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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
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Bioquímica
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JoVE Journal Bioquímica
Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

DOI:
10.3791/56474-v

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10:37 min

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November 15, 2017

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, , , , , , ,
1St. Jude Proteomics Facility,St. Jude Children’s Research Hospital, 2Departments of Structural Biology and Developmental Neurobiology,St. Jude Children’s Research Hospital, 3Heilongjiang University of Chinese Medicine

Capítulos

  • 00:05Título
  • 01:00Preparation of Cells/Tissues
  • 01:45Protein Extraction, Quality Control Western Blotting, In-solution Digestion, and Peptide Desalting
  • 04:45TMT Labeling of Peptides
  • 05:50Extensive High-resolution, Basic pH LC Prefactionation
  • 06:43LC-MS/MS Preparation and Parameters
  • 07:41Results: Deep Proteome Profiling Analysis
  • 08:45Conclusion

Summary

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Tadução automática

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Tags

Proteome ProfilingIsobaric LabelingLiquid ChromatographyMass SpectrometryQuantificationProtein IdentificationProtein QuantitationBiomarker DiscoveryCell LysisProtein DigestionAcetonitrileLys-C ProteaseDTTHEPES

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