Frequently Asked Questions

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/proteins/phosphorylation sites of interest.

For SysQuant phosphoprotein enrichment, we use metal affinity chromatography as it has the best overall performance for enrichment of phosphorylated peptides relative to the sample requirements. We use 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.

For SysQuant workflows in general we typically use LC-MS/MS (MS2) running on UHPLC-Orbitrap Fusion Tribrid, ThermoScientific.

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 and 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.

• PS offers its SysQuant customers a bioinformatics and data interpretation consultancy service.
• This service is designed to enable our SysQuant customers to unlock the valuable insight contained within their data.
• As a consultancy service the details and outputs are discussed with our customers on a case by case basis, to meet the needs of the customer and are typically charged at a daily rate.  Please contact us for more details on this.

Using the ability to multiplex samples with Tandem Mass Tags, TMTcalibrator uniquely combines fluids and tissues or cells lines to bias biomarker discovery towards disease-related biomarkers. The use of an excess of disease tissue sample ensures that all representative biomarkers are present in each analysis and forces the mass spectrometer to mostly select tissue-derived peptides for analysis. Because the same peptide present in the body fluid will also be selected for analysis at the same time, due to the isobaric TMT labelling, we substantially increase sensitivity and reduce the frequency of peptides from the normal higher abundance fluid proteins.  

In biomarker discovery studies TMTcalibrator requires two types of sample;

• The peripheral fluid (CSF, Plasma) in which you want to look for disease associated biomarkers
• A calibrant sample (tissue or cells) that is associated to the pathophysiology of the disease

In validation studies, synthetic or recombinant peptides can also be used as the calibrant sample.

Yes, synthetic or recombinant peptides can be used when using TMTcalibrator for validation studies or as an absolute qualification method.

TMTcalibrator usually requires around 50 – 100 micrograms of protein from each fluid sample and around 800 micrograms of protein for the calibration sample. If combining TMTcalibrator with phosphopeptide enrichment we require approximately 10 times this amount for both fluids and the calibrant.

For TMTcalibrator we typically use LC-MS/MS (MS2) running on UHPLC-Orbitrap Fusion Tribrid, ThermoScientific.

Yes. We have successfully combined TMTcalibrator with metal affinity enrichment of phosphopeptides. In principle any enrichment method can be followed, though careful consideration must be given when using immunoprecipitation methods. Please contact us discuss your specific requirements.

It is possible to add an include list to any of our mass spectrometry methods however for TMTcalibrator an include list would not normally be required. This is because the calibrator trigger provides excellent protein coverage without the need to run include list that can reduce the total number of peptides and proteins identified.

TMTcalibrator typical performance stats;

• Approx 500,000 data points, 50,000 peptides, 4,000 proteins.

The peptides and proteins that are identified in TMTcalibrator originate from three groups;

• Fluid proteins
• Tissue proteins
• Tissues proteins that have filtered into the fluid.

It is the proteins within this final group that are the best candidate fluid biomarkers of disease.

Sensitivity of TMTcalibrator will vary from peptide to peptide and is influenced by the efficiency of trypsin digestion of the parent protein and the ease of ionisation of the TMT-labelled peptide relative to other peptides eluting at the same time. We have been able to detect peptides from proteins known to be at concentrations of around 100 pg/ml in CSF and similar low abundance targets in plasma such as myelin basic protein, neurogranin and PDGF receptor alpha.

The standard data output of TMTcalibrator 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.

• PS offers its TMTcalibrator customers a bioinformatics and data interpretation consultancy service.
• This service is designed to enable our TMTcalibrator customers to unlock the valuable insight contained within their data.
• As a consultancy service the details and outputs are discussed with our customers on a case by case basis, to meet the needs of the customer and are typically charged at a daily rate. Please contact us for further details.

While all samples types, across all therapeutic areas can be analyzed in a TMT-MS3 study we particularly recommend it for plasma biomarker discovery and other highly complex samples where quantitative accuracy is the most important factor.

Sample multiplexing in TMT-MS3 studies is achieved using TMT-11 and TMT-18 plex tags. When sample numbers exceed 11 or 18 samples, one of the channel is used as a pooled reference bridging channel, such that, for example each TMT 18-plex set contains 17 study samples plus the reference sample.

The standard TMT labelling protocol requires a minimum of 100 µg total protein per sample. We also request a minimum of 50µg total protein for sample quality control to assess the suitability of samples for analysis.

Yes, TMT based workflows are compatible with a wide range of proteomic techniques. Please contact us discuss your specific requirements. If you are specifically interested in phosphopeptide (or other PTM's) enrichment please see our SysQuant workflow.

TMT-MS3 studies are run on either a UHPLC-Orbitrap Fusion Tribrid or a UHPLC-Orbitrap Exploris 480, both from ThermoScientific. Please contact us discuss your specific requirements.

Yes, it is possible to add an include list to the mass spectrometry method in TMT-MS3 studies. Please contact us discuss your specific requirements.

The standard data output of TMT-MS3 is QuantSheet, a Microsoft Excel file. We can also provide other formats such as .CSV or .txt on request. Please contact us to discuss your specific requirements.

• PS offers its TMT-MS3 customers a bioinformatics and data interpretation consultancy service.
• This service is designed to enable our TMT®MS3 customers to unlock the valuable insight contained within their data.
• As a consultancy service the details and outputs are discussed with our customers on a case by case basis, to meet the needs of the customer and are typically charged at a daily rate.  Please contact us for further details.

Selective Reaction Monitoring (SRM) is a targeted mass spectrometry assay format that enables absolute biomarker quantitation.

