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Sample Preparation Steps
Sample Preparation Steps
o Protein extraction
o Protein extraction
o Protein purification (acid precipitation, alcohol precipitation,
o Protein purification (acid precipitation, alcohol precipitation,
salting precipitation, immunoprecipitation, HPLC, etc)
salting precipitation, immunoprecipitation, HPLC, etc)
o Protein concentration measurement (Bradford, BCA, Lowry, etc)
o Protein concentration measurement (Bradford, BCA, Lowry, etc)
o SDS-PAGE electrophoresis
o SDS-PAGE electrophoresis
o In gel digestion
o In gel digestion
o In solution digestion
o In solution digestion
o Peptide labeling (iTRAQ, TMT, etc)
o Peptide labeling (iTRAQ, TMT, etc)
o Desalting with Stage-tips
o Desalting with Stage-tips
Discovery Proteomics
Discovery Proteomics
o Protein identification (MS1 scanning followed by data-dependent MS2 fragmentation with HCD or CID)
o Protein identification (MS1 scanning followed by data-dependent MS2 fragmentation with HCD or CID)
o PTM identification (fragmentation with HCD, CID, or ETD)
o PTM identification (fragmentation with HCD, CID, or ETD)
o Global protein quantitation: label-free method, labelling method (iTRAQ, TMT, SILAC)
o Global protein quantitation: label-free method, labelling method (iTRAQ, TMT, SILAC)
Targeted Proteomics
Targeted Proteomics
o Selection of peptides in Skyline
o Selection of peptides in Skyline
o Commercial synthesis of labeled peptides
o Commercial synthesis of labeled peptides
o Method establishment and optimization (MRM on Quantiva, PRM on Fusion Lumos or Fusion)
o Method establishment and optimization (MRM on Quantiva, PRM on Fusion Lumos or Fusion)
Platforms for Data Acquisition
Platforms for Data Acquisition
o Lumos
o Lumos
o Fusion
o Fusion
o Elite
o Elite
o Velos
o Velos
o Quantiva
o Quantiva
Data Treatment
Data Treatment
o Database searching (Proteome Discoverer, Maxquant, MSFragger, etc)
o Database searching (Proteome Discoverer, Maxquant, MSFragger, etc)
o Data analysis (Skyline for targeted quantitative analysis, Perseus for quantitation, SAS and SPSS for statistical analysis)
o Data analysis (Skyline for targeted quantitative analysis, Perseus for quantitation, SAS and SPSS for statistical analysis)
o Functional analysis (IPA, PANTHER, DAVID, STRING, Blast2go, etc)
o Functional analysis (IPA, PANTHER, DAVID, STRING, Blast2go, etc)
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