Summary of Study ST004062

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002550. The data can be accessed directly via it's Project DOI: 10.21228/M81K02 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST004062
Study Title	Enantioselective Protein Affinity Selection Mass Spectrometry (EAS-MS)
Study Summary	We report an enantioselective protein affinity selection mass spectrometry screening approach (EAS-MS) that enables the detection of weak binders, informs on selectivity, and generates orthogonal confirmation of binding. After method development with control proteins, we screened 31 human proteins against a designed library of 8,210 chiral compounds. A total of 16 binders to 12 targets, including many proteins predicted to be “challenging to ligand”, were discovered and confirmed in orthogonal biophysical assays. Seven binders to six targets bound in an enantioselective manner, with KD values ranging from 3 to 50 µM. Binders for four targets (DDB1, WDR91, WDR55, and HAT1) were selected for in-depth characterization using X-ray crystallography. In all four cases, the mechanism for enantioselectivity was readily explained. We conclude EAS-MS can be used to identify and characterize selective and weakly binding ligands for novel protein targets with unprecedented throughput and sensitivity.
Institute	University of Toronto
Last Name	peng
First Name	hui
Address	80 st george street, toronto, ON, M5S3H6, Canada
Email	hui.peng@utoronto.ca
Phone	6476730580
Submit Date	2025-07-17
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2025-07-23
Release Version	1

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Project:

Project ID:	PR002550
Project DOI:	doi: 10.21228/M81K02
Project Title:	Enantioselective Protein Affinity Selection Mass Spectrometry (EAS-MS)
Project Summary:	We report an enantioselective protein affinity selection mass spectrometry screening approach (EAS-MS) that enables the detection of weak binders, informs on selectivity, and generates orthogonal confirmation of binding. After method development with control proteins, we screened 31 human proteins against a designed library of 8,210 chiral compounds. A total of 16 binders to 12 targets, including many proteins predicted to be “challenging to ligand”, were discovered and confirmed in orthogonal biophysical assays. Seven binders to six targets bound in an enantioselective manner, with KD values ranging from 3 to 50 µM. Binders for four targets (DDB1, WDR91, WDR55, and HAT1) were selected for in-depth characterization using X-ray crystallography. In all four cases, the mechanism for enantioselectivity was readily explained. We conclude EAS-MS can be used to identify and characterize selective and weakly binding ligands for novel protein targets with unprecedented throughput and sensitivity.
Institute:	University of Toronto
Last Name:	peng
First Name:	hui
Address:	80 st george street, toronto, ON, M5S3H6, Canada
Email:	hui.peng@utoronto.ca
Phone:	6476730580

Subject:

Subject ID:	SU004208
Subject Type:	Other abiotic sample

Factors:

