Data-Independent Acquisition
Data Independent Acquisition (DIA), a new trend in Mass spectrometry proteomics, provides higher protein coverage than Data Dependent Acquisition (DDA) methods. In contrast to DDA, which fragments only peptide precursors meeting certain cutoffs, the DIA approach fragments all peptides across a predefined peptide precursor window during the entire nanoLC gradient.
In data analysis, DIA searches all the MS2 precursor data against a spectral library database using sophisticated software such as DIA-NN or Spectronaut. In contrast, DDA data analysis searches only selected MS2 data against a protein database. Although the DIA method results in more hits, the analysis is less robust, consumes more time, and generates more false positives than DDA. A comparison of the two methods is shown below:
| DDA | DIA | |
|---|---|---|
| Sample type | All protein samples | All protein samples |
| Number of sample | Unlimited | Unlimited |
| MS2 Precursors | Selected precusors | All precursor |
| Data analysis | Protein database search | Spectral library-based / Library-free search |
| Quantitation | ++ | + |
| Protein coverage | + | ++ |
| Reproducibility | + | ++ |
| Main drawbacks | Miss low abundant peptides | Data analysis not robust False positives |
Main applications of DIA proteomics:
- Study objectives: Comparing protein expression levels between experimental groups
- Sample type: All protein samples
DIA experimental procedure
- Sample preparation: Sample clean up, protein extraction, measure/adjust protein concentration, and trypsin digestion
- nanoLCMSMS: Peptides separated by ThermoFisher Ultimate 3000 & MSMS by Orbitrap Exploris
- DIA data analysis: Library based or library free analysis
- Data report
Sample Info
Sample type: We accept in-solution, in-gel samples or IP beads for this service.
Protein amount: 5-200 µg of total protein from each sample.
Protein concentration: 5-20 mg/ml is preferred for in-solution samples..
Buffer: Please feel free to submit samples in whichever buffer that you would normally use. Please inform our scientists about buffer composition. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.
Biohazardous Materials: We do NOT accept samples containing any biohazardous materials. If your samples contain any biohazardous materials, please make sure to deactivate them completely using the appropriate procedure and declare them in the order form. In addition, we do NOT accept any samples containing Level 4 biohazardous materials even if they are deactivated. Please view here for the rank of biohazardous materials.
Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.
Pricing
| Services Description | Academic/Governmental Labs | Industrial/Commercial Labs | ||
|---|---|---|---|---|
| Code | Price | Code | Price | |
| DIA Experiment | 335A | call for price | 335N | call for price |
Turnaround time is 7-9 days. Price covers the following:
- Reduction, alkylation and Trypsin digestion
- NanoLC-MS/MS with DIA settings
- DIA data analysis against one species in NCBI or SwissProt database
- Data report
Price does NOT cover sample preparation