{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Msf":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD051902/20240310_AL_ADC_GNF_MMAE.msf","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD051902/20240328_AL01_GNF_Biotin.msf","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD051902/20240328_AL02_MMAE_Exatecan.msf"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD051902/20240328_AL02_MMAE_Exatecan.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD051902/20240310_AL_ADC_GNF_MMAE.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD051902/20240328_AL01_GNF_Biotin.raw"],"Fasta":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD051902/Uniprot_Human_IgG.fasta"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["nemes@umd.edu"],"submitter":["Peter Nemes"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":[""],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD051902"],"tissue":["Cell Culture"],"sample_protocol":["All purified ADCs were reduced using 20 mM dithiothreitol (DTT) final concentration with a 30 minute incubation at 60 °C. The sample was then alkylated with 60 mM iodoacetamide (IAA) for 20 minutes in dark at room temperature. Alkylation was quenched with 20 mM DTT, and the sample was digested with 1:25 (w/w) 1 µg/µL Trypsin: ADC for 12 hours at 37°C. Pierce Peptide Desalting Spin Columns (Thermo Fisher, Waltham, MA) were used for desalting the digested samples, following the manufacturer’s specifications. The eluted peptides were then lyophilized.  The amount of peptide in the antibody digest was measured using a colorimetric assay (Pierce Quantitative Peptide Assay, Thermo Scientific, Waltham, MA). Then the concentration of the sample was adjusted to 0.1 µg/µL using 0.1% formic acid in water."],"repository":["Pride"],"modification":[""],"quantification_method":["Not available"],"data_protocol":["Proteome Discoverer 3.0 (Thermo Scientific) was utilized to analyze the primary MS–MS/MS data, employing SEQUEST to search against the human IgG sequence containing the heavy and light chains of the protein (UniProt). The search parameters used trypsin as the digestion enzyme, allowing for a maximum of 2 missed cleavages. Cysteine carbamidomethylation was set as a fixed modification, while methionine oxidation was considered variable, alongside variable modification of 349.1373 Da for GlcNAc-Fuc, 445.2247 Da for biotin, 1266.7727 Da for MMAE, and 2160.9117 Da for Exatecan conjugation. Identification considered at least 1 proteotypic peptide, with a mass tolerance of 10 ppm for the precursor ion and 0.6 Da for the fragment ion. Peptides and protein identifications were filtered to less than 1% false discovery rate (FDR), compared against a reversed-sequence decoy database."],"omics_type":["Proteomics"],"labhead":["Peter Nemes"],"instrument_platform":[""],"labhead_affiliation":["Department of Chemistry & Biochemistry, University of Maryland at College Park"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"publication":["10.1021/ACSCHEMBIO.6C00229"],"submitter_mail":["nemes@umd.edu"],"submitter_affiliation":["University of Maryland, College Park"],"submitter_country":["United States"],"additional_accession":[]},"is_claimable":false,"name":"Orthogonal Conjugation via Endoglycosidase and Microbial Transglutaminase Enables Efficient Synthesis of Dual-Payload Antibody-Drug Conjugates","description":"Antibody drug conjugates (ADCs) are a rapidly growing field of therapeutics which combines the highly specific targeting ability of an antibody with a cytotoxic drug to deliver the payload in a targeted manner to cancer cells. Traditional ADC construction used stochastic conjugation methods resulting in heterogeneous products with subpopulations having mixed stability and pharmacokinetics. Consequently, recent efforts have favored site-specific conjugation methods that produce homogeneous ADCs with improved therapeutic indexes. Here we report a highly efficient site-specific conjugation method that combines an endoglycosidase and microbial transglutaminase to construct homogeneous and well defined antibody drug conjugates. We found an Endo-S2 treated IgG antibody, which conserves the inner-most GlcNAc residue at the conserved N-glycosylation site, is an excellent substrate for mTG conjugation activity. We further demonstrate an Fc glycan remodeling strategy with Endo-S2 can be used for loading of a second payload to construct a dual-drug