MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v05/MSV000091168/primary200OK0000Ardeshir BayatJonathan Blackburnhttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=3f23f8e9c1c0441392a77b498d408211ardeshir.bayat@uct.ac.zajonathan.blackburn@uct.ac.zaMassIVE405MOD:00110 - "A protein modification that effectively converts an L-cysteine residue to L-cysteine methyl disulfide."ProteomicsQ ExactiveHomo Sapiens (ncbitaxon:9606)University of Cape TownInstitute of Infectious Disease & Molecular Medicine and Department of Integrative Biomedical Sciences, UCT.Keloid disease (KD) is a common connective tissue disorder of unknown aetiopathogenesis with ill-defined treatment. Keloid scars present as exophytic fibroproliferative reticular lesions postcutaneous injury, and even though KD remains neoplastically benign, keloid lesions behave locally aggressive, invasive and expansive. To date, there is limited understanding and validation of biomarkers identified through combined proteomic and genomic evaluation of KD. Therefore, the aim in this study was to identify putative causative candidates in KD by performing a comprehensive proteomics analysis of subcellular fractions as well as the whole cell, coupled with transcriptomics data analysis of normal compared with KD fibroblasts. We then applied novel integrative bioinformatics analysis to demonstrate that NF-kB-p65 (RELA) from the cytosolic fraction and CAPN2 from the whole-cell lysate were statistically significantly upregulated in KD and associated with alterations in relevant key signaling pathways, including apoptosis. Our findings were further confirmed by showing upregulation of both RELA and CAPN2 in KD using flow cytometry and immunohistochemistry. Moreover, functional evaluation using real-time cell analysis and flow cytometry demonstrated that both omeprazole and dexamethasone inhibited the growth of KD fibroblasts by enhancing the rate of apoptosis. In conclusion, subcellular fractionation and metaproteogenomic analyses have identified, to our knowledge, 2 previously unreported biomarkers of significant relevance to keloid diagnostics and therapeutics.Subcellular Fractionation and Metaproteogenomic Identification and Validation of Key Differentially Expressed Molecular Targets for Keloid Disease.Child mortality associated with reasons for not breastfeeding.Kidzeru Elvis B EB, Sinkala Musalula M, Chalwa Temwani T, Matobole Relebohile R, Alkelani Madeha M, Ghasemishahrestani Zeinab Z, Mbandi Stanley K SK, Blackburn Jonathan J, Tabb David L DL, Adeola Henry Ademola HA, Khumalo Nonhlanhla P NP, Bayat Ardeshir AHenderson Peggy P, Martines José J, de Zoysa Isabelle Ikeloids, other disease, human disease, Dmikkgamma, IKK-gamma, disorders, CG16910, dIKK-gamma, keloid, non-neoplastic, IKKgamma, disease, DmIKKgamma, DmelCG16910, IKK, Keloids, diseases, dIKK, DmIKK-gamma, Kenny, Diseases, disease or disorder, condition, disorder, diseases and disorders, Homo sapiens disease, medical condition., dmIKKgamma, IKK[[gamma]], IKKg, cheloid, KEY, Keysodium salt, Water, 5730502P04Rik, ammonium formate, Size, 13C-labeled, MeCN, cadmium salt, Product, PhrB photolyase