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Quick and efficient purification and identification of large peptides with puriFlash® MS
During numerous discussions regarding the puriFlash® MS, the upper mass limit of this detector (2000 m/z for the puriFlash® MS L) often comes up. This parameter is particularly significant when dealing with high molecular weight compounds, such as large peptides.
To speed up the identification of these substances, our system features an algorithm that helps determine the exact molecular mass of the compound, known as charge deconvolution. With an accompanying application, we’ll demonstrate just how straightforward it is to obtain this information.
### Charge Deconvolution: A Simple Way to Enhance Signal Strength
In mass spectrometry, peptides are detectable with an ESI source under "soft" conditions. These conditions allow peptides to ionize multiple times, making them visible across the puriFlash® MS's m/z range. By knowing the charge of the various peaks in the mass spectrum, an algorithm can automatically deduce the original molecular mass of the peptide.
The Advion deconvolution algorithm uses a combination of techniques, including predictive charge envelope generation, overlap calculations with actual MS data employing a maximum entropy goodness-of-fit approach, and artifact removal through peak significance determination. This innovative approach enables rapid deconvolution over a wide mass range in mere seconds. Additionally, it provides a neutral mass output where the absolute signal intensity directly correlates with the MS raw data signal intensity and the analyte concentration in solution.
In practice, performing this operation with the puriFlash® MS is quite simple:
1. Acquire the mass spectrum of the compound using the "Advion Data Express" software:
2. For better visualization, remove the background signal:
3. Perform deconvolution:
Upon clicking the "deconvolve" button, a window will appear where you can specify:
- The input mass range.
- The output mass range (to display after deconvolution).
- The charge range – by default, it includes all sensible charges for masses up to approximately 40 kDa. Larger masses may require an expanded range. Reducing to only the charge states in the envelope minimizes noise and artifacts. The charge range can be determined using manual mode.
- Adjust the adduct mass if an adduct other than H+ is expected to dominate. Negative adduct masses can be used for negative charges.
After clicking "OK," the deconvolved spectrum will be displayed.
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### Application Example
Initially, we 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. The method was subsequently optimized to shorten the purification cycle.
**System:** 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 dissolved and prepared in solution)
**Injection 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:**
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### Conclusion
Each peak was 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 MS analysis of the first fraction indicates that the compound is Cytochrome C with a molecular weight of ~11.8 kDa.**
**Peak 2: MS Spectrum**
**Result After Charge Deconvolution:**
**The result after MS analysis of the second fraction reveals that the compound is lysozyme, with a molecular weight of ~14.3 kDa.**
**Peak 3: MS Spectrum**
**Result After Charge Deconvolution:**
**The result after MS analysis of the third fraction shows that the compound is myoglobin, with a molecular weight of ~17 kDa.**
**Peak 4: MS Spectrum**
**Result After Charge Deconvolution:**
**The result after MS analysis of the final fraction confirms that the compound is chymotrypsinogen, with a molecular weight of ~25.6 kDa.**
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### Optimization
Once the different peaks were identified, the purification method was refined to achieve faster purification while maintaining the required level of purity, using puriFlash® Monolith columns.
**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%.
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### Conclusion
The puriFlash® MS facilitates rapid compound identification and, combined with the performance of puriFlash® Monolith phases, can significantly reduce purification time. In addition to being compatible with the puriFlash® instrument range, the puriFlash® MS is highly versatile:
- Coupled with HPLC or purification systems.
- Integrated with an express plate for analyzing TLC spot samples.
- Equipped with a solid injection source (ASAP).
- Installed with a direct analysis open port source (OPSI).
- Identifies large molecules quickly and easily via post-run processing using charge deconvolution.
For further details, visit our website at [www.flash-chromatographie.com](http://www.flash-chromatographie.com) or feel free to reach out to us directly.