{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075140/ChanJ_ID_CPOS-230228-BY-16841_Results.xlsx","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075140/ChanJ_ID_CPOS-230228-BY-16841_Results_Peptide.xlsx"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075140/checksum.txt"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075140/ACE2_DMSOd6.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075140/ACE2_MB_32.raw"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075140/2.p","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075140/1.p"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["liuli71@hku.hk"],"submitter":["Kong Hung SZE"],"technology_type":["Mass Spectrometry","Top-down proteomics","Gel-based experiment","Bottom-up proteomics"],"disease":["Covid-19"],"software":[""],"submitter_keywords":["Human","Allosteric inhibitor","Angiotensin-converting enzyme 2","Covid19","Benzothiazole","Formaldehyde crosslinking","Sarbecovirus","Entry inhibtor","Mb-32"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD075140"],"sample_protocol":["Formaldehyde (FA) crosslinking was performed on a sample mixture of 10 µL 18mg/ml human ACE2 (Cat Q9BYF1-1, ACROBiosystems Group, DE, USA) with 0.6 µL 15.5mM MB-32 and 0.9 µL formaldehyde (final FA concentration ~3%) in 30 oC shaker for 30 minutes. The sample was vortex every minute during this period. FA crosslinking reaction was quenched by adding 10µL of 4M Tris and incubated for 30 minutes in the water bath at 30 oC. A control FA crosslinking experiment was performed with the same conditions and steps on a sample with human ACE2 only. The samples were subjected to an SDS-PAGE gel-purification by running in a stacking 3% (3 cm wide) and resolving 8% gel. The gel was run with all protein stacked forming as a band at the boundary between the stacking and resolving gel. After Commassie Blue staining, each band was excised and subjected to in-gel digestion. Briefly, gel slices were subjected to reduction and alkylation by 10 mM TCEP and 55 mM 2-chloroacetamide, respectively. Protein digestion was performed by incubating with trypsin (1.0 ng/µL) overnight at 37 oC. Subsequent tryptic  peptides  were  extracted  from  the  gel  with  50  %  ACN/5%  FA  and  100%  ACN sequentially. The peptide extracts were pooled together and speedvac dried. The Peptides were desalted using C18 StageTips for LC-MS/MS analysis with Dionex Ultimate3000 nanoRSLC system coupled to Thermo Fisher Orbitrap Fusion Tribid Lumos mass spectrometer. Peptides were separated on a commercial C18 column (75 μm i.d. × 50cm length  × 2μm particle size) coupled to a NanoTrap column (75 μm i.d. × 2cm length × 3μm particle size) (Thermo Fisher). Separation was attained using a linear gradient of increasing buffer B (80% ACN and 0.1% FA) and declining buffer (0.1% FA) at 300 µL/min. Buffer B was increased to 27.5% B in 30 min and ramped to 44% B in 16 min followed by a quick ramp to 95% B, where it was held for 3 min before a quick ramp back to 3% B, where it was held and the column was re-equilibrated. Mass spectrometer was operated in positive polarity mode with capillary temperature of 300 °C. Full MS survey scan resolution was set to 120 000 with an automatic gain control (AGC) target value of 5 × 104, maximum ion injection time (IT) of 30ms, and for a scan range of 400−1500 m/z. A data-dependent top 10 method was operated during which higher-energy collisional dissociation (HCD) was used. Spectra were obtained at 30000 MS2 resolution with a custom normalized AGC target of 200% and maximum ion injection time (IT) of 80 ms, 1.6 m/z isolation width, and normalized collisional energy of 30. Preceding precursor ions targeted for HCD were dynamically excluded of 22 s."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Raw mass spectrometry data were processed using MaxQuant 1.6.14.0. Raw data was searched against Human Swissprot FASTA database (May 2022) containing 20,373 entries using settings as below: oxidized methionine (M), acetylation (Protein N-term) were selected as dynamic modifications, and carbamidomethyl (C) as fixed modifications with minimum peptide length of 7 amino acids was enabled. Confident proteins were identified using a target-decoy approach with a reversed database, strict false-discovery rate 1% at peptide and peptide spectrum matches (PSMs) level; minimum ≥1 unique peptide, ≥2 peptide spectral matches (PSMs). Additional FA crosslinking related modifications were searched by using the mass of MB-32 (558.22) plus a 2 Carbon (24) bridge on 4 fixed amino acid types of aspartic acid, tyrosine, arginine and lysine by using PEAKS Studio 11."],"omics_type":["Proteomics"],"labhead":["Dr. Liu Li"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Department of Microbiology, The University of Hong Kong, Hong Kong, SAR, China"],"species":["Homo Sapiens (human)","Sarbecovirus"],"publication":["Not available"],"submitter_mail":["konghungsze@gmail.com"],"submitter_affiliation":["Department of Microbiology, University of Hong Kong"],"submitter_country":["Hong Kong"],"additional_accession":[]},"is_claimable":false,"name":"Human Angiotensin-Converting Enzyme 2-Specific Benzothiazole Allosteric Inhibitor  against Pan-Sarbecoviruses","description":"Emerging  SARS-CoV-2  variants  of  concern  (VOCs)  have  demonstrated  exceptional transmissibility and a strong dependence on the cellular receptor human angiotensin-converting enzyme 2 (hACE2). In this study, we present MB-32, a benzothiazole-based small molecule allosteric inhibitor of hACE2, which exhibits broad-spectrum antiviral activity against multiple SARS-CoV-2   VOCs,   SARS-CoV-1,   and   bat/pangolin-derived  sarbecoviruses.   MB-32 effectively inhibits lung infections and prevents contact transmission of the SARS-CoV-2 Omicron in animal models. We subsequently studied the binding site of MB-32 on hACE2 using  formaldehyde (FA) crosslinking coupled with mass spectrometry. To investigate FA crosslinking  modifications, we utilized the mass of 558.22 Da, which is derived from the monoisotopic mass of  MB-32 (534.22 Da) combined with a 2-carbon (24 Da) bridge, using PEAKS Studio 11. Focusing on four fixed amino acid type—saspartic acid, tyrosine, arginine, and lysine—we detected  a  558.22  Da  adduct  in  the  combined  mass  of  two  peptides  containing  Y83 (EQSTLAQMY83PLQEIQNLTVK) and K94 (EQSTLAQMYPLQEIQNLTVK94), with high confidence scores of 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