ADC.","dates":{"publication":"2026-05-29","submission":"2024-04-30"},"accession":"PXD051902","cross_references":{"TAXONOMY":["NEWT:3555","NEWT:71647","NEWT:35554","NEWT:32046","NEWT:544496","NEWT:2042546","NEWT:259447","NEWT:45351","NEWT:445974","NEWT:43179","NEWT:180454","NEWT:5722","NEWT:1129","NEWT:55153","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:10036","NEWT:498019","NEWT:1351","NEWT:1438992","NEWT:1352","NEWT:2649997","NEWT:1147161","NEWT:224326","NEWT:1333499","NCBITaxon:79857","NEWT:1096976","NEWT:95648","NEWT:1589","NEWT:135622","NEWT:383379","NEWT:10029","NEWT:913645","NEWT:317447","NEWT:4688","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:4442","NEWT:192875","NEWT:59729","NEWT:2164133","NEWT:1316931","NEWT:224308","NEWT:3347","NEWT:511145","NEWT:931281","NEWT:77133","NEWT:145481","NCBITaxon:79824","NCBITaxon:4563","NEWT:5755","NEWT:44689","NEWT:5759","NEWT:1736231","NEWT:1392","NEWT:498217","NEWT:2242","NEWT:11320","NEWT:286","NEWT:391619","NEWT:246196","NEWT:287","NEWT:246197","NEWT:10239","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:5507","NEWT:55571","NEWT:35500","NEWT:1140","NEWT:1143","NEWT:4896","NEWT:1390","NEWT:11557","NEWT:1094343","NEWT:1336795","NEWT:644042","NEWT:1773","NEWT:1182590","NEWT:3712","NEWT:82380","NEWT:935293","NEWT:375146","NEWT:263","NEWT:52283","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:408169","NEWT:47946","NEWT:3702","NEWT:243277","NEWT:7067","NEWT:2850","NEWT:408172","NEWT:408170","NEWT:3708","NEWT:332648","NEWT:536231","NEWT:1436733","NEWT:460519","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:9483","NEWT:1513458","NCBITaxon:40559","NEWT:84588","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:300852","NEWT:1502","NEWT:376686","NEWT:95486","NEWT:508771","NEWT:1883446","NEWT:253","NCBITaxon:2759","NEWT:1233435","NEWT:93061","NCBITaxon:4932","NEWT:644223","NEWT:235443","NEWT:108458","NEWT:272623","NEWT:272624","NEWT:983964","NEWT:118499","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:476272","NEWT:3747","NEWT:195051","NEWT:3988","NEWT:1255228","NEWT:649908","NEWT:410289","NEWT:373153","NEWT:352472","NEWT:1071661","NEWT:360094","NEWT:470","NEWT:41364","NEWT:1313","NEWT:39491","NCBITaxon:5811","NEWT:29491","NEWT:2014887","NEWT:33952","NEWT:571256","NEWT:40483","NEWT:9031","NEWT:1872122","NEWT:7091","NEWT:108931","NEWT:1055524","NEWT:9778","NEWT:150475","NEWT:303","NEWT:7111","NEWT:347515","NEWT:9534","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:185579","NEWT:941442","NEWT:13076","NEWT:7108","NEWT:40479","NEWT:317","NEWT:1006581","NEWT:885318","NEWT:39488","NEWT:550","NEWT:4081","NEWT:273068","NEWT:554","NEWT:98334","NEWT:451516","NEWT:36185","NEWT:1225786","NEWT:7574","NEWT:1715256","NEWT:575412","NEWT:929793","NEWT:29204","NEWT:28112","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:643680","NCBITaxon:6157","NEWT:6239","NEWT:162425","NEWT:216257","NEWT:102169","NEWT:9986","NEWT:4054","NEWT:1268063","NEWT:1263854","NEWT:118503","NEWT:2059687","NEWT:160488","NEWT:28104","NEWT:407821","NCBITaxon:2","NEWT:985076","NEWT:1215323","NEWT:986","NEWT:52641","NEWT:7159","NEWT:28532","NEWT:353152","NEWT:27448","NEWT:1194669","NEWT:243230","NEWT:1080772","NEWT:8479","NEWT:6063","NEWT:49240","NEWT:104395","NEWT:6289","NEWT:6287","NEWT:300641","NEWT:727","NEWT:9796","NEWT:725","NEWT:469008","NEWT:256318","NEWT:206411","NCBITaxon:6191","NEWT:596153","NEWT:1836","NEWT:80863","NEWT:105231","NEWT:52638","NEWT:4097","NEWT:884204","NEWT:9785","NEWT:6279","NEWT:1123869","NEWT:9544","NEWT:7370","NEWT:83906","NCBITaxon:780","NCBITaxon:1502","NEWT:6282","NEWT:1134506","NEWT:38783","NEWT:4006","NEWT:6426","NCBITaxon:5476","NEWT:6669","NEWT:9935","NEWT:89453","NEWT:51515","NEWT:51511","NEWT:8845","NEWT:92867","NEWT:92866","NEWT:5334","NEWT:51750","NEWT:202950","NEWT:382352","NEWT:413071","NEWT:632957","NEWT:9925","NEWT:89462","NEWT:8839","NEWT:4232","NEWT:2758385","NEWT:11298","NEWT:171101","NEWT:714","NEWT:421932","NEWT:312017","NEWT:196627","NEWT:870435","NEWT:5315","NEWT:9913","NEWT:105841","NEWT:337330","NEWT:2666255","NEWT:2285","NEWT:1392998","NEWT:380394","NEWT:981087","NEWT:180066","NEWT:226900","NEWT:326423","NEWT:452467","NEWT:36111","NEWT:326424","NEWT:1416333","NEWT:1678078","NEWT:749906","NEWT:418985","NEWT:749907","NEWT:145263","NEWT:694569","NEWT:27606","NEWT:9739","NEWT:59202","NEWT:9975","NEWT:51953","NEWT:3197","NEWT:860688","NEWT:13443","NEWT:36329","NEWT:62615","NCBITaxon:3044782","NEWT:72407","NEWT:349741","NEWT:157295","NEWT:2096","NEWT:9721","NEWT:137221","NEWT:9963","NEWT:1617910","NEWT:565050","NEWT:2785785","NEWT:411901","NEWT:105884","NCBITaxon:9615","NEWT:58334","NEWT:1193501","NEWT:58331","NEWT:6689","NEWT:70448","NEWT:698936","NEWT:9940","NEWT:521001","NEWT:192011","NEWT:1813887","NEWT:224911","NCBITaxon:11552","NEWT:6945","NEWT:184922","NEWT:6703","NEWT:2","NEWT:157546","NEWT:474186","NEWT:38942","NEWT:307972","NEWT:149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