Impact of phosphorylation on thermal stability of proteins

Abstract

Reversible protein phosphorylation regulates virtually every cellular process and is arguably the most wellstudied post-translational modification. Still, less than 3% of the phosphorylation sites identified in humans have annotated functions. Functionally-relevant phosphorylation sites are known to trigger conformational changes to proteins and/or to regulate their interactions with other proteins, nucleic acids and small molecules -all of which can be reflected in the thermal stability of a protein. Thus, combining thermal proteome profiling (TPP) with phosphoproteomics (phospho-TPP) provides a way to assess the functional relevance of identified phosphorylation sites on a proteome-wide scale by comparing the melting behavior of a protein and its phosphorylated form(s). We performed phospho-TPP experiments in HeLa cells with an optimized protocol, and conclude that phosphorylation does affect protein thermal stability, but to a much lesser extent than previously reported.

Identification of phosphosites that alter protein thermal stability

“…In contrast to the Huang et al dataset, we find that the stability of phosphopeptides correlates well with the stability of their respective proteins (R 2 =0.79 for mean R s comparisons) ( Fig. 1d), suggesting that most phosphosites do not alter protein stability as also observed by Potel et al 3 . As expected, the stability of non-modified peptides present in the phosphopeptide-enriched samples also correlated well with their proteins (R 2 = 0.90 for mean R s comparisons), indicating that R s measurements in the phosphopeptide samples and protein samples can be reliably compared ( Supplementary Fig.…”

“…To minimize technical noise derived from sample preparation, peptide samples should be labeled and mixed prior to phosphopeptide enrichment (Supplementary Discussion and accompanying manuscript 3 ). Because scaling-up isobaric chemical labeling increases reagent costs substantially, we have developed an alternative approach to identify phosphosites that alter thermal stability, that we call Dali (Fig.…”

Systematic Identification of Protein Phosphorylation-Mediated Interactions

“…Finally, we were able to evaluate the hits from our study in comparison to two previous thermal protein profiling (TPP) studies performed in human cells 19,20 . When looking at the functional score distributions, all three studies were observed to be enriched for functional phosphoproteins ( Supp Fig 2a ).…”

“…Experimental approaches for high-throughput identification of protein phosphosite functions have been largely lacking. More recently, a promising approach has been to use a modified thermal proteome profiling (TPP) approach where the TPP protocol is conducted in conjunction with phosphoproteomics to identify functional phosphosites [18][19][20][21] . This method has proven useful to directly determine whether a phosphosite is likely to be functional or not.…”

SARS-CoV-2 infection remodels the host protein thermal stability landscape

“…Many physiological changes to the functional state of a protein are re ected in altered protein thermostability 8,9 . Thermal proteome pro ling (TPP) measures protein thermal stability in situ on a proteome-wide scale and is a powerful tool for identifying proteins with altered biophysical states in living cells: protein-protein interactions (PPIs), the reorganization of protein complexes 10,11 , posttranslational modi cations 12,13 , or the interaction of proteins with co-factors, small molecules, or nucleic acids 14 . The method quanti es protein abundance changes in the same samples, offering a comprehensive insight into the physiological changes that occur during cellular perturbations.…”