Scaled-Down Thermal Profiling and Coaggregation Analysis of the Proteome for Drug Target and Protein Interaction Analysis

Abstract

Thermal proteome profiling (TPP), an experimental technique combining the cellular thermal shift assay (CETSA) with quantitative protein mass spectrometry (MS), identifies interactions of drugs and chemicals with endogenous proteins. Thermal proximity coaggregation (TPCA) profiling extended TPP to study the intracellular dynamics of protein complexes. In TPP and TPCA, samples are subjected to multiple denaturing temperatures, each requiring over 100 μg of proteins, which restricts their applications for rare cells and precious clinical samples. We developed a workflow termed STASIS (scaled-down thermal profiling and coaggregation analysis with SISPROT) that scales down the required protein to as low as 1 μg per temperature. This is achieved by heating and centrifugation using the same PCR tube, processing samples with the SISPROT technology (simple and integrated spintip-based proteomics technology), and tip-based manual fractionation of TMT-labeled peptides. We evaluate the STASIS workflow with starting protein quantities of 10, 5, and 1 μg per temperature prior to heating, identifying between 4000 and 5000 proteins with 6 h of acquisition time. Importantly, we observed a high correlation in the T m of proteins with minimal difference in TPCA performance for predicting protein complexes. Moreover, STASIS could identify the targets of methotrexate and panobinostat with high precision with 1 μg of proteins per temperature. In conclusion, STASIS is a robust cost-effective technique for target deconvolution and extended TPCA to rare primary cells and precious clinical samples for the analysis of protein complexes.

Protein painting for structural and binding site analysis via intracellular lysine reactivity profiling with o-phthalaldehyde

“…The fractionation on the C18 membrane is performed by the acetonitrile concentration fractionation method, called a tip-based manual fractionation. 34 C18 is filled in a 200 μL pipette tip, and the number of C18 is equal to the total amount of peptides to be fractionated. The C18 device was activated with methanol and 80% (v/v) ACN and 0.5% (v/v) acetic acid, followed by 1% (v/v) FA and 5 mM ammonium formate to wash off the excess methanol solution.…”

High-throughput drug target discovery using a fully automated proteomics sample preparation platform

“…16,17 Like TPP, both PISA and iTSA assays rely on TMT quantification, which necessitates offline fractionation steps and a substantial amount of expensive TMT reagents. 18 Recently, label-free data independent acquisition (DIA) quantification was employed in iTSA to further increase throughput. 15 Overall, for large-scale drug target identification using TPP, there is an urgent need for an automated and high-throughput sample preparation method.…”

Development and evaluation of data analysis approaches to increase the specificity and performance of thermal shift assays for assessment of protein-small molecule interactions

“…41,43 With DDA, solutions to minimize co-fragmentation of co-eluting peptides include sample HPLC-or SPE-based fractionation, narrowing isolation windows, and using MS 3 -based quantitation of TMT-derived ion abundances,. 21,[41][42][43][44][45][46][47] Recently introduced, SPE tip-based manual fractionation involves the implementation of thermal proteome profiling with some modifications: mainly, the heating and centrifugation occur on the same PCR tube followed by sample processing on a multimode SPE tip and fractionation on a different SPE tip with a C18 membrane. 47 MS 3based DDA quantitation methods isolate fragments after MS 2 fragmentation, which results in increased quantitation accuracy at the cost of a reduction in proteomic coverage.…”

“…21,[41][42][43][44][45][46][47] Recently introduced, SPE tip-based manual fractionation involves the implementation of thermal proteome profiling with some modifications: mainly, the heating and centrifugation occur on the same PCR tube followed by sample processing on a multimode SPE tip and fractionation on a different SPE tip with a C18 membrane. 47 MS 3based DDA quantitation methods isolate fragments after MS 2 fragmentation, which results in increased quantitation accuracy at the cost of a reduction in proteomic coverage. 6 Another option to remediate ratio compression effects is to employ a OnePot approach introduced by Gaetani et al 28 The OnePot approach consists of a physical pooling all temperature-challenged sample aliquots prior to isobaric labeling of the sample.…”