Finding New Biological Insights

New Tools for TMT® Data Analysis

A new set of bioinformatics tools to improve data integration, select regulated features and map to biological processes

Proteomics experiments generate highly complex data matrices and must be planned, executed and analyzed with extreme care to ensure the most accurate and relevant knowledge can be obtained. We take a modular approach allowing clients to enter and exit the pipeline at any stage, whilst ensuring seamless integration of each module. Our team of highly qualified and experienced scientists, bioinformaticians and biostatisticians will work with you throughout to provide a comprehensive service – from initial careful study design and planning through to detailed interpretation of your results. 

Module Software Tool Output
Computational MS Proteome Discoverer Peptide sequence and TMT® quantitation
Data Assembly and Pre-processing SQuaT,CalDIT,DIANA Normalized quantitative values and functional annotation at peptide and protein level
Statistical and Exploratory Analysis of Expression FeaST Visualization of data quality, class identifier model, biomarker candidate lists
Functional Analysis FAT Identification of biological processes and cellular components showing variance

The Integrated Bioinformatics Pipeline

Bioinformatics Diagrams

Computational MS, QC and data integration are standard components. Feature selection and functional analysis are optional components and strongly recommended for clients with limited experience of processing proteomics data

Data Integration and Pre-Processing

We have developed separate modules to integrate and process Proteome Discoverer output data for each of our core workflows. SQuaT (SysQuant®), CalDIT (TMTcalibrator™) and DIANA (TMT®MS3) perform similar functions including isotopic correction, removal of peptides lacking TMT® quantitative values, data normalization within each TMT®10plex, calculation of expression ratio and functional annotation. The output is used for feature selection (FeaST) and is included in the QuantSheet™, an Excel file that is provided to our clients.

Statistical and Exploratory Analysis

The Feature selection module FeaST takes the output from SQuaT, CalDIT or DIANA and applies data normalization between TMT®10plexes to remove batch effects before calculating relative fold-change and significance of differential expression between groups (p-value, adjusted p-value). FeaST also performs quality assessment to remove any outlier samples and exploratory analysis before applying multivariate statistical models (LIMMA)  to the processed data matrix to identify the main peptide and protein features that drive separation between experimental groups. Each iteration of the model removes features exhibiting variance due to technical or confounding clinical features (age, gender etc.) unrelated to the key biological question.

Box and Whisker PlotsBox and Whisker Plots

Box and Whisker Plots - Before normalization (left image) and after batch effect removal (right image).

Functional Analysis Tool

The Functional Analysis Tool is an optional, bespoke bioinformatics package that provides biological context around regulated proteins and peptides within each experiment. Analysis is performed following data processing by FeaST to reveal detailed information on regulatory and signaling pathways affected by disease or treatment aiding compound prioritization. In the case of fluid biomarkers, the tool can identify which aspects of disease biology are represented in the proteomics data, providing detailed knowledge of disease and drug mechanisms and supporting selection of pharmacodynamics markers of drug mechanisms. Outputs can include biological pathway and Gene Ontology enrichment and protein interaction network maps. More specialist analyses include kinase substrate and functional domain enrichments. As each experiment is different, the functional analysis package is tailored to individual requirements in consultation with the client.

Enrichment Analysis Volcano PlotsEnrichment Analysis Volcano Plots

Enrichment Analysis Volcano Plots - Enrichment of kinase substrates based on phosphopeptide expression (left figure). Enrichment of microRNA substrates based on protein expression (right figure).

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