activity, Laboratory, dermatofibrosarcoma, ccl, H(2)O, BOUND WATER, magnesium formate, Capillary, mg/ml, oxidane, zinc salt, Steels, Solvent, Long Term, P270, HF1-b, WATER, HOH, l(1)AA33, 5730420M11Rik, dmTAF[[II]]230, actin-dependent regulator of chromatin subfamily F member 1, Polypeptides, DMU42699, KL receptor activity, protein polypeptide chains, M(3L)i, cobalt(II) formate dihydrate, DmelCG12298, CH3-C#N, GRP1, Beds, Grp1, B1, Line, SCO5, 1, 2, 3, SCRAMBLED, SCO1, NUP96, Analysis, Research Activity, Gsfsow3, Laboratory Research, epithelium, SMC-2, Republic of South Africa, integumentum commune, Priorities, 5730497N03Rik, average, SET, C79325, TFIID TAF250, Analyses, cel, Cytoskeletal, DmelCG13176, M, PTPSTEP, entire skin, ORF19, proteins, W, Sinusoidal Bed, adult mouth, SUPPRESSOR OF AUXIN RESISTANCE 3, AI047805, Bed, DmelCG4299, Stainless Steels, set, h, column, l(1)B2/13.1, anatomical tube, ACN, Acn, sample, D1, Fam91a1, Otf11, Bs, Homo sapiens disease, D9, Stainless steel, AV220772, M(3)RpS17, skin and subcutaneous tissue, ELD, nickel salt, GPH, hOSA1, dTAF[[II]]230, c-SP4, l(1)LB9, cobalt (+2) salt, STRUBBELIG, TAF200, Longterm Effect, Research Priorities, Sinusoids, not genetically inherited, Spectrum Analysis, Oral region, PBT, DmelCG42865, H2O, sodium (4:1:1) salt, cavitas oris, magnesium salt, Dm1, M(3)i(55), anatomische Mund, Sizes, dipyrimidine photolyase (photosensitive), 1110049F14Rik, familial dermatofibrosarcoma protuberans (subtype), END, Hydrogen Oxide, Research and Development, rpS17, pbt, HLA-DR-associated protein II, DI-2, CG11628, Oral part of face, I-2Dm, Dioxide Snow, formate, OSA1, Spectrometry, cromium (+3), Mouth, Striatum-enriched protein-tyrosine phosphatase, beta Trypsin, valva (Spanish, Activities, I-2PP1, Nuclear Membrane, TAF-IBETA, Keloids, Taf250, Dmel_CG13885, duct, vestibule of mouth, ammonium (4:1) salt, TAF-Ibeta, entire integument, methyl cyanide, Carbon Dioxide Snow, oral cavity, lead (+2) salt, glutamic acid side-chain, Fibroblast, TAF230, Smc2l1, krk1, region of skin, Nuclear Membranes, nickel formate dihydrate, GRP1/cytohesin 1, lead formate, Peptidomics, Effects, Otf-11, aluminum salt, protein-containing complex, thomson, 17alpha)-isomer, CG7826, agua, mass-to-charge ratio, CG11633, M(3)i, Gene Products, CG7835, disease or disorder, CG42273, Capillary Bed, Sample Sizes, 3.1, PH3, Skn-1a, anatomical systems, dTAF[[II]]250, FAM39E, skin, 2310061K05Rik, Acinus, thallium (+1) salt, Longterm, cell, Sinusoidal Beds, ligand, SPR-1, mei-1794, beta-Trypsin, rubidium salt, BAF250, F23A5.3, 2pp2a, Sp4, primary dermal fibroblast, Long-Term, l(3)dtOA4, methanoic acid, SKIN, CG10574, Solution, acinusL, oral vestibule, dTAF250, skin organ, 2PP2A, acinusS, Steel, BRG1-associated factor 250, M(3)i[55], dSET, dSet, peptidos, Development and Research, SP4, oral fissure, capn8, PTHB1, LB5, Pharmaceuticals, Products, proto-oncogene c-Kit, metastatic dermatofibrosarcoma protuberans (subtype), Snow, Oct-11a, SMARCF1, mouse embryonic fibroblast, 1-(14)C-labeled, BM029, Dry, TRH, Trh, Proteins, disorders, skin zone, stepk, BG:DS00004.13, potassium formate, HF-1b, copper (+2) salt, Rnt, acetonitrile, buffer, Cell, mKIAA0670, dTAF230, lLB5, polypeptide, skin