Subject type: Other abiotic sample; Subject species: - (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Protein name
SA469542	TLE4_ChemDivA04_2	protein	-
SA469543	TLE4_ChemDivA04_3	protein	-
SA469544	TLE4_ChemDivA05_1	protein	-
SA469545	TLE4_ChemDivA05_2	protein	-
SA469546	AASSLKR-GFP_Chiral6kA01_3	protein	AASSLKR-GFP
SA469547	AASSLKR-GFP_Chiral6kA04_3	protein	AASSLKR-GFP
SA469548	AASSLKR-GFP_Chiral6kA04_1	protein	AASSLKR-GFP
SA469549	AASSLKR-GFP_Chiral6kA03_3	protein	AASSLKR-GFP
SA469550	AASSLKR-GFP_Chiral6kA03_2	protein	AASSLKR-GFP
SA469551	AASSLKR-GFP_Chiral6kA03_1	protein	AASSLKR-GFP
SA469552	AASSLKR-GFP_Chiral6kA02_3	protein	AASSLKR-GFP
SA469553	AASSLKR-GFP_Chiral6kA02_2	protein	AASSLKR-GFP
SA469554	AASSLKR-GFP_Chiral6kA02_1	protein	AASSLKR-GFP
SA469555	AASSLKR-GFP_ChemDivC04_3	protein	AASSLKR-GFP
SA469556	AASSLKR-GFP_Chiral6kA01_2	protein	AASSLKR-GFP
SA469557	AASSLKR-GFP_Chiral6kA01_1	protein	AASSLKR-GFP
SA469558	AASSLKR-GFP_Chiral6kA05_2	protein	AASSLKR-GFP
SA469559	AASSLKR-GFP_ChemDivC04_2	protein	AASSLKR-GFP
SA469560	AASSLKR-GFP_ChemDivC04_1	protein	AASSLKR-GFP
SA469561	AASSLKR-GFP_ChemDivC03_3	protein	AASSLKR-GFP
SA469562	AASSLKR-GFP_ChemDivC03_2	protein	AASSLKR-GFP
SA469563	AASSLKR-GFP_ChemDivC03_1	protein	AASSLKR-GFP
SA469564	AASSLKR-GFP_ChemDivC02_3	protein	AASSLKR-GFP
SA469565	AASSLKR-GFP_ChemDivC02_2	protein	AASSLKR-GFP
SA469566	AASSLKR-GFP_Chiral6kA05_1	protein	AASSLKR-GFP
SA469567	AASSLKR-GFP_Chiral6kA06_2	protein	AASSLKR-GFP
SA469568	AASSLKR-GFP_Chiral6kA05_3	protein	AASSLKR-GFP
SA469569	AASSLKR-GFP_Chiral6kA09_3	protein	AASSLKR-GFP
SA469570	AASSLKR-GFP_ChemDivA01_1	protein	AASSLKR-GFP
SA469571	AASSLKR-GFP_ChemDivA01_2	protein	AASSLKR-GFP
SA469572	AASSLKR-GFP_Chiral6kA11_3	protein	AASSLKR-GFP
SA469573	AASSLKR-GFP_Chiral6kA11_2	protein	AASSLKR-GFP
SA469574	AASSLKR-GFP_Chiral6kA11_1	protein	AASSLKR-GFP
SA469575	AASSLKR-GFP_Chiral6kA10_3	protein	AASSLKR-GFP
SA469576	AASSLKR-GFP_Chiral6kA10_2	protein	AASSLKR-GFP
SA469577	AASSLKR-GFP_Chiral6kA10_1	protein	AASSLKR-GFP
SA469578	AASSLKR-GFP_Chiral6kA09_2	protein	AASSLKR-GFP
SA469579	AASSLKR-GFP_Chiral6kA06_1	protein	AASSLKR-GFP
SA469580	AASSLKR-GFP_Chiral6kA09_1	protein	AASSLKR-GFP
SA469581	AASSLKR-GFP_Chiral6kA08_3	protein	AASSLKR-GFP
SA469582	AASSLKR-GFP_Chiral6kA08_2	protein	AASSLKR-GFP
SA469583	AASSLKR-GFP_Chiral6kA08_1	protein	AASSLKR-GFP
SA469584	AASSLKR-GFP_Chiral6kA07_3	protein	AASSLKR-GFP
SA469585	AASSLKR-GFP_Chiral6kA07_2	protein	AASSLKR-GFP
SA469586	AASSLKR-GFP_Chiral6kA07_1	protein	AASSLKR-GFP
SA469587	AASSLKR-GFP_Chiral6kA06_3	protein	AASSLKR-GFP
SA469588	AASSLKR-GFP_ChemDivC01_3	protein	AASSLKR-GFP
SA469589	AASSLKR-GFP_ChemDivC02_1	protein	AASSLKR-GFP
SA469590	AASSLKR-GFP_Chiral6kA04_2	protein	AASSLKR-GFP
