An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation

Sabatier, Pierre; Beusch, Christian M.; Saei, Amir Ata; Aoun, Mike; Moruzzi, Noah; Coelho, Ana Lúcia; Leijten, Niels; Nordenskjöld, Magnus; Micke, Patrick; Maltseva, D. V.; Tonevitsky, Alexander G.; Millischer, Vincent; Villaescusa, J. Carlos; Kadekar, Sandeep; Gaetani, Massimiliano; Altynbekova, Kamilya; Kel, Alexander; Berggren, Per‐Olof; Simonson, Oscar E.; Grinnemo, Karl‐Henrik; Holmdahl, Rikard; Rodin, Sergey; Zubarev, Roman A.

doi:10.1038/s41467-021-26879-4

Cited by 27 publications

(34 citation statements)

References 67 publications

(72 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Obviously, substrates not demonstrating a shift in solubility will be missed in SIESTA-PISA 38 . There are estimates that the solubility of only a small fraction of substrates changes significantly upon post-translational modification 38,51,61,73 ; however, the substrates undergoing larger solubility changes should be of higher biological importance. Another complication is that some acceptor Thr and Ser residues on substrates might be occupied by other common modifications, such as phosphorylation.…”

Section: Discussionmentioning

confidence: 99%

Mapping the GALNT1 substrate landscape with versatile proteomics tools

Saei

Lundström

Lyu

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

O-GalNAc type glycosylation is a common post-translational modification (PTM) of proteins catalyzed by polypeptide GalNAc transferases, but the substrate specificity of these transferases is poorly understood. Here we develop a strategy based on integral thermal proteome solubility profiling to identify and prioritize the protein substrates of polypeptide N-acetylgalactosaminyltransferase 1 (GALNT1). Combined with glycoprotein enrichment followed by HCD and soft EThcD gas-phase fragmentation technique, we uncover hundreds of novel GALNT1 substrates in two model human cell lines. GALNT1-mediated O-glycosylation is more common on Thr than Ser residues, with a strong preference for Pro at positions +3 and +4 in respect to O-glycosylation. These results implicate GALNT1 in potentially regulating proteins in several diverse pathways, including some unexpected processes, such as TCA cycle and DNA transcription. This study depicts a roadmap for identification of functional substrates for glycosyltransferases, facilitating fundamental insight into the role of glycosylation in homeostasis and disease.

show abstract

Section: Discussionmentioning

confidence: 99%

Mapping the GALNT1 substrate landscape with versatile proteomics tools

Saei

Lundström

Lyu

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition, the cell experiment did not require extensive filtering that may result in protein loss. On the other hand, the cell experiment required normalization of each PISA dataset by separately measured protein abundance, as in PISA-Express 25 , and thus demands multiplexing of a larger number of samples. In order to meet this increased demand, we metabolically labeled proteins in cells with light and heavy Stable Isotope Labeling by Amino acids in Cell culture (SILAC) 31 reagents and additionally used TMT16-plex labeling of tryptic peptides.…”

Section: Rest-pisa Is Amenable To Intact Cellsmentioning

confidence: 99%

“…Some time ago we have introduced Proteome Integral Solubility Alteration (PISA) assay as a high-throughput analogue of TPP 23 . PISA analysis was used for deconvoluting the action mechanism of redox modulating anti-cancer compounds 24 and profiling stem cell transitions 25 . Here we hypothesize that the ∆Sm solubility shift in PISA will reflect the degree of target engagement by a drug in a cell lysate or intact cell at certain time past drug removal by filtration (for lysate) or washing (for cells).…”

Section: Introductionmentioning

confidence: 99%

System-wide profiling by proteome integral solubility alteration assay of drug residence times for target characterization

Sabatier

Beusch

Meng

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Most drugs used in the clinic and drug candidates target multiple proteins, and thus detailed characterization of their efficacy targets is required. While current methods rely on quantitative measurements at thermodynamic equilibrium, kinetic parameters such as the residence time of a drug on its target provide a better proxy for efficacy in vivo. Here, we present Residence Time Proteome Integral Solubility Alteration (ResT-PISA) assay which provides monitoring temporal protein solubility profiles after drug removal (off-curve) in cell lysate or intact cells, quantifying the lifetime of drug-target interaction. A compressed version of the assay measures the integral under the off-curve enabling the multiplexing of binding affinity and residence time assessments into a single proteomic analysis. We introduce a combined scoring system for three parametric dimensions to improve prioritization of targets. By providing complementary information to other characteristics of drug-target interaction, ResT-PISA approach will be useful in drug development and precision medicine.

show abstract

“…Therefore, TPP is now widely used in diverse applications, such as the study of protein–protein interactions, 12 metabolite–protein interactions, 13 , 14 determination of the enzyme substrates, 15 and studying cellular mechanisms. 16 − 20 …”

Section: Introductionmentioning

confidence: 99%

Ion-Based Proteome-Integrated Solubility Alteration Assays for Systemwide Profiling of Protein–Molecule Interactions

2022

Self Cite

View full text Add to dashboard Cite

Unbiased drug target engagement deconvolution and mechanism of action elucidation are major challenges in drug development. Modification-free target engagement methods, such as thermal proteome profiling, have gained increasing popularity in the last several years. However, these methods have limitations, and, in any case, new orthogonal approaches are needed. Here, we present a novel isothermal method for comprehensive characterization of protein solubility alterations using the effect on protein solubility of cations and anions in the Hofmeister series. We combine the ion-based protein precipitation approach with Proteome-Integrated Solubility Alteration (PISA) analysis and use this I-PISA assay to delineate the targets of several anticancer drugs both in cell lysates and intact cells. Finally, we demonstrate that I-PISA can detect solubility changes in minute amounts of sample, opening chemical proteomics applications to small and rare biological material.

show abstract

An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation

Cited by 27 publications

References 67 publications

Mapping the GALNT1 substrate landscape with versatile proteomics tools

Mapping the GALNT1 substrate landscape with versatile proteomics tools

System-wide profiling by proteome integral solubility alteration assay of drug residence times for target characterization

Ion-Based Proteome-Integrated Solubility Alteration Assays for Systemwide Profiling of Protein–Molecule Interactions

Contact Info

Product

Resources

About