plus hypodermis, Wash2, Wash1, MS, native protein, Temperatures, spe, I-2PP2A, KIT ligand receptor activity, C18, cupric formate, chemical analysis, Long Term Effects, Dm I-2, TAF[[II]]250/230, condition, BcDNA:RE44119, 5alpha, aluminum formate, Sinusoidal, Skin, dJ142L7.2, Mass Spectrum Analyses, cryg3, XKrk1, skin benign neoplasm, Taf[[II]]250, AU020952, MRD14, BAF250A, lithium formate, RPE, Pharmaceutic Preparations, underdeveloped, ensemble, PZ32, M(3)q, Dub, Magnetic, hydrogen phosphorus, ethanenitrile, CD117, CG13885, SWI|SNF complex protein p270, anon-EST:Posey48, primary structure of sequence macromolecule, DmelCG11628, Longterm Effects, Epoc-1, p. pigmentosa retinae, ME-IV, dermatofibrosarcoma protuberans, Gene Proteins, matrix-associated, Capillary Beds, ARID domain-containing protein 1A, C-Kit, hydrogen phosphide, Rima oris, Ssm, xkl-1, M(3L)i[55], primary structure of sequence macromolecule., deoxyribonucleate pyrimidine dimer lyase (photosensitive), CG6883, [OH2], Pharmaceutical Products, the integument, Rps17, IPP2A2, pelt, Activity, NCMe, determination, Skn-li, leg, RPS17, zinc formate, lead salt, Xkl-1, protein, Protein D123, Nuclear Envelopes, Foundation, pigmented epithelium, SUB, M(3)67, peptide, BAF250a, Gsfsco1, ammonium tetraformate, peptido, exact), diseases, BANF, Pharmaceutical Product, SWI|SNF-related, diseases and disorders, Min, protein aggregate, Gsfsco5, Effect, medicament, KIF20A, cheloid, DmelCG42273, SOW3, DFSP, Mass Spectrum Analysis, AI255214, 3-hydroxy-5-estrane-17-carbonitrile, human disease, peptides, vestibulum oris, TAF-I, Envelope, l(3)67BDo, 2610036I19Rik, mouth cavity, 2610510L13Rik, dermoid system, hypoplasia, pigmented retina, min, mAPC, CCR-like, copper, experimental design, DNA cyclobutane dipyrimidine photolyase activity, anatomical valve, keloid, IGAAD, fSAP152, lithium salt, CG16705, DmelCG10574, Ice, RUN, Run, Pharmaceutical, l(2)k08110, Carbon, Carbon Dioxide, dihydridooxygen, TEAB, blood capillary, Research Priority, dihydrogen oxide, Sl, Dmel_CG6883, ammonium (2:1) salt, Long-Term Effects, Sinusoid, phapii, PRE, Tube, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, aqua, Osa homolog 1, M(3)67C, Dmel_CG7826, StF-IT-1, Tr-kit, Th, integumental organ, TAFII-250, CT37173, TAF250/230, AA33, Spectroscopy, TUBE, HDC07747, TAFII250, batch, retinal pigment, BC033609, Diseases, 3.1.3.48, SMC2L1, kl1-A, cavum oris, B120, Pharmaceutic, bHLHe13, KIT, WASH, nickel (+2) salt, retinal pigment layer, Dmel_CG7835, Lines, Mnb, MNB, tyrosine-protein kinase Kit, ammonium salt, Step, CAPE, Rp S17, cobaltous formate, hydrogen hydroxide, kit, CG4299, AW124434, CG17603, proPO-AE, TAF[[II]], Stainless, mKIAA0493, acqua, disease, phr A photolyase activity, vertebrate epidermis, Sample, DNA-photoreactivating enzyme, STEP, SR3-5, pharmaceuticals, copper salt, Polypeptide, Wasser, dermal system, i2pp2a, AT1G11140, dermis plus epidermis plus hypodermis, other disease, CG1849, d230, cesium salt, SCF receptor activity, Gene, dTAFII250, ACINUS, Spectrum Analyses, photoreactivating enzyme activity, EfW1, PHAPII, epidermis, CG12298, Buffer, formic acid, cytohesin/GRP1, CAP-E, hELD, M(3)S33, potassium