Discovery Proteomics: TMTcalibrator

Advantages

• The presence of  tissue trigger channels in the TMT based experiment significantly increases the disease specific protein identifications in the other fluid channels.
• Ability to enrich for specific protein subsets, such as secreted proteins, phosphopeptides and other post translational modifications and membrane proteins.
• Due to the unbiased nature of the analysis, we report all proteins and associated PTM’s that are detectable,  unlike antibody or aptamer/affimer based technologies where the data is biased to the make-up of the selected analysis panel.
• By using an upfront high abundance protein depletion stage, Super Depletion, we can quantify many more lower abundant proteins in plasma and serum.
• Multiplexing multiple samples in a single TMT based mass spectrometry run significantly increases the sensitivity of protein detection.
• Compatibility with a wide variety of sample matrices, including plasma, serum, cell lysates, CSF, exosomes etc.
• Available for human, rodent, monkey, pig and other higher animal species.
• With TMT, multiple samples can be analysed on one mass spectrometric run.  Using a common reference channel per plex we can quantify samples from 14 to 100+ sample cohorts in multiple mass spectrometric runs.
• Analysis package includes full computational proteomic analysis and bioinformatics covering extensive data interpretation, pathway analysis and biological relevance.

Application	Service	Description
Functional Biomarker Discovery	TMTcalibrator 11-plex TMTcalibrator 18-plex	Incorporates a specific tissue/cell trigger into the TMT assay to enhance detection of tissue derived proteins in fluid matrix Relative protein quantification Combine with abundant protein depletion in  plasma Generally doubles detection rate of proteins in fluid matrix
Targeted Biomarker Assays	TMTcalibrator 11-plex TMTcalibrator 18-plex	Calibrator channels used to provide relative or absolute concentration standards Multi-point calibration curve within the same spectrum as peptide sequence Combine with LC-MS3 for most accurate quantification Further sensitivity enhancements with target immunoprecipitation, off-line fractionation and high-spike trigger channels

Magnifying the search for circulating Biomarkers

Looking for functional  biomarkers (those related to relevant biology) in body fluids faces several challenges. Fluid-derived proteins are generally present at low concentrations, whilst higher abundance proteins restrict the amount we can load into mass spectrometers. 

To overcome those limitations we invented TMTcalibrator where we use the isobaric labelling with TMT or TMTpro to mix sources enriched with biomarkers (e.g. diseased tissue lysates) and peripheral fluids (Figure 1). By adding the content of tissue and fluid channels in a single analytical sample we substantially increase visibility of highly relevant peptides and proteins and improve the rate of discovery of valuable biomarkers.

TMTcalibrator can be combined with other workflows such as phosphopeptide enrichment, peptidomics and triggering with synthetic peptides depending on your specific needs.

Sample requirements for TMTcalibrator analysis (per 18-plex experiment)

Application	Plasma/Serum (per sample)	CSF (per sample)	Tissue Trigger (total protein)
Standard LC-MS2	100ul	250ul	1.3 mg
LC-MS2 with upfront Super Depletion	250ul	Not applicable	1.3 mg
SysQuant phosphoproteomics	Generally not applicable	600ul	7.4 mg

Case studies:

1. “Comprehensive Quantitative Profiling of Tau and Phosphorylated Tau Peptides in Cerebrospinal Fluid by Mass Spectrometry Provides New Biomarker Candidates”.  A Proteome Sciences paper co-authored with University of Gothenburg.  Combined use of a brain trigger and phosphopeptide enrichment alongside standard proteomics to identify several thousand proteins and unique phosphorylation events related to Alzheimer’s disease biology that can be monitored in cerebrospinal fluid (CSF). This paper includes the first report of quantification for over 30 phosphorylation sites on Tau in Alzheimer’s disease and control individual CSF.   
2. “Combined tissue and fluid proteomics with Tandem Mass Tags® to identify low-abundance protein biomarkers of disease in peripheral body fluid: An Alzheimer’s Disease case study”.  A Proteome Sciences authored paper co-authored with University of Gothenburg and University College London describing application of the TMTcalibrator approach to identify biomarkers of neuroinflammation through use of cultured microglial cells as the trigger in a human CSF cohort. Also demonstrates translational  potential as mouse BV2 cells were used in this case.
3. “A Novel Method For Discovery of Peripheral Blood Biomarkers in Idiopathic Pulmonary Fibrosis Using Extensive Depletion and TMTcalibrator™ Tissue-Enhanced Plasma Proteomics” A Proteome Sciences poster presentation co-authored with Pliant Therapeutics showing the powerful combination of combining a fibrotic lung tissue trigger and Super Depletion of abundant plasma proteins to quantify over 5,600 proteins in plasma, including analysis of post-translationally modified collagens.

An example report is available upon request.

For FAQ’s on Unbiased Protein Biomarker Discovery based Proteomics please follow this link.

What’s included	Incoming sample QC to check sufficient protein in samples for a satisfactory analysis Protein isolation, peptide cleavage and TMT labelling Mass spectrometry-based peptide and protein ID Computational proteomics and full bioinformatics analysis Comprehensive report summarising the proteins, peptides and if applicable the peptide-PTM’s.  Data interpretation, pathway analysis and biological relevance An Excel based QuantSheet detailing qualitative and quantitative protein  identification data. Provision of all raw data if required Contact us for an example copy of our final analysis report.
Material required	This is sample and assay specific and will be detailed in the agreed Statement of Work - please contact us to discuss your specific needs so we can start the Statement of Work preparation
Typical turnaround time	Generally 6 to 8 weeks from receipt of up to 36 samples and submission of the comprehensive analytical report and associated data.