SA469591	AASSLKR-GFP_ChemDivC01_2	protein	AASSLKR-GFP
SA469592	AASSLKR-GFP_ChemDivA05_2	protein	AASSLKR-GFP
SA469593	AASSLKR-GFP_ChemDivA08_2	protein	AASSLKR-GFP
SA469594	AASSLKR-GFP_ChemDivA08_1	protein	AASSLKR-GFP
SA469595	AASSLKR-GFP_ChemDivA07_3	protein	AASSLKR-GFP
SA469596	AASSLKR-GFP_ChemDivA07_2	protein	AASSLKR-GFP
SA469597	AASSLKR-GFP_ChemDivA07_1	protein	AASSLKR-GFP
SA469598	AASSLKR-GFP_ChemDivA06_3	protein	AASSLKR-GFP
SA469599	AASSLKR-GFP_ChemDivA06_2	protein	AASSLKR-GFP
SA469600	AASSLKR-GFP_ChemDivA06_1	protein	AASSLKR-GFP
SA469601	AASSLKR-GFP_ChemDivA05_3	protein	AASSLKR-GFP
SA469602	AASSLKR-GFP_ChemDivA05_1	protein	AASSLKR-GFP
SA469603	AASSLKR-GFP_ChemDivB01_1	protein	AASSLKR-GFP
SA469604	AASSLKR-GFP_ChemDivA04_3	protein	AASSLKR-GFP
SA469605	AASSLKR-GFP_ChemDivA04_2	protein	AASSLKR-GFP
SA469606	AASSLKR-GFP_ChemDivA04_1	protein	AASSLKR-GFP
SA469607	AASSLKR-GFP_ChemDivA03_3	protein	AASSLKR-GFP
SA469608	AASSLKR-GFP_ChemDivA03_2	protein	AASSLKR-GFP
SA469609	AASSLKR-GFP_ChemDivA03_1	protein	AASSLKR-GFP
SA469610	AASSLKR-GFP_ChemDivC01_1	protein	AASSLKR-GFP
SA469611	AASSLKR-GFP_ChemDivA02_2	protein	AASSLKR-GFP
SA469612	AASSLKR-GFP_ChemDivA02_1	protein	AASSLKR-GFP
SA469613	AASSLKR-GFP_ChemDivA01_3	protein	AASSLKR-GFP
SA469614	AASSLKR-GFP_ChemDivA08_3	protein	AASSLKR-GFP
SA469615	AASSLKR-GFP_ChemDivA02_3	protein	AASSLKR-GFP
SA469616	AASSLKR-GFP_ChemDivB01_2	protein	AASSLKR-GFP
SA469617	AASSLKR-GFP_ChemDivB05_2	protein	AASSLKR-GFP
SA469618	AASSLKR-GFP_ChemDivB01_3	protein	AASSLKR-GFP
SA469619	AASSLKR-GFP_ChemDivB08_3	protein	AASSLKR-GFP
SA469620	AASSLKR-GFP_ChemDivB08_2	protein	AASSLKR-GFP
SA469621	AASSLKR-GFP_ChemDivB07_3	protein	AASSLKR-GFP
SA469622	AASSLKR-GFP_ChemDivB07_2	protein	AASSLKR-GFP
SA469623	AASSLKR-GFP_ChemDivB07_1	protein	AASSLKR-GFP
SA469624	AASSLKR-GFP_ChemDivB06_3	protein	AASSLKR-GFP
SA469625	AASSLKR-GFP_ChemDivB06_2	protein	AASSLKR-GFP
SA469626	AASSLKR-GFP_ChemDivB06_1	protein	AASSLKR-GFP
SA469627	AASSLKR-GFP_ChemDivB05_3	protein	AASSLKR-GFP
SA469628	AASSLKR-GFP_ChemDivB08_1	protein	AASSLKR-GFP
SA469629	AASSLKR-GFP_ChemDivB05_1	protein	AASSLKR-GFP
SA469630	AASSLKR-GFP_ChemDivB03_1	protein	AASSLKR-GFP
SA469631	AASSLKR-GFP_ChemDivB04_3	protein	AASSLKR-GFP
SA469632	AASSLKR-GFP_ChemDivB02_2	protein	AASSLKR-GFP
SA469633	AASSLKR-GFP_ChemDivB02_1	protein	AASSLKR-GFP
SA469634	AASSLKR-GFP_ChemDivB02_3	protein	AASSLKR-GFP
SA469635	AASSLKR-GFP_ChemDivB03_2	protein	AASSLKR-GFP
SA469636	AASSLKR-GFP_ChemDivB03_3	protein	AASSLKR-GFP
SA469637	AASSLKR-GFP_ChemDivB04_1	protein	AASSLKR-GFP
SA469638	AASSLKR-GFP_ChemDivB04_2	protein	AASSLKR-GFP
SA469639	AASSSDH-GFP_Chiral6kA03_3	protein	AASSSDH-GFP
SA469640	AASSSDH-GFP_Chiral6kA03_2	protein	AASSSDH-GFP
SA469641	AASSSDH-GFP_Chiral6kA03_1	protein	AASSSDH-GFP