salt, scfr, polypeptide chain, reduced, dmTAF1, Taf230, farmaco, p63, 14C-labeled, SWI-like protein, p65, Mass, R-citramalyl-CoA lyase activity, stratum pigmentosa retinae, tiny, SCFR, valve, Mass Spectroscopy, Pro-Mega, TAF250, Drugs, study, Union of South Africa, Taf200, Fdc, MOS3, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, PRECOCIOUS, Research, Runt, ipp2a2, Envelopes, phosphane, l(2)SH2 0323, Minute, Taf1p, eau, calcium formate, CTRCT2, Fin16, non-neoplastic, giant cell fibroblastoma, Neural-specific protein-tyrosine phosphatase, DmelCG3922, taf-ibeta, pharyngeal valve, disorder, cromium (+3) salt, Long-Term Effect, SRF9, Preparation, TAF, DmelCG16705, sodium formate, F23A5_3, HHT1, TCEP, small, keloids, DYRK1, TAF[[II]]250, CG42865, nickel formate, Edg, 3H-labeled, protein complex, 弁 (Japanese, HF1B, DmelCG1849, igaad, Cell Lines, strontium formate, l(3)84Ab, medical condition, strontium salt, Peptide, group, STRUBBELIG-RECEPTOR FAMILY 9, MODIFIER OF SNC1, Priority, capillary vessel, Oct11, skin region, natural protein, p230, water, Protein, Research Activities, AI551343, deoxyribonucleic photolyase activity, I2PP2A, sequence, TFIID, Nuclear, Dyrk1, Skin-1a, BP1081, tegument, (3alpha, l(2)SH0323, ACETONITRILE, buccal cavity, cyanomethane, CG13176, CRYG3, CYH1, Membranes, Mass Spectrum, c-KIT, C1orf4, TAF[[II]]230, AW545314, acetic acid ammonium salt, PRSS, photolyase activity, HT-1080, CC1, CL-2, vertebrate integument, CL-1, Tripcellim, TAF[II]250, l(1)19Ea, Membrane, sample population, chromic formate, CG3922, cl-2, Protein Gene Products, Drug, P235, Trypure, portion of skin, dSET/TAF-Ibeta, Preparations, c-kit, 2610030F17Rik, DmelCG17603, BRG1-associated factor 250a, integument, concentration, 2-carboxyethyl, Ccl, CCL, l(3)10512, calcium salt, Peptid, assay, ORW1, SCH, AA407739, T19D16.8, SCM, Pharmaceutical Preparation, TAF1, 3-OHECNkeloids, other disease, human disease, Dmikkgamma, IKK-gamma, disorders, CG16910, dIKK-gamma, keloid, non-neoplastic, IKKgamma, disease, DmIKKgamma, DmelCG16910, IKK, Keloids, diseases, dIKK, DmIKK-gamma, Kenny, Diseases, disease or disorder, condition, disorder, diseases and disorders, Homo sapiens disease, medical condition., dmIKKgamma, IKK[[gamma]], IKKg, cheloid, KEY, KeyOradexon, Protein kinase lysine-deficient 1, Surrogate Endpoints, Laboratory, GRASP65, Physical, growth and development, Long Term, trauma, dmTAF[[II]]230, Enhancer-Binding Protein, Bio Informatics, Immunogold-Silver Techniques, Biological, Programmed, Omeprazole Magnesium, symptoms, 10.5, Analysis, Profilings, H 168-68, Cell Sortings, 16alpha-methyl-9alpha-fluoro-1-dehydrocortisol, 10.9, treatment, average, F, 21-trihydroxy-16-methylpregna-1, TFIID TAF250, Analyses, cel, DmelCG13176, PRKWNK1, (11beta, Decaspray, NF-kappa B, V, Omapren, Nuclear Factor Kappab, Computational, Gene Expression Analysis, Intrinsic Pathway Apoptosis, Immunogold-Silver Technic, "Other musculoskelet/connectiv" EXACT [SNOMEDCT_2005_07_31:268126004], Immunolabeling Technique, Homo sapiens disease, Methylfluorprednisolone, CXXC finger protein 9, Computational Molecular Biologies, "Connective tissue disorder" EXACT [NCI2004_11_17:C26729], single