Label-free quantitation
Differential protein expression analyses
(a) identify the absolute abundances of the proteins, (using APEX software or simple spectral counting)
(b) measure the differential expression of proteins, for multiple datasets (using APEX software or simple spectral counting).
1. Peptide hits validation
Validate the peptide hits returned by the mass-spectrometry peptide identification algorithms
- Deliverables:
- reports generated by peptideProphet
- Tools Used:
- peptideProphet: (Nesvhizskii et. al. ) used to validate peptide hits:
- pepXML output file that has list of validated peptide hits.
- pepXML output file that has list of validated peptide hits.
- peptideProphet: (Nesvhizskii et. al. ) used to validate peptide hits:
2. Protein hits validation
Validate the protein hits using the pepXML peptideProphet output
- Deliverables:
- reports generated by iProphet
- Tools Used:
- iProphet: (Nesvhizskii et. al. ) used to identify high-confident protein hits
- protXML output file that has list of validated protein hits.
- iProphet: (Nesvhizskii et. al. ) used to identify high-confident protein hits
3. Differential protein expression analyses
Identify differentially expressed proteins as well as absolute protein counts
- Deliverables:
- Excel file that has list of differentially expressed proteins (for more than 1 sample) or just absolute protein counts (for a single sample)
- Tools Used:
- APEX: (Marcotte et. al.)
- APEX: (Marcotte et. al.)
Note: This step includes analysis needs peptide and protein identification and validation, if the user does not have this data/protein hit lists from mass-spectrometry search algorithm run on raw-data. If this is not available, we can do this for you. Please see Protein Identification and Validation.