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Enrichment and Liquid Chromatography-Mass Spectrometry Analysis of Trastuzumab and Pertuzumab Using Affimer Reagents. Anal Chem. 2021 93(40):13597-13605 10.1021/acs.analchem.1c02807"],"curator_keywords":["Human proteome project","Biology/disease-driven human proteome project (b/d-hpp)","Antibodies (b/d-hpp)"],"submitter_affiliation":["University of Groningen"],"submitter_country":["Netherlands"],"doi":["10.6019/PXD027103"],"pubmed_abstract":["Trastuzumab and pertuzumab are monoclonal antibodies used in the treatment of human epidermal growth factor receptor-2 (HER2)-positive breast cancer. Therapeutic proteins may undergo chemical modifications that may affect the results of bioanalytical assays, as well as their therapeutic efficacy. Modifications may arise during production and storage, as well as after administration to patients. Studying in vivo biotransformation of monoclonal, therapeutic antibodies requires their enrichment from plasma to discriminate them from endogenous antibodies, as well as from other plasma proteins. To this end, we screened Affimer reagents for selectivity toward trastuzumab or pertuzumab. Affimer reagents are alternative binding proteins possessing two variable binding loops that are based on the human protease inhibitor stefin A or phytocystatin protein scaffolds. Affimer reagents were selected from an extensive library by phage display. The four best-performing binders for each therapeutic antibody were prioritized using a microtiter plate-based approach combined with liquid chromatography-mass spectrometry (LC-MS) in the selected reaction monitoring (SRM) mode. These Affimer reagents were immobilized via engineered 6-His or Cys tags to Ni<sup>2+</sup>- or maleimide beads, respectively. Recovery values of 70% and higher were obtained for both trastuzumab and pertuzumab when spiked at 100, 150, and 200 μg/mL concentrations in human plasma followed by trypsin digestion in the presence of 0.5% sodium deoxycholate and 10 mM dithiothreitol (DTT). Notably, the maleimide beads showed undetectable unspecific binding to endogenous immunoglobulin G (IgGs) or other plasma proteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enrichment method was applied to samples from stress tests of the antibodies at 37 °C to mimic in vivo conditions."],"pubmed_title":["Enrichment and Liquid Chromatography-Mass Spectrometry Analysis of Trastuzumab and Pertuzumab Using Affimer Reagents."],"pubmed_authors":["Olaleye Oladapo O, Spanov Baubek B, Ford Robert R, Govorukhina Natalia N, van de Merbel Nico C NC, Bischoff Rainer R"],"description_synonyms":["Plasma, Fresh, potassium, Fresh Frozen Plasmas, Fresh Frozen, Blood Plasma, Frozen Plasma, Fresh Frozen Plasma, Peptidomics, Protein., Blood, Proteins, portion of plasma, Gene, Plasmas, Protein Gene Products, Frozen Plasmas, Gene Proteins, Blood Plasmas, Protein, Gene Products, maleimide, silver (+1) salt, silver (+1) (2:1:1) salt, plasma"],"data_synonyms":["Applications Software, Open Source Softwares, Open Source, Software Applications, human being, Computer Software, Man (Taxonomy), Source Software, Software Application, Open, Modern, Engineering, Open Source Software, Computer Programs and Programming, Computer, Data Base., Source Softwares, human, Solution, EG:196F3.2, Software Tools, Computer Software Application, Tool, Programs, Human, Computer Programs, Program, Computer Applications, Applications, Software Tool, Applications Softwares, Tools, Homo sapiens, Modern Man, Computer Applications Software, Computer Applications Softwares, Software Engineering, Softwares, DmelCG32810, CG14784, Software, CG32810, Man, Computer