organism signaling, screening, Cell Sorting, dTAF[[II]]230, Viral, Injury and Wounds, connective tissue disease, Procelac, Biologies, dexametasona, TAF200, Monitorings, Up-Regulation (Physiology), Immunogold Technique, Longterm Effect, Dexpak, Physical Traumas, Cicatrization, Computational Molecular, DmIKKgamma, HSAN2, signaling (initiator) caspase activity, Immunolabeling Technic, Zema-Pak, induction of apoptosis, Marker, dIKK, Computational Molecular Biology, Immunolabeling Technics, Technics, Transcription Factor NF kB, Caspase-Dependent, fluormethylprednisolone, Decaject-L.A., Hexadrol, End Points, NFkB, Subcellular, Upregulation, Transcriptome Profilings, Cortisumman, Ozurdex, 20-dione, IKK-gamma, 5-methoxy-2-(((4-methoxy-3, signs, Immunogold Silver Techniques, Immunologic, RPS4, Laboratory Marker, Fluorescence-Activated Cell, Intrinsic Pathway Apoptoses, Gene Expression Analyses, Keloids, Taf250, 21-trihydroxy-16-methyl-, H 168 68, Calonat, Research-Related Injuries, eve2, Extrinsic Pathway, DexPak, NF kB, H 16868, "Connective tissue dis." EXACT [SNOMEDCT_2005_07_31:268048008], TAF230, Fibroblast, Capa2, CG2328, Immunoglobulin Enhancer-Binding Protein, Gasec, Research Related Injuries, Protein kinase with no lysine 1, Immunogold-Silver, NF-kB, Immunoglobulin, Peptidomics, Effects, Clinical Marker, 5430411C10Rik, 5-methoxy-2-{[(4-methoxy-3, Gastrimut, apoptotic programmed cell death, Transcription Factor, Injuries and Wounds, dIKK-gamma, activation of apoptosis, Gene Expression Profilings, Fluorescence-Activated, Aim, AIM, mRNA Differential Displays, Fluorescence-Activated Cell Sortings, Microfluorometry, Gene Expression Pattern Analysis, Wounds, omeprazol, Fractions, PPP1R167, DmIKK-gamma, disease or disorder, Scars, CCG2, even, Decaject, Decadron, Technique, Immunogold Silver Technics, Aeroseb-Dex, Cytofluorometry, DNMT1, Transcription, Injury, 16alpha)-, dTAF[[II]]250, Clinical, DNMT1_HUMAN, Flow Cytofluorometries, Longterm, cell, Receptor Up-Regulation, Transcriptomics, Long-Term, tissue disease, Monitoring, API6, dTAF250, NFKB3, Diodex, Subcellular Fraction, Classic Apoptosis, execution phase of apoptotic process, Flow, Flow Microfluorometries, Cytometry, SVP65, Biologic, Immunoglobulin Enhancer Binding Protein, Sortings, Apoptosis, DNA (cytosine-5)-methyltransferase 1, Auxiron, cell suicide, findings, Immunogold Technic, Serum Markers, apoptotic cell death, disorders, Immunohistocytochemistry, BG:DS00004.13, Flow Cytometries, MCMT, Cell, Immune Marker, dTAF230, Differential Display, IKKgamma, Transcript Expression Analyses, traumatic injury, Wounds and Injury, Surrogate End Point, Trauma, Audazol, chemical analysis, Long Term Effects, xrela, TAF[[II]]250/230, condition, Sodium, Eve, EVE, m-calpin, Flow Microfluorometry, 1-dehydro-16alpha-methyl-9alpha-fluorohydrocortisone, mRNA Differential Display, Biologic Markers, Immunolabeling, CXXC9, Taf[[II]]250, Gene Expression Monitoring, Immunocytochemistry, Pregna-1, CT-2, apoptosis, mRNA, Surrogate, Dmikkgamma, Endpoints, disorder of connective tissue, postnatal growth, wound, severe cutaneous adverse reaction, Indurgan, Differential Displays, Understanding, AI326419, CG16910, NF-kappa