Deep thermal profiling for detection of functional proteoform groups

Abstract

The complexity of the functional proteome extends considerably beyond the 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 applied thermal proteome profiling with deep peptide coverage to detect functional proteoform groups in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detected 15,846 proteoforms, capturing differently spliced, cleaved and post-translationally modified proteins expressed from 9,290 genes. We identified differential co-aggregation of proteoform pairs and established links to disease biology. Moreover, we systematically made 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 proteoform groups.

Proteoform-level deconvolution reveals a broader spectrum of ibrutinib off-targets

“…This work demonstrates the potential of proteoform-level deconvolution to identify new targets of drugs, using ibrutinib as a case study. Proteoforms can be identified from untargeted thermal proteomics data and in relevant cellular contexts by applying our previous methods (23), and here we illustrate that proteoforms can also be distinguished with respect to their drug binding abilities, leveraging the thermal impact of a drug for treatment-specific proteoform identification. This enables deeper interpretation of the functional implications of drug activity, and paves the way for identification of specific proteoforms and the roles they perform.…”

“…Obtained proteoform signal intensities were then normalized per temperature using variance stabilizing normalization, and relative fold changes to the lowest measured temperature were formed. Differential melting curve analysis was performed using NPARC, as previously published and described in the context of this analysis (23,38).…”

Subcellular thermal profiling enables the deep functional exploration of the mitochondrial proteome

“…Recently, an approach combining TPP with deep fractionation for high sequence coverage was developed to detect functional proteoform groups 29 . We applied this method to the ultra-pure mitochondrial data by computing pairwise similarities between melting curves of peptides mapped to the same gene, followed by the construction of similarity networks and community detection for the assignment of proteoform groups (Fig.…”

“…It has also been used to study the effect of post-translational modifications (PTM) [24][25][26] and different cellular states, like cell cycle progression 27,28 on protein thermal stability. Finally, this method has been recently applied for the detection of functional proteoform groups 29 , where proteins encoded by the same gene but differing in their final form (e.g. due to alternative splicing, proteolytic cleavage, or PTMs 30 ) can have distinct functions.…”

Cyclic Ion Mobility-Mass Spectrometry and Tandem Collision Induced Unfolding for Quantification of Elusive Protein Biomarkers

“…Recent reports have indicated strong and varied links between cellular proteoform expression levels and human disease. − Such proteoforms are now routinely analyzed using a variety of high-resolution tandem mass spectrometry (MS) techniques, including those incorporating a wide range of separation technologies and MS-based imaging modalities. − While most proteoform populations are defined based on variations in post-translational modification or sequence, there is a growing recognition that structural proteoforms can be similarly linked to discrete biological functions and disease states. , Examples of diseases where such structural proteoforms play central roles include many forms of cancer , and protein misfolding disorders, including Alzheimer’s Disease …”