JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F2_bio2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F4_bio1.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_E6_Thermo_RIPA_6Murea_noSDS_2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F6AndF7_bio2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F0_bio1.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F3_bio2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F5_bio1.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6__0117Thermo_HpHStageTip_F1_bio1.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F1_bio2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F2_bio1.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F4_bio2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_E5_50ug_4du30min_200ulTip_bio2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F0_bio2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F6AndF7_bio1.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F3_bio1.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_E6_Thermo_RIPA_6MUrea_0.2%25SDS_bio2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_E6_Thermo_RIPA_6MUrea_0.2%25SDS_bio1.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_4E6_Thermo_HpHStageTip_F5_bio2.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_E5_50ug_4du30min_200ulTip_bio1.rawhttps://storage.jpostdb.org/JPST002017/files/UreaDTT_E6_Thermo_RIPA_6Murea_noSDS_1.rawprimary200OKProteomicsMingliang YeHomo Sapiens (human)https://repository.jpostdb.org/entry/JPST002017Dalian Institute of Chemical PhysicsjPOSTCell surface proteins are responsible for many critical functions. Systematical profiling of these proteins would provide a unique molecular fingerprint to classify cells and provide important information to guide immunotherapy. Cell surface biotinylation method is one of the effective methods for cell surface proteome profiling. However, classical workflows suffer the disadvantage of poor sensitivity. In this work, we presented an optimized protocol which enabled identification of more cell surface proteins from a smaller number of cells. When this protocol was combined with a tip based fractionation scheme, 4510 proteins, including 2055 annotated cell surface-associated proteins, were identified with only 20 microgram protein digest, showing the superior sensitivity of the approach. To enable process 10 times fewer cells, a pipet tip based protocol was developed, which led to the identification of about 600 cell surface-associated proteins. Finally, the new protocol was applied to compare the cell surface proteomes of two breast cancer cell lines, BT474 and MCF7. It was found that many cell surface-associated proteins were differentially expressed. The new protocols were demonstrated to be easy to perform, time-saving, and yielding good selectivity and high sensitivity. We expect this protocol would have broad applications in the future. SIGNIFICANCE: Cell surface proteins confer specific cellular functions and are easily accessible. They are often used as drug targets and potential biomarkers for prognostic or diagnostic purposes. Thus, efficient methods for profiling cell surface proteins are highly demanded. Cell surface biotinylation method is one of the effective methods for cell surface proteome profiling. However, classical workflows suffer the disadvantage of poor sensitivity. In this work, we presented an optimized protocol which enabled identification of more cell surface proteins from a smaller number of starting cells. The new protocol is easier to perform, time-saving and has less protein loss. By using a special pipet tip, sensitive and in-depth cell surface proteome analysis could be achieved. In combination with label-free quantitative MS, the new protocol can be applied to the differential analysis of the cell surface proteomes between different cell lines to find genetically- or drug-induced changes. We expect this protocol would have broad application in cell surface protein studies, including the discovery of diagnostic marker proteins and potential therapeutic targets.Sensitive profiling of cell surface proteome by using an optimized biotinylation method.Li Yanan Y, Wang Yan Y, Mao Jiawei J, Yao Yating Y, Wang Keyun K, Qiao Qinglong Q, Fang Zheng Z, Ye Mingliang Mplan specification, allergic