B Complex, CXXC-type zinc finger protein 9, Longterm Effects, Classic Apoptoses, 5-dimethylpyridin-2-yl)methyl]sulfinyl}-1H-benzimidazole, dexamethasone, signalling process, "Connective tissue disease" EXACT [SNOMEDCT_2005_07_31:201432001], apoptotic program, Connective Tissue Disorder, "connective tissue disorder" EXACT [CSP2005:0729-7208], Immunogold-Silver Technics, commitment to apoptosis, Immunolabeling Techniques, Antra, Decacort, CLEC2C, Data Analyses, "disorder of connective tissue (disorder)" EXACT [SNOMEDCT_2005_07_31:105969002], Immune Markers, Biological Markers, Viral Marker, Azium, Prazidec, kappa B Enhancer Binding Protein, 9-fluoro-11, HSN1E, determination, CANPL2, Wound, postnatal development, Biochemical, Endpoint, KDP, Erythrocyte 65 kDa protein, Serum, type I programmed cell death, Technic, injury, Xrel1, Molecular Biologies, Transcript Expression Analysis, Readability, Laboratory Markers, Techniques, 17, diseases, m-calpain, Extrinsic Pathway Apoptoses, Immunogold-Silver Technique, Gene Expression Monitorings, CANPml, Ceprandal, diseases and disorders, OMP, Flow Microfluorimetry, Effect, Ulcesep, Omeprazole Sodium, cheloid, Research-Related, IKKg, OMZ, Kinase deficient protein, KEY, Key, Dexason, human disease, primary disorder of connective tissue, DmelCG2328, Biology, Fluorescence Activated Cell Sorting, Decameth, Bio-Informatic, CANP2, Nuclear Factor-Kappab, omeprazolum, Omebeta, Decalix, keloid, Ig-EBP-1, Immune, Markers, connective, Bio-Informatics, Classic, Viral Markers, Classical Apoptosis, disease management, Therapies, connective tissue disorders, Immunogold Technics, Long-Term Effects, Ulceral, Flow Cytofluorometry, Therapy, cellular suicide, Scar, Fluorescence-Activated Cell Sorting, Surrogate Endpoint, Caspase-Dependent Apoptosis, 5-dimethyl-2-pyridinyl)methyl)sulfinyl)-, mCANP, Biochemical Markers, TAFII-250, TAF250/230, Biologic Marker, 16alpha)-9-fluoro-11, Microfluorimetry, Gene Expression, Immunogold, Immunogold Techniques, TAFII250, SCAR, connective tissue disease or disorder, PSK, Intrinsic Pathway, Extrinsic Pathway Apoptosis, Diseases, SYT, Kenny, Dexacortal, Millicorten, disease or disorder of connective tissue, VI, Physical Trauma, 9alpha-fluoro-16alpha-methylprednisolone, ADCADN, Epistemology, 20.35, Cytofluorometries, Bioinformatic, UNQ203/PRO229, Corson, growth pattern, non-developmental growth, DNA (cytosine-5-)-methyltransferase 1, RELA, Cell Death, CG17603, TAF[[II]], Treatments, NF kappa B Complex, 4-diene-3, disease, data analysis, DmelCG16910, induction of apoptosis by p53, Biochemical Marker, NF kappaB, SR3-5, connective tissue diseases, Omeprazole, Decaject L.A., Expression Analysis, Research-Related Injury, rela-a, Classical, Apoptoses, Synaptotagmin I, 14.10, Sanamidol, other disease, disease of connective tissues, d230, NF-kappaB, Clinical Markers, Capa-2, P65, caspase-dependent programmed cell death, Gene, Caspase Dependent Apoptosis, dTAFII250, EfW1, Ig EBP 1, Hexadecadrol, Surrogate End Points, Surrogate Markers, hWNK1, dmTAF1, Taf230, p63, OMEP, p65, Nuclear Factor kappa B, dmIKKgamma, Type I, IKK[[gamma]], TAF250, Transcription Factor NF-kB, Biomarker, study, Taf200, connective