Deep thermal proteome profiling for detection of proteoforms and drug sensitivity biomarkers

Abstract

The complexity of the functional proteome extends significantly beyond the protein coding genome resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases, but challenging to perform systematically. Here, we apply thermal proteome profiling with deep peptide coverage to detect functional proteoforms in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detect 15,846 proteoforms, capturing differently spliced, post-translationally modified, and cleaved proteins expressed from 9,290 genes. We identify differential coaggregation of proteoform pairs and establish links to disease biology. Moreover, we systematically make use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach thus provides a powerful and unique tool for systematic detection and functional annotation of proteoforms.

Hierarchical Gaussian process models explore the dark meltome of thermal proteome profiling experiments

“…This would make it possible, e.g. to cluster peptides replicates based on their melting behaviours, thus allowing proteoforms detection, as pioneered by Kurzawa et al [14].…”

“…These proteoforms correspond to possible molecular modifications affecting the protein product of a single gene, among which are found genetic variations, alternative splicing and post-translational modifications (PTMs) [15]. Different proteoforms can have different cellular localisations and/or functions, which will be reflected in differences in melting behaviours [14]. Thus, measured peptides mapped to a single protein entry can originate from different proteoforms.…”

“…Here, the quantitative MS measurements of all tryptic peptides corresponding to a single gene entry in the used proteome database are combined to obtain the protein-level melting curve. However, the consideration of peptide-level melting curves from these MS measurements is also possible and is especially of interest for the study of post translational modifications (PTMs) [11] and the detection of proteoforms [14]. Proteoforms encompass all possible molecular modifications (among which genetic variations, alternative splicing and PTMs) affecting the protein product of a single gene [15].…”

Integration of Thermal Proteome Profiling with phosphoproteomic and transcriptomic data via mechanistic network models decodes the molecular response to PARP inhibition

“…We believe that generalizing this framework and extending it to additional data types such as solubility profiling or subcellular proteomics will be a useful step forward. Moreover, TPP data o en contain more than just binary stabilization/destabilization information, such as effect size or distinct drug response curves, which could be used to improve modeling assumptions or downstream interpretation of the network 28 .…”