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Collection:

Collection ID:	CO004201
Collection Summary:	This study includes binding screening data for 31 protein-compound pairs. Detailed information on the 31 proteins is provided in the “Protein name” column of the study design table. The protocol file titled 'Collection information' describes the purification methods and procedures for each protein. After purification, all proteins were stored at –80 °C until use.
Collection Protocol Filename:	Collection_information.pdf
Sample Type:	Protein

Treatment:

Treatment ID:	TR004217
Treatment Summary:	no treatment

Sample Preparation:

Sampleprep ID:	SP004214
Sampleprep Summary:	Chemicals were incubated with purified recombinant His-tagged human proteins on 96-well plates, in the binding buffer (150 mM NaCl, 50 mM Tris-HCl, 0.1 mM TCEP, 0.5% (v/v) glycerol, 0.01% (v/v) Triton X-100, and pH 7.5). 5 µL of Ni-NTA magnetic beads was added to the binding buffer, adjusted to a final volume of 250 µL per sample. All EAS-MS screenings were performed in triplicate. Incubation was carried out on a rotary mixer at 4 oC for 30 min. After incubation, the 96-well plate was placed on a magnetic plate to separate the beads, and the incubation buffer was removed. The beads were then washed twice with 100 µL of washing buffer 1 (150 mM NaCl, 50 mM Tris-HCl, 0.1 mM TCEP, 0.5% glycerol, 0.01% Triton X-100, 5 mM imidazole, and pH 7.5), followed by one wash with 100 µL of washing buffer 2 (150 mM NaCl, 50 mM Tris-HCl, 5 mM imidazole, and pH 7.5). The beads and washing buffer 2 were transferred to a new plate. After removing the washing buffer 2, 120 µL of methanol was added to each well to denature proteins and release chemical ligands. The methanol extracts separated from magnetic beads were directly subjected to LC-MS analysis.120 µL of methanol was added to each well to denature proteins and release chemical ligands. The methanol extracts separated from magnetic beads were directly subjected to LC-MS analysis.

Sampleprep ID:

SP004214

Sampleprep Summary:

Chemicals were incubated with purified recombinant His-tagged human proteins on 96-well plates, in the binding buffer (150 mM NaCl, 50 mM Tris-HCl, 0.1 mM TCEP, 0.5% (v/v) glycerol, 0.01% (v/v) Triton X-100, and pH 7.5). 5 µL of Ni-NTA magnetic beads was added to the binding buffer, adjusted to a final volume of 250 µL per sample. All EAS-MS screenings were performed in triplicate. Incubation was carried out on a rotary mixer at 4 oC for 30 min. After incubation, the 96-well plate was placed on a magnetic plate to separate the beads, and the incubation buffer was removed. The beads were then washed twice with 100 µL of washing buffer 1 (150 mM NaCl, 50 mM Tris-HCl, 0.1 mM TCEP, 0.5% glycerol, 0.01% Triton X-100, 5 mM imidazole, and pH 7.5), followed by one wash with 100 µL of washing buffer 2 (150 mM NaCl, 50 mM Tris-HCl, 5 mM imidazole, and pH 7.5). The beads and washing buffer 2 were transferred to a new plate. After removing the washing buffer 2, 120 µL of methanol was added to each well to denature proteins and release chemical ligands. The methanol extracts separated from magnetic beads were directly subjected to LC-MS analysis.120 µL of methanol was added to each well to denature proteins and release chemical ligands. The methanol extracts separated from magnetic beads were directly subjected to LC-MS analysis.

Chromatography:

Chromatography ID:	CH005108
Chromatography Summary:	Ultrapure water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B) were used as the two mobile phases.
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Accucore C18 (100 x 2.1mm,2.6um)
Column Temperature:	40
Flow Gradient:	Gradient elution started with 5% B, increased to 80% at 1.5 min, then reached to 100% at 3 min, held for 1.1 min, and finally returned to 5% B over 1.5 min.
Flow Rate:	0.3 mL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% methanol; 0.1% formic acid
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN006721
Analysis Type:	MS
Chromatography ID:	CH005108
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST004062_AN006721_Results.txt
Units:	intensity