MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v03/MSV000085483/primaryOK2000000Liu Yanghttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=d96336a446ac489dab95c3db005a5803lyang10@nd.eduMassIVE432UNIMOD:4 - "Iodoacetamide derivative."UNIMOD:27 - "Pyro-glu from E."UNIMOD:7 - "Deamidation."UNIMOD:1 - "Acetylation."UNIMOD:28 - "Pyro-glu from Q."UNIMOD:35 - "Oxidation or Hydroxylation."ProteomicsQ ExactiveHomo Sapiens (ncbitaxon:9606)University of Notre DameIdentifying the target proteins of small-molecule drug candidates is important for determining their molecular mechanisms of action. Porous membranes derivatized with such small molecules may provide an attractive target-identification platform due to a high protein-capture efficiency during flow through membrane pores. This work employs carbonic anhydrase II (CAII) binding to immobilized 4-(2-aminoethyl)benzenesulfonamide (AEBSA) to examine the efficiency and selectivity of affinity capture in modified membranes. Selective elution of captured protein, tryptic digestion, tandem mass spectrometry analysis, and label-free quantification (LFQ) identify CAII as the dominant AEBSA target in diluted serum or cell lysate. CAII identification relies on determining the ratio of protein LFQ intensities in sample and control experiments, where free AEBSA added to the control loading solution limits CAII capture. Global proteomics shows that the spiked CAII is the only protein with a log₂ ratio consistently >2, and the detection limit for CAII identification is 0.004 wt % of the total protein in 1:4 diluted human serum or 0.024 wt % of the total protein from breast cancer cell lysates. The same approach also identifies native CAII in human kidney cell lysate as an AEBSA target. Comparison of affinity capture using membranes, Affi-Gel 10 resin or M-270 Dynabeads derivatized with AEBSA suggests that only membranes allow identification of low-abundance CAII as a target.Membrane-Based Affinity Purification to Identify Target Proteins of a Small-Molecule Drug.Yang Liu L, Bui Loan L, Hanjaya-Putra Donny D, Bruening Merlin L MLProtein Gene Products, Gene Products, Proteins, ASS, fold, Gene Proteins, Gene, Protein., Ass-1, AA408052, Proteinsmall, Membrane Tissue, Products, membrane, Product, drug, Proteins, integral to membrane, Gene, neutral molecular compounds, membranous organ component, Membrane Tissues, reduced, isolation, integral component of membrane, Protein, Pharmaceutical Product, whole membrane, Gene Products, Pharmaceutic, tiny, molecule, molecula, Drugs, transmembrane, membrane of organ, molecules, underdeveloped, Pharmaceutic Preparations, Tissues, membrane region, Tissue, hypoplasia, purification, Molekuel, Membrane, Protein Gene Products, Drug, isolation and purification, Gene Proteins, Preparations, drugs, Pharmaceuticals., medicine, Pharmaceutical, region of membrane, Preparation, Pharmaceutical Products, Pharmaceutical PreparationAT1G11140, human being, Product, Tandem, Peptidomics, Blood, Gene, protein, neutral molecular compounds, Serum, protein-containing complex, Mass Spectrometry Mass Spectrometry, prevention, CG12298, Productivity, Human, CA-II, SUB, Membrane Tissues, protein polypeptide chains, reduced, polypeptide chain, Homo sapiens, DmelCG12298, integral component of membrane, Pharmaceutical Product, Gene Products, SCRAMBLED, Low, tiny, protein aggregate, prevention and control, molecule, KIF20A, Man, mammary carcinoma cell, mammary adenocarcinoma cell, molecula, Drugs, me75, molecules, AI131712, AI131712., Man (Taxonomy), reference sample, Tissues, MS2, ligand, mei-1794, membrane region, Tissue, hypoplasia, breast epithelial cancer cell, breast carcinoma cell, proteins, Mass Spectrometry, Molekuel, breast adenocarcinoma cell, free, D17Mit170, T1, Solution, preventive measures, drugs, lysate, medicine, Pharmaceutical, succinylethylenediaminocarboxymethyl agarose N-hydroxysuccinimide ester, sample, region of membrane, Lvtw-5, mammary tumor cell, SRF9, Ca2, Preparation, Controlled, Pharmaceuticals, small, Products, Membrane Tissue, Carbonate dehydratase II, membrane, Car-2, Controlling, preventive therapy, cou, protein complex, drug, Modern, Proteins, STRUBBELIG, integral to membrane, mammary gland carcinoma cell, membranous organ component, Blood Serum, Tl3, Tl2, STRUBBELIG-RECEPTOR FAMILY 9, CAC, Carbonic Anhydrase Isoenzyme C, Lr, 4.2.1.1, native protein, natural protein, Carbonic anhydrase C, Protein, whole membrane, HEL-76, Pharmaceutic, tandem MS, Carbonic anhydrase II, transmembrane, membrane of organ, underdeveloped, Pharmaceutic Preparations, MS/MS, prophylaxis, mammary cancer cell, Dub, Ltw-5, Carbonic Anhydrase C, Mass Spectrometry-Mass Spectrometry, Membrane, sample population, human, AEBSNH2, Protein Gene Products, Drug, CAII, Car2, Gene Proteins, Preparations, breast tumor cell, control, label, Modern Man, Bra, Pharmaceutical Products, T19D16.8, Serums, SCM, humans, Pharmaceutical Preparationsmall, Membrane Tissue, Products, membrane, Product, drug, Proteins, integral to membrane, Gene, neutral molecular compounds, membranous organ component, Membrane Tissues, reduced, isolation, integral component of membrane, Protein, Pharmaceutical Product, whole membrane, Gene Products, Pharmaceutic, tiny, molecule, molecula, Drugs, transmembrane, membrane of organ, molecules, underdeveloped, Pharmaceutic Preparations, Tissues, membrane region, Tissue, hypoplasia, purification, Molekuel, Membrane, Protein Gene Products, Drug, isolation and purification, Gene Proteins, Preparations, drugs, Pharmaceuticals., medicine, Pharmaceutical, region of membrane, Preparation, Pharmaceutical Products, Pharmaceutical Preparation0PXD019432falseMembrane-Based Affinity Purification to Identify Target Proteins of a Small-Molecule DrugWe employ MaxLFQ to relatively quantify fold changes of proteins in two groups.Wed May 27 19:51:00 BST 2020MSV00008548332867494