We recommend a classical SRM assay for:

• Absolute biomarker quantitation
• A bespoke list of candidates generated from the literature a biomarker discovery project that did not use TMT
• A bespoke list of post-translational modifications on particular protein(s)
• Customers looking to obtain specific measurements of endogenous as well as transgene proteins in pre-clinical models.

SRM is particularly suited to the measurement of candidates that do not have commercially available antibodies especially post-translational modifications, protein isoforms and species specific proteins.

SRM assays are typically developed for between 5 to 50 biomarker candidates.

There are six stages in the SRM Assay Development process:

• Phase 1 - Selection of proteotypic peptides
• Phase 2 - Ordering AQUA peptides (if required)
• Phase 3 - SRM assay development and validation
• Phase 4 - Optimization of sample preparation (if required)
• Phase 5 - Biomarker qualification in small cohort
• Phase 6 - Biomarker validation in larger cohort

All sample types including cell and tissue lysates and body fluids such as CSF and plasma across all therapeutic areas can be analyzed in a SRM Assay.

LOD, LOQ, precision and linear working range.

SRM Assays are developed and ran on a UHPLC-TSQ Vantage Triple Stage Quadrupole LC/MS Mass Spectrometer from ThermoScientific.

Quantitative values for each analyte are provided normally in a simple spreadsheet format.

• PS offers its SRM customers a biostatistics consultancy service.
• As a consultancy service the details and outputs are discussed with our customers on a case by case basis, to meet the needs of the customer and are typically charged at a daily rate. Please contact us for further details.

TMT-SRM is a targeted mass spectrometry assay format that enables the rapid validation of biomarker candidates.

TMT-SRM is particularly suited to candidates identified in TMT discovery workflows (SysQuant , TMTcalibrator, TMT-MS2, TMT-MS3) providing a faster and less expensive approach to biomarker validation than classical SRM.

Development of a TMT-SRM assay offers a bespoke approach to validate your candidates in one single assay without the need to purchase multiple antibodies for Western Blotting or ELISA kits.TMT-SRM is particularly suited to the validation of proteins and post-translational modifications that do not have commercially available antibodies.

TMT®SRM assays are typically developed for between 5 and 50 biomarker candidates.

There are four stages in the TMT-SRM Assay Development process:

• Phase 1 - Selection of proteotypic peptides
• Phase 2 - TMT-SRM assay development and validation
• Phase 3 - Biomarker qualification in small cohort
• Phase 4 - Biomarker validation in larger cohort

All sample types including cell and tissue lysates and body fluids such as CSF and plasma across all therapeutic areas can be analyzed in a TMT-SRM Assay.

LOD, LOQ, precision and linear working range.

TMT-SRM Assays are developed and ran on a UHPLC-TSQ Vantage Triple Stage Quadrupole LC/MS Mass Spectrometer from Thermo Scientific.

Quantitative values for each analyte are provided normally in a simple spreadsheet format.

• PS offers its TMT-SRM customers a biostatistics consultancy service.
• As a consultancy service the details and outputs are discussed with our customers on a case by case basis, to meet the needs of the customer and are typically charged at a daily rate. Please contact us for further details.

TMT-MS2 is our standard quantitative proteomics workflow for the analysis of cells and tissues when phosphopeptide enrichment is not required. The workflow combines the multiplexing power of tandem mass tags (TMT), with state-of-the-art mass spectrometry (MS) and bioinformatics to provide the broadest and deepest analysis of the proteome with excellent precision.

While all samples types, across all therapeutic areas can be analysed in a TMT-MS2 study we particularly recommend it for the analysis of cell and tissue lysates, in applications that require the broadest coverage of the proteome.

In highly complex samples such as plasma quantitation using MS2 can become comprised. To overcome this a further round of fragmentation MS3 is applied. Whilst using MS3 is slower and so reduces total proteome coverage, the gain in quantitative accuracy can reveal biomarkers that would have otherwise been missed. 

Sample multiplexing in TMT-MS2 studies is achieved using either TMT-11 and TMTpro-18plex. When sample numbers exceed 18 samples, one of the channels is used as a pooled reference bridging channel, such that each TM1-18plex set contains 17 study samples plus the reference sample. 

The standard TMT labelling protocol requires a minimum of 100 µg total protein per sample. We also request a minimum of 50 µg total protein for sample quality control to assess the suitability of samples for analysis. 

Yes, TMT workflows are compatible with a wide range of proteomic techniques. Please contact us discuss your specific requirements. If you are specifically interested in phosphopeptide enrichment please see our SysQuant workflow.

TMT-MS2 studies are run on either a UHPLC-Orbitrap Fusion Tribrid or a UHPLC-Orbitrap Exploris 480, both from ThermoScientific. Please contact us discuss your specific requirements. 

Yes, it is possible to add an include list to the mass spectrometry method in TMT-MS2 studies. Please contact us discuss your specific requirements.

The standard data output of TMT-MS2 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.

• PS offers its TMT-MS2 customers a bioinformatics and data interpretation consultancy service.
• This service is designed to enable our TMT-MS2 customers to unlock the valuable insight contained within their data.
• As a consultancy service the details and outputs are discussed with our customers on a case by case basis, to meet the needs of the customer and are typically charged at a daily rate.

TMT-MS3 is our standard workflow for plasma biomarker discovery and other highly complex samples where quantitative accuracy is the most important factor. The workflow combines the multiplexing power of tandem mass tags (TMT), with state of the art mass spectrometry (MS), MS3 fragmentation & bioinformatics to provide the most accurate quantitative results while retaining good coverage.