Program, Application, Computer Software Applications, CG14783"],"pubmed_title_synonyms":["Perjeta, Trastuzumab-qyyp, 2C4, Trastuzumab beta, Indicator., Omnitarg, RG-1273, Reagent, Trastuzumab qyyp, Herceptin, rhumab-2C4, Reagents, Trazimera, Reagents and Indicators, Indicators, beta, Trastuzumab, rhumab 2C4"],"sample_synonyms":["BPBS, Fresh, Frozen Plasma, Fresh Frozen Plasma, BZRP, DBI, Blood, Spectrum Analyses, abdominal muscles, Plasmas, buffer, Spectrum Analysis, Buffer, PBS, PBR, Spectroscopy, Frozen Plasmas, MS, PKBS, Mass, silver (+1) salt, Analysis, eagle-Barrett syndrome, Mass Spectrum Analyses, Mass Spectroscopy, Mass Spectrum Analysis, Plasma, Mass Spectrum, potassium, Fresh Frozen Plasmas, Fresh Frozen, absence of, Blood Plasma, Painful Bladder Syndrome, with urinary tract Abnormality and cryptorchidism, PRSS, Analyses, beta-Trypsin, Tripcellim, Spectrometry, portion of plasma, IBP, pk18, mDRC, beta Trypsin, MBR, Trypure, Painful bladder syndrome, Blood Plasmas, Mass., PTBR, maleimide, Bladder pain syndrome, silver (+1) (2:1:1) salt, plasma, Bladder Pain Syndrome"],"pubmed_abstract_synonyms":["Erbb-2, 2C4, Urogastrone Receptor, dmBest1, FON1, Liquid Chromatography Mass Spectrometry, Receptors, Ultra Performance Liquid Chromatography-Mass Spectrometry, Tumor, FLORAL ORGAN NUMBER 1, Deoxycholic Acid, DmelCG6264, Epidermal Growth Factor Receptor Family Protein, ErbB-1, HPLC-MS, Kiaa3023, Gamma Globulin, Method, Protease Inhibitor, RG-1273, 1, 2, 3, cancer of the breast, 2-amino-3-mercaptopropanoic acid, BEST1_HUMAN, (R*, treatment, BC, Disodium Salt, Fresh Frozen, Ultra Performance Liquid Chromatography Mass Spectrometry, C, Polyacrylamide, SDS-PAGE electrophoresis, catabolism, Moods, Bdr, Epidermal Growth Factor Receptor, Epidermal Growth Factor Receptor Kinase, SNAP-25, Peptidohydrolase Inhibitor, cancer of breast, TGF-alpha, 3beta-Isomer, Breast Malignant Neoplasms, plasma, Magnesium (2:1) Salt, Tumors, EGF Receptors, Herceptin, MLN19, Procedure, antibodies, Dihydroxycholanoic Acid, Hydrolase Inhibitor, GENA70, secretion, (2R)-2-amino-3-sulfanylpropanoic acid, END, TU15B, Urogastrone, c-neu, 5beta, Carcinomas, Antagonists, Sodium Dodecyl Sulfate-PAGE, High Pressure Liquid Chromatography Mass Spectrometry, erbB 1 Proto Oncogene Protein, dbest1, Spectrometry, TGF-alpha Receptor, erbB-1 Proto-Oncogene Protein, Methodological, \"mammary neoplasm\" RELATED [], human, ErbB-1 Receptor, 12beta-Isomer Deoxycholic Acid, Transforming-Growth Factor alpha, Sodium Salt, B cell receptor accessory molecule complex, 5alpha-Isomer, breast cancer, Breast Tumors, GT, Indicator, B lymphocyte receptor complex, Breast Malignant Tumors, Endopeptidase, Peptide Peptidohydrolase Inhibitor, Sodium Dodecyl Sulfate-PAGEs, cancer, 4-Dimercapto-2, FLORAL DEFECTIVE 10, Fresh, 5alpha Isomer, Cleland Reagent, rac-Dithiothreitol, Fresh Frozen Plasma, Immunoglobulin, Family Receptors, Endopeptidase Inhibitors, threo-1, Neoplasms, Cleland's reagent, BEST1, number, L-Zystein, Transforming Growth Factor alpha Receptor, Mammary Cancers, 4-dimercapto-, SUPERMAN, Gel Electrophoresis, R*)-, protein-containing complex, SDS polyacrylamide gel electrophoresis of proteins, c-erbB-1 Protein, c erbB 1 Protein, Human, Breast Malignant Tumor, 12-dihydroxy-, Breast Tumor, Polyacrylamide Gel Electrophoresis, Gene Products, breast tumor, Peptide Hydrolase, 12alpha)-, HER-2/neu, Man, Technique, erbB-1, UPLC-MS, F15E12_6, 12beta Isomer, Immunoglobulin GT, Mammary Neoplasm, VMD2, ligand, E-920, Mammary Carcinoma, Monosodium Salt, 3beta Isomer, Spectrometries, Hcys, BMD, Antagonist, Study, Protease Antagonists, CYSTEINE, Malignant Tumor of