of 11.19% and a median analytical CV (n=4) of 7.81% at the peptide level was achieved. Analytical CV is based on the comparison of 4 reference samples analysed across four TMT® 10plex sets.
The standard data output of SysQuant® is QuantSheet™, a Microsoft Excel file. We can also provide other formats such as .CSV or .txt on request. Please discuss specific requirements with our sales team.