tissue disorder, Microfluorometries, "Connective tissue disease" EXACT [MTH:NOCODE], Biological Marker, Complex, Up Regulation, Inhibitron, Elgam, connective tissue, Taf1p, Treatments., Transcript Expression, Transcriptome Analysis, apoptosis activator activity, Scarring, non-neoplastic, Immunologic Markers, lysate, data processing, IKK, Fraction, Traumas, disorder, Long-Term Effect, TAF, Expression Analyses, Factor-Kappab, programmed cell death by apoptosis, Immunologic Marker, Connective Tissue Disorders, SytI, Cytometries, DNA MTase HsaI, keloids, Profiling, Molecular Biology, TAF[[II]]250, Transcriptome, DNMT, Dexacortin, End Point, l(3)84Ab, medical condition, Magnesium, Bioinformatics, 1H-Benzimidazole, development, Emeproton, 2.7.11.1, Belmazol, Factor NF-kB, Dexasone, p230, DNA methyltransferase HsaI, Programmed Cell Death, Losec, TFIID, Nuclear, Solurex, dexamethasonum, CG13176, Maxidex, apoptosis signaling, omeprazole, Serum Marker, TAF[[II]]230, Transcriptome Profiling, Olit, HSN2, Danlox, disease of connective tissue, l(2)46Ce, TAF[II]250, Transcriptome Analyses, EBP-1, l(2)46Cg, Surrogate Marker, Injuries, Ultop, l(2)46CFj, l(2)46CFh, DmelCG17603, Therapeutic, l(2)46CFp, Data, Sorting, Prilosec, Treatment, assay, E(eve), growth, GOLPH5, V-rel reticuloendotheliosis viral oncogene homolog A (Avian), l(2)46CFg, Desec, m.HsaI, TAF10PXD039729falseProteogenomic Identification and Validation of Key Differentially Expressed Molecular Targets for Keloid DiseaseThe study incorporated 5 keloid disease (KD) and 5 normal skin (NS) samples, and due to challenges in obtaining primary samples, we had a sample size of 3 biological replicates for FKN, 3 for DFSP made up of 2 primary cells and 1 cell line, and 2 Fibrosarcoma cell lines FS-CL-1 (HT-1080 [HT1080], CCL-121, ATCC, Manassas, VA, USA) and FS-CL-2 (HT-1080-Luc2 CCL-121-LUC2). Fibroblast subcellular proteins were isolated by employing the Qproteome Cell Compartment Kit (QIAGEN Pharmaceutical). Also, different cell lysis or cell extraction (CE) buffers specific for the different subcellular compartments, including CE1 for cytosolic proteins, and CE2, CE3 and CE4 for membrane, nuclear and cytoskeletal proteins respectively. Proteomics service took place at the Centre for Proteomic and Genomic Research (CPGR) in Cape Town, South Africa. Extracted proteins were transported on dry ice and samples were logged and stored at -80 degrees C until further processing. Briefly, HILIC beads (ReSyn Biosciences, HLC010) were aliquoted into a new tube and the shipping solution removed. Beads were then washed with 250 uL wash buffer [15% Acetonitrile, 100 mM Ammonium acetate (Sigma 14267) pH 4.5] for one minute. This was repeated once. The beads were then resuspended in loading buffer (30% ACN, 200 mM Ammonium acetate pH 4.5) to a concentration of 2.5 mg/mL. A total of 25 ug of protein (or the entire sample) from each tube was transferred to a 96-well protein LoBind plate (Merck, 0030504.100). Protein was reduced with tris (2-carboxyethyl) phosphine (TCEP; Sigma 646547) which was added to a final concentration of 10 mM TCEP and incubated at 60 degrees C for one hour. Samples were