Breast, Epidermal Growth Factor Urogastrone, Peptide Peptidohydrolase Inhibitors, FREE CYSTEINE, Kybella, Choleic Acid, Receptor Tyrosine-protein Kinase erbB-1, (2R)-2-amino-3-mercaptopropanoic acid, SPS1, SDS PAGE, Neu, NEU, Cancer, RP50, vitelliform macular dystrophy 2 (Best disease, 3-butanediol, EGF Receptor, Breast Carcinoma, Human Mammary Neoplasm, Breast Carcinomas, Epidermal Growth Factor Receptor Family Proteins, degradation, ErbB, ERBB, 5alpha-Isomer Deoxycholic Acid, Affects, HER-2|neu, Proteins, Proto-Oncogene Protein, Clelands Reagent, High Pressure Liquid Chromatography-Mass Spectrometry, Synaptosomal-associated 25 kDa protein, \"breast tumor\" EXACT [NCI2004_11_17:C2910], Dithiothreitol, Perjeta, Frozen Plasmas, native protein, Neoplasm, 3beta-Isomer Deoxycholic Acid, NGL, Human Mammary Carcinoma, Sodium, Library, ATCMPG1, MET, DTL, ATCMPG2, Cleland's, potassium, DTT, best, storage, c-erbB2, sodium dodecyl sulphate–polyacrylamide gel electrophoresis, Polyglobin, Monopotassium Salt, (R)-2-amino-3-mercaptopropanoic acid, Cancers, Peptidohydrolase Inhibitors, 2-Amino-3-mercaptopropionic acid, beta, plan specification, Cleland's Reagent, Gene Proteins, Trastuzumab-qyyp, Peptidase, Antiprotease, Epidermal Growth Factor, HER Family, LC-MS, E920, Cancer of Breast, maleimide, Receptor, Peptide Hydrolase Inhibitors, sequestering, BEST, silver (+1) (2:1:1) salt, breast, biochemical pathways, IgG Antibody, malignant tumor of the breast, Endopeptidase Inhibitor, E 920, Blood, Peptide Hydrolase Inhibitor, mammary neoplasm, High Performance Liquid Chromatography Mass Spectrometry, Human Mammary Neoplasms, protein, B-cell receptor complex, High Performance Liquid Chromatography-Mass Spectrometry, CD340, 12beta-Isomer, Techniques, Liquid Chromatography-Mass, \"neoplasm of breast (disorder)\" EXACT [SNOMEDCT_2005_07_31:126926005], 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Clients, Blood Plasmas, Breast, Chromatography-Mass Spectrometries, mammary tumor, HHT1, Proto-oncogene, Monoammonium Salt, Epidermal Growth Factor Receptor Protein Tyrosine Kinase, Edg, protein complex, Trastuzumab qyyp, mKIAA3023, PAPT, Deoxycholate, Client, Peptide, Breast Malignant Neoplasm, Dithiotreitol, count in organism, Mammary Carcinomas, Protein, Epidermal Growth Factor Receptor Protein-Tyrosine Kinase, DL-threo-1, Proto-oncogene c-ErbB-1 Protein, HER Family Receptor, (3alpha, bestrophin), Zystein, Trastuzumab, opsonin activity, Plasma, Dbest, Breast Neoplasm, biodegradation, 2-amino-3-sulfanylpropanoic acid, portion of plasma, L-2-Amino-3-mercaptopropionic acid, ERBB1, FLO10, HERP, Protein Gene Products, EGF, Therapeutic, SNAP, Modern Man, Breast Cancer, Chromatography-Mass Spectrometry, 7S Gamma Globulin, B cell receptor activity, Allerglobuline, Treatment, ORW1, variable, HER2, HER1"],"name_synonyms":["Protein Gene Products, Plasma, Fresh, Frozen Plasmas, Gene Proteins, Fresh Frozen Plasmas, Fresh Frozen, Blood Plasma, Frozen Plasma, Fresh Frozen Plasma, Protein., Blood Plasmas, ligand, Blood, Protein, Gene Products, Proteins, portion of plasma, Gene, Plasmas, plasma"],"additional_accession":[]},"is_claimable":false,"name":"Unspecific Binding of Beads to Plasma Proteins","description":"Nickel and maleimide beads were incubated in plasma and analysed with shotgun proteomics to determine the amount of plasma proteins bound to