Acetic Acid Is Needed To Prepare the PepCalMix Peptides in the SWATH® Acquisition Performance Kit


日付: 11/09/2017
カテゴリー: Academia Omics , Pharma CRO , Standards and Reagents

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For research use only. Not for use in diagnostic procedures.


Issue Description

Replacing 5% acetic acid with 0.1% formic acid, may result in many of the higher m/z peptides disappearing and the remaining peptides may be lower in abundance. In the example below, not all of the 20 PepCalMix peptides can be seen, and some peptides show very low abundance (see peaks at retention times of 16-18 min).
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Resolution

It is essential to use 5% acetic acid when preparing the PepCalMix peptides for nano-LC or infusion. Typically the highest purity of acetic acid is recommended (> 99.7-99.9 %). In addition, HPLC-grade water of (99.9% purity) is also recommended.

These are two ways to prepare the PepCalMix samples:

A) Infusion: The PepCalMix vial contains 50 pmol of each of the 20 different stable isotope-labeled peptides in 50 μL of solvent. This stock solution contains 1 pmol/μL of each peptide.
1. When opening a new vial of the stock solution, the stock solution (1 pmol/μL) should be aliquotted into 5-10 μL volumes and then frozen for future use.
2. Add 5 μL of the PepCalMix stock solution and 95 μL of 10% Buffer B (10% acetonitrile : 5% acetic acid) to the vial.
***Note: Acetic acid is essential for the stability of the higher mass peptides and must not be substituted.***
3. Using a vortexer, mix the solution for a minimum of 30 s.
4. Using a centrifuge, spin the vial to bring the liquid to the bottom of the vial before opening.

B) Nano LC injection: This procedure is used to prepare the PepCalMix stock solution (1 pmol/μL) from the vial provided in the SWATH® Performance Kit.
1. When opening a new vial of the stock solution, aliquot the stock solution (1 pmol/μL) into 5-10 μL volumes and then frozen for future use.
2. Add 99 μL of the dilution buffer (5% acetic acid : 2% acetonitrile) to a vial, and then add 1 μL of the PepCalMix stock solution to prepare a 10 fmol/μL solution.
***Note: Acetic acid is essential for the stability of the higher mass peptides and must not be substituted.***
3. Using a vortexer, mix the solution for a minimum of 30 s.
4. Using a centrifuge, spin the vial to bring the liquid to the bottom of the vial before opening.
5. Pipette the solution into an autosampler vial.

To see a discussion in the SCIEX Community, please follow this link: https://sciex.com/community/application-discussions/proteomics/swath/optimizing-swath-performance/troubleshooting-spread-of-peptide-elution-is-compressed