reaction, Biotinylations, Study, method, Techniques, Methodological Studies, method used in an experiment., Procedures, sensitive, Method, Studies, total expressed protein, Methodological, Procedure, cell bound, sensitivity, Proteomes, Technique, Methodological Study, cell associatedplan specification, allergic reaction, Biotinylations, Study, method, Techniques, Methodological Studies, method used in an experiment., Procedures, sensitive, Method, Studies, total expressed protein, Methodological, Procedure, cell bound, sensitivity, Proteomes, Technique, Methodological Study, cell associatedIntegral Membrane Proteins, Nup32D, Biological Markers, Viral Marker, PLIP, Surrogate Endpoints, Product, determination, Laboratory, Surface Proteins, RCB1904, Biochemical, Endpoint, protein, Membrane-Associated Proteins, Serum, Integral, Type 2A-interacting protein, Long Term, cell associated, Techniques, Laboratory Markers, Method, dJ69E11.3, Biological, Pharmaceutical Product, Line, protein aggregate, Effect, HTATIP, nup154, ERV-R envelope protein, Surface, µg, breast epithelial cancer cell, breast carcinoma cell, procedures, breast adenocarcinoma cell, free, MCF7 cell, Biotinylations, allergic reaction, SU, Immune, Markers, Methodological Studies, cPLA2, medicine, Pharmaceutical, Viral Markers, Surface Protein, Membrane Associated Proteins, associated, TM, TIP, Membrane Protein, Long-Term Effects, Envelope polyprotein, MCF 7 cell, HERV-T Env protein, anatomical protrusion, Viral, wide/broad, Surrogate Endpoint, ZC2HC5, ug, Longterm Effect, Biochemical Markers, HTATIP1, Procedure, tip, ANAC091, Biologic Marker, HERV-T_19q13.11 provirus ancestral Env polyprotein, Transmembrane protein, Putative MAPK-activating protein PM10, Marker, Pharmaceutic, TIP60, Lines, Cell Membrane Protein, Immunotherapies, End Points, mammary cancer cell, Membrane-Associated, Cell Membrane Proteins, Methodological, LNKN-1, Immunologic, ERV3 envelope protein, Laboratory Marker, study protocol, ERV-3 envelope protein, Methodological Study, MCF7, HERV-R_7q21.2 provirus ancestral Env polyprotein, wide, Specificity and Sensitivity, spine, Biochemical Marker, label, tlp, cell bound, TIP41, Proteomes, Integral Membrane, Procedures, Effects, Clinical Markers, Clinical Marker, number, Gene, broad, protein-containing complex, presence, ESA1, protrusion, Surrogate End Points, Surrogate Markers, method, sensitive, Membrane-Associated Protein, method used in an experiment, Arabidopsis NAC domain containing protein 91, Gene Products, Studies, zk, Technique, sensitivity, mammary carcinoma cell, mammary adenocarcinoma cell, Drugs, Cell Membrane, Biomarker, ERV3-1 envelope protein, l(2)10432, Clinical, Longterm, Biological Marker, Long-Term, Study, DmelCG4579, ms(2)zk, drugs, Immunologic Markers, HERV-R envelope protein, Sensitivity, mammary tumor cell, Long-Term Effect, l(2)01501, D8Wsu49e, Preparation, Immunologic Marker, Biologic, MCF-7 cell, Pharmaceuticals, Products, protein complex, BcDNA:LD21772, drug, Proteins, Serum Markers, Cell Lines, total expressed protein, End Point, Cell Surface, Cell Surface Protein, mammary gland carcinoma cell, Surface protein, Cell, Immune Marker, count in organism, native protein, Surrogate End Point, Protein, Long Term Effects, chemical analysis, Membrane Proteins, techniques, Cell Surface Proteins, Membrane Associated Protein, Biologic Markers, nup32D, Serum Marker, Pharmaceutic Preparations, Protein., Surrogate, Endpoints, Specificity, CG4579, CDA08, Membrane, Surrogate Marker, Longterm Effects, Protein Gene Products, plan specification, Drug, Gene Proteins, Preparations, breast tumor cell, Integral Membrane Protein, cardinality, TCV-interacting protein, assay, Pharmaceutical Products, methodology, Pharmaceutical Preparation, Immune Markersplan specification, allergic reaction, Biotinylations, Study, method, Techniques, Methodological Studies, method used in an experiment., Procedures, sensitive, Method, Studies, total expressed protein, Methodological, Procedure, cell bound, sensitivity, Proteomes, Technique, Methodological Study, cell associatedfalseSensitive profiling of cell surface proteome by using an optimized biotinylation methodSensitive profiling of cell surface proteome by using an optimized biotinylation methodThu Feb 02 00:00:00 GMT 2023PXD039794960630707948