cooled to room temperature (RT) and then alkylated with methylmethanethiosulphonate (MMTS; Sigma 208795) which was added to a final concentration of 10 mM MMTS and incubated at RT for 15 minutes. HILIC magnetic beads were added at an equal volume to that of the sample and a ratio of 5:1 total protein. The plate was then incubated at RT on the shaker at 900 RPM for 30 minutes for binding of protein to beads. After binding, the beads were washed four times with 500 uL of 95% ACN for one minute. For digestion, Trypsin (Promega PRV5111), was made up in 50 mM Triethylammonium bicarbonate buffer (TEAB) and added at a ratio of 1:12.5 total protein and the plate was then incubated at 37 degrees C on the shaker for 4 hours. After digestion, the plate was placed on a magnet to separate the beads from the supernatant. The supernatant containing peptides was removed and dried down. Samples were then resuspended in LC loading buffer: 0.1% formic acid (FA; Sigma 56302), 2.5% CAN. LCMS analysis was conducted using a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) coupled to a Dionex Ultimate RS3000 nano-UPLC system. Data was acquired using: Xcalibur v4.1.31.9, Chromeleon v6.8 (SR13), Orbitrap MS v2.9 (build 2926) and Thermo Foundations 3.1 (SP4). Peptides were dissolved in 0.1% Formic Acid (FA; Sigma 56302), 2% Acetonitrile (ACN) and loaded on a C18 trap column (PepMap100, 300um x 5mm x 5um). An estimate of approximately 400 ng of peptide was injected for each sample. Samples were trapped onto the pre-analytic column and washed for 3 minutes before the valve was switched and peptides eluted onto the analytical column. Chromatographic separation was performed with a Waters nanoEase (Zenfit) M/Z Peptide CSH C18 analytic column (186008810, 75 um x 25 cm x 1.7 um). The solvent system employed was solvent A: LC water (Burdick and Jackson BJLC365), 0.1% FA and solvent B: ACN, 0.1% FA. All data acquisition employed Proxeon stainless steel emitters (Thermo Fisher TFES523). The multi-step gradient for peptide separation was generated at 300 nL/min as follows: time change 5 min, gradient change: 2 to 5% Solvent B, time change 40 min, gradient change 5 to 18% Solvent B, time change 10 min, gradient change 18 to 30% Solvent B, time change 2 min, gradient change 30 to 80% Solvent B. The gradient was then held at 80% solvent B for 10 min before returning it to 2% solvent B and conditioning the column for 15 min. The mass spectrometry was operated in positive ion mode with a capillary temperature of 320 degrees C with the applied electrospray voltage of 1.95 kV. Based on the study design, each fraction was run as a block randomised set with a fraction specific pool run after, at the start, middle and end of each block. The fractions were run in clusters to minimise carry over of proteins across sub-cellular locations during data acquisition. In between each fraction block, a pool produced from each fraction pool to represent a complete sample pool was run to monitor system performance during the sequence of acquisition. Blanks were utilised to minimise carry over from QCs throughout the sequence of acquisition.Sun Jan 29 05:18:00 GMT 2023MSV00009116839122141