Discovery and Interrogation of Functional Protein Modifications by Hotspot Thermal Profiling

Hundreds of protein posttranslational modification types have been reported across diverse organisms, however we still lack methods to systematically predict, or even prioritize, which modification sites may perturb protein function under specific cellular contexts. This protocol describes a method to detect the effects of site-specific protein phosphorylation on the thermal stability of thousands of native proteins in live cells. This mass spectrometry-based protocol measures shifts in overall protein stability in response to site-specific phosphorylation sites. The resulting dataset can enable discovery of intrinsic changes to protein structure as well as extrinsic changes to protein-protein, and protein-metabolite interactions, and can help prioritize site-specific study in a high-throughput and unbiased fashion. This approach takes several days complete, can be performed with multiple samples in parallel and is applicable to diverse organisms, cell types and posttranslational modifications.


Introduction
Recent advances in global profiling methods have greatly accelerated the discovery and study of endogenous protein posttranslational modification (PTM) 1 , which represents a central signaling mechanism in all aspects of cellular function. Chief among these technologies are liquid chromatography-mass spectrometry (LC-MS) proteomics platforms, which are typically integrated with front-end, PTM-specific enrichment protocols to detect modified peptides and proteins from biological samples. Coupled with data-dependent and de-novo PTM discovery algorithms, mass spectrometry-based studies have discovered tens of thousands unique PTM sites on proteins from diverse organisms [2][3][4] . More recently, quantitative proteomic methods have enabled the dynamics of these sites to be studied in diverse cell states and responses to stimuli [5][6][7] . A critical question that remains, however, is how minute changes to chemical structure alters the biophysical properties of a much larger protein, and how these changes lead to signaling and phenotypic consequences. The traditional approach to address this problem involves identifying specific modification sites associated with a phenotype of interest, and then laboriously studying the properties of mutant proteins that attempt to mimic the modified protein one-by-one. Because we lack general methods with which to predict, or even prioritize, which modification sites are likely to be functional in the proteome, the downstream study of PTMs is intrinsically inefficient and low-throughput.
Here we sought to address these limitations by developing a quantitative proteomic method that enables direct, high-throughput interrogation of altered protein stability in response to endogenous, site-specific posttranslational modifications for thousands of proteins in parallel. This method, Hotspot Thermal Profiling (HTP), couples PTM-specific enrichments and isotopic labeling with thermal proteomic profiling to globally detect and quantitatively measure the thermal stability of endogenous proteins and their site-specific, modified proteoforms, which we refer to herein as "modiforms," from in vitro andinsitusamples. We establish and apply this approach to a ubiquitous yet diminutive PTM -phosphorylation -and demonstrate that global thermal profiling of the modified proteome enables specific detection of both inter-and intramolecular interactions on wide range of proteins.
Our approach is applicable to study posttranslational modifications in diverse organisms and cell types. Before embarking on this protocol, one should first optimize enrichment strategies for the PTM of interest using the appropriate cell lines. Often a large amount of bulk cell lysates is needed for sufficient enrichment. Since our protocol has ten temperature fractions and each fraction is enriched independently before pooling, variable input proteome quantities should be tested for optimal results.
Other step-by-step experimental considerations are described in the protocol. In Situ Thermal Pulse 7. Expose each 400 mL of cell lysates to a steady temperature between 37°C to 67°C for 3 mins in parallel (eg. aliquot #1 at 37°C for 3 mins, aliquot #2 at 40°C for 3 mins, etc.) |Caution| Cells must be exposed to temperature treatment immediately after collection from plates and all aliquots must be treated in parallel to avoid sample variation due to time delay. 8. After heat treatment, cool and incubate cells at 25°C for another 3 mins. 9. Immediately lyse cells by rapid freeze-thawing three times with liquid nitrogen.
10. Remove insoluble proteins and cell debris by centrifugation at 17,000gfor 10 mins.
11. Set aside a small amount of supernatant from the 37°C aliquot for protein quantification by  enzyme cleavage specificity at C-terminal lysine and arginine residues with 3 missed cleavage sites permitted; static modification of +57.02146 on cysteine (carboxyamidomethylation), +229.1629 on N-terminal and lysine for TMT-10-plex tag; 4 total differential modification sites per peptide, including oxidized methionine (+15.9949), and phosphorylation (+79.9663) on serine, threonine, and tyrosine (only for phospho-enriched samples); primary scoring type by XCorr and secondary by Zscore; minimum peptide length of six residues with a candidate peptide threshold of 500; minimum of one peptide per protein and half-tryptic peptide specificity; Δmass cutoff = 10 p.p.m. with modstat, and trypstat settings; false-discovery rates of peptide (sfp) at 1%. Perform TMT quantification using the isobaric labeling 10-plex labeling algorithm, with a mass tolerance of 5

Anticipated Results
Depending on LC-MS/MS instrumentation and settings this method should yield >4,000 proteins from combined replicates of the bulk, unmodified proteome and >10,000 TMT-labeled phosphopeptides from the phospho-enriched proteome.