Jul 10, 2024
Proteomic Sample Preparation
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Proteomic Sample Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5x4og47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 10, 2024
Last Modified: July 10, 2024
Protocol Integer ID: 103178
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Proteomic Sample Preparation
**Sample Storage and Preparation** - Received 12 samples (3 of each WT CYT, GS CYT, WT EZR, and GS EZR) and kept at -80°C until processing. - Spike samples with undigested bovine casein at a total of either 1 or 2 pmol as an internal quality control standard.
**Reduction and Alkylation** - Reduce samples for 15 minutes at 80°C. - Alkylate with 20 mM iodoacetamide for 30 minutes at room temperature.
**Protein Trapping** - Supplement samples with a final concentration of 1.2% phosphoric acid and 636 μL of S-Trap (Protifi) binding buffer (90% MeOH/100 mM TEAB). - Trap proteins on the S-Trap micro cartridge.
**Digestion** - Digest trapped proteins using 20 ng/μL sequencing grade trypsin (Promega) for 1 hour at 47°C.
**Elution** - Elute digested proteins using the following solutions in sequence: - 50 mM TEAB - 0.2% FA - 50% ACN/0.2% FA
**Lyophilization** - Lyophilize all samples to dryness.
**Resuspension** - Resuspend samples in 12 μL of 1% TFA/2% acetonitrile with 12.5 fmol/μL of yeast ADH.
**Study Pool QC (SPQC) Creation** - Create a study pool QC (SPQC) by combining equal volumes of each sample.