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Quick and efficient purification and identification of large peptides with puriFlash® MS
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During numerous discussions about the puriFlash® MS, the maximum mass limit of this detector (2000 m/z for the puriFlash® MS L) often comes up. This parameter is particularly crucial when dealing with high molecular weight compounds, which are frequently encountered with large peptides.
To expedite the identification of such compounds, our system features an algorithm that enables the determination of their molecular masses through charge deconvolution. This process is straightforward and significantly aids in simplifying the identification process.
Let me walk you through an example to demonstrate just how simple this process can be.
### Charge Deconvolution: A Simplified Approach to Enhancing Signal Clarity
In mass spectrometry, peptides typically appear under "soft" ionization conditions using an ESI source. These peptides can ionize multiple times, making them detectable across the m/z range of the puriFlash® MS.
Using an algorithm, the charge states of the various peaks in the mass spectrum can be analyzed to automatically deduce the original molecular mass of the peptide.
The deconvolution algorithm employed by Advion combines predictive charge envelope generation with overlap calculations using actual MS data, applying a maximum entropy goodness-of-fit approach, followed by artifact removal based on peak significance. This innovative method allows for rapid deconvolution over a broad mass range within seconds. Additionally, it provides a neutral mass output where the signal intensity directly correlates with the MS raw data signal intensity and the analyte concentration in solution.
Practically speaking, performing this operation with the puriFlash® MS is quite straightforward. Here’s how:
1. **Acquire the Mass Spectrum**: Use the "Advion Data Express" software to capture the mass spectrum of your compound.
2. **Remove Background Noise**: For better visualization, eliminate the background noise from the signal.
3. **Perform Deconvolution**: Click on the "Deconvolute" button to initiate the process.
Upon clicking the "Deconvolute" button, a window will appear where you can specify:
- Input mass range.
- Output mass range (to be displayed after deconvolution).
- Charge range – defaults include all sensible charges for masses up to about 40 kDa, though this may need adjustment for larger masses. Narrowing down to specific charge states reduces noise and artifacts. The charge range can be determined manually.
- Adjust the adduct mass if an adduct other than H+ is expected to dominate; a negative adduct mass can be used for negative charges.
After inputting these details and clicking "OK," the deconvolved spectrum will be displayed.
### Application Example
We initially conducted a reverse-phase purification of four peptides with molecular weights ranging from 10 to 27 kDa. Since we were unsure of the elution order of the different peptides, they were collected and later identified via direct injection into the puriFlash® MS. Subsequently, the method was refined to minimize the duration of the purification process.
**System Specifications**:
- puriFlash® 5.250P
- Solvents:
- A: Water + 0.1% AF
- B: Acetonitrile + 0.1% AF
- Column: PM-30C18-F0025
- Flow Rate: 15 mL/min
- Injection Mode: Liquid (lyophilized compounds reconstituted and dissolved)
- Injected Volume: 150 µL
- Compounds:
- Cytochrome c from horse heart (P/N: 1J484A)
- Lysozyme from chicken egg white, salt-free (P/N: 6D6694)
- Myoglobin Type I, horse skeletal muscle (P/N: 294440)
- Chymotrypsinogen A, from bovine pancreas (P/N: IYQ650)
**Gradient**:
| Time (min) | A (%) | B (%) |
|------------|-------|-------|
| 00:00 | 75.0 | 25.0 |
| 23:00 | 47.0 | 53.0 |
| 23:03 | 0.00 | 100 |
| 30:00 | 0.00 | 100 |
**Results**:
### Conclusion
The different peaks were collected in separate tubes for identification using the puriFlash® MS in direct injection mode.
**MS Conditions**:
- Solvent Makeup: Water (20%) / MeOH (80%) + 0.1% AF
- Flow Rate: 0.2 mL/min
- Source Type: ESI
**Source Parameters**:
- Mode: Positive
- Capillary Temperature: 200°C
- Capillary Voltage: 160 V
- Source Voltage Offset: 30 V
- Source Voltage Span: 5 V
- Source Gas Temperature: 250°C
- ESI Voltage: 3500 V
The resulting MS spectra are as follows:
**Peak 1: MS Spectrum**
**Result After Charge Deconvolution**
The result after analyzing the first fraction by MS indicates that the compound is Cytochrome C with a molecular weight of approximately 11.8 kDa.
**Peak 2: MS Spectrum**
**Result After Charge Deconvolution**
The result after analyzing the second fraction by MS shows that the compound is lysozyme, with a molecular weight of approximately 14.3 kDa.
**Peak 3: MS Spectrum**
**Result After Charge Deconvolution**
The result after analyzing the third fraction by MS shows that the compound is myoglobin, with a molecular weight of approximately 17 kDa.
**Peak 4: MS Spectrum**
**Result After Charge Deconvolution**
The result after analyzing the final fraction by MS shows that the compound is chymotrypsinogen, with a molecular weight of approximately 25.6 kDa.
### Optimization
Once the different peaks were identified, the purification method was further optimized to enhance efficiency while maintaining the necessary level of purity, utilizing puriFlash® Monolith columns.
**Optimized Gradient**:
| Time (min) | A (%) | B (%) |
|------------|-------|-------|
| 00:00 | 75.0 | 25.0 |
| 07:00 | 47.0 | 53.0 |
| 07:03 | 0.00 | 100 |
| 11:00 | 0.00 | 100 |
The purification step was reduced by 60%.
### Conclusion
The puriFlash® MS facilitates rapid compound identification and, when combined with the puriFlash® Monolith phase, helps reduce purification time significantly.
Moreover, the puriFlash® MS is highly versatile, capable of coupling with HPLC or purification systems, analyzing TLC plate spots, supporting solid injection sources, enabling direct analysis via open ports, and identifying large molecules through post-run charge deconvolution.
For more information, visit our website at [www.flash-chromatographie.com](http://www.flash-chromatographie.com) or feel free to reach out to us directly.
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