them","dates":{"publication":"2022-02-17","submission":"2021-07-05"},"accession":"PXD027103","cross_references":{"TAXONOMY":["NEWT:68239","NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2042546","NEWT:148942","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:57577","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:77173","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:661410","NEWT:488003","NEWT:224326","NEWT:376619","NEWT:3765","NEWT:3767","NEWT:433727","NEWT:135622","NEWT:672127","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:3760","NEWT:36630","NEWT:381198","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:44250","NEWT:317447","NEWT:5537","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:12637","NEWT:2164133","NEWT:2767846","NEWT:314344","NEWT:108061","NEWT:224308","NEWT:49928","NEWT:160621","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:1230383","NEWT:77133","NEWT:1310165","NEWT:114727","NEWT:5516","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:33554","NEWT:1310173","NEWT:37919","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:65121","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:28174","NEWT:550537","NEWT:284812","NEWT:980415","NEWT:3701","NEWT:3702","NEWT:66440","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:34878","NEWT:10886","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:215451","NEWT:460519","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:2709072","NEWT:2499525","NEWT:142786","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:1385720","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:272831","NEWT:987059","NEWT:491","NEWT:460523","NEWT:133894","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","NEWT:291146","NEWT:29466","NEWT:233271","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:3735","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:445","NEWT:2003758","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:108931","NEWT:219799","NEWT:219798","NEWT:548","NEWT:150475","NEWT:256737","NEWT:51350","NEWT:8694","NEWT:8692","NEWT:542","NEWT:394503","NEWT:13076","NEWT:219785","NEWT:451515","NEWT:8673","NEWT:550","NEWT:10804","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:1225786","NEWT:771870","NEWT:8665","NEWT:51338","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:311339","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:2585209","NEWT:643680","NEWT:214092","NEWT:40697","NEWT:13095","NEWT:162425","NEWT:242124","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:595495","NEWT:80833","NEWT:479096","NEWT:28108","NEWT:28104","NEWT:1114957","NEWT:568703","NEWT:568708","NEWT:52641","NEWT:353152","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NEWT:51329","NCBITaxon:1763","NEWT:443906","NEWT:13037","NEWT:519","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:13064","NEWT:7375","NEWT:300641","NEWT:39655","NEWT:38323","NEWT:206411","NEWT:80869","NEWT:214053","NEWT:80863","NEWT:665890","NEWT:52639","NEWT:52638","NEWT:367774","NEWT:8697","NEWT:861109","NEWT:8695","NEWT:1196866","NEWT:6039","NEWT:510516","NEWT:1045775","NEWT:1123869","NCBITaxon:1335626","NEWT:7370","NEWT:83906","NEWT:99488","NEWT:1134506","NEWT:255470","NEWT:762903","NEWT:27697","NEWT:86185","NEWT:9935","NEWT:266940","NEWT:287889","NEWT:1028307","NEWT:52603","NEWT:92867","NEWT:92866","NEWT:1344414","NEWT:202950","NEWT:741158","NEWT:1293091","NEWT:202956","NEWT:315750","NEWT:99490","NEWT:632957","NEWT:9925","NCBITaxon:9606","NEWT:1436183","NEWT:1479235","NEWT:4232","NEWT:9922","NEWT:416348","NEWT:105812","NEWT:11298","NEWT:4236","NEWT:398031","NEWT:196627","NEWT:523840","NEWT:1076878","NEWT:200308","NEWT:200302","NEWT:9917","NEWT:95912","NEWT:9913","NEWT:9915","NEWT:105841","NEWT:1170227","NEWT:2666255","NEWT:999810","NEWT:28995","NEWT:1392998","NEWT:1392997","NEWT:114796","NEWT:1150621","NEWT:1085791","NEWT:745531","NEWT:226900","NEWT:231490","NEWT:5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