Apr 01, 2022
Version 2
Design and preparation of synthetic reference peptides for APP/Aβ TOMAHAQ proteomics V.2
- Hankum Park1,2,3,
- Frances V Hundley1,2,
- Harper JW1,2
- 1Department of Cell Biology, Harvard Medical School Boston, MA 02115, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
- 3Current affiliation: Soeul National University, School of Dentistry
Protocol Citation: Hankum Park, Frances V Hundley, Harper JW 2022. Design and preparation of synthetic reference peptides for APP/Aβ TOMAHAQ proteomics. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6bqk1gqe/v2Version created by Frances V Hundley
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: March 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59928
Keywords: ASAPCRN
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Abstract
TOMAHAQ-based targeted proteomics relies on heavy-labeled reference peptides for multi-plexed quantification of peptides of interest within a set of samples. The APP amyloid precursor protein is thought to be proteolytically processed within the endolysosomal system by β-secretase and ϒ-secretase to yield various forms of Aβ. To quantitatively track APP products, we describe a protocol for design and generation of synthetic reference peptides for TOMAHAQ analysis. We also include reference peptides for the extracellular and cytosolic domains.
Attachments
Design APP peptides corresponding to extracellular and cytosolic regions based on data available in Peptide Atlas which are predicted to have favorable LC-MS properties. See attached Table.
Design half-tryptic peptides based on the cleavage sites for BACE1 and ϒ-secretase. See attached table.
Order commercial synthesis of peptides from Biomatik and Thermo Fischer Scientific, or similar.
Reconstitute the peptides with 3% acetonitrile/0.1% formic acid, and quantify the concentration using Pierce Quantitative Fluorometric Peptide Assay.
Mix 10 μL of 200 μM peptide in 200 mM EPPS buffer (pH 8.5) with 1 μL of 5 μg/μL super-heavy TMT reagent (TMTsh). Incubate at 25 °C for 1 hr then check the labeling efficiency by LC-MS.
Add extra TMTsh to under-labeled peptides until > 95% labeling on both N-terminus and lysine residues is reached.
Desalt the reaction mixture using C18 StageTip.
Resuspend the peptides in 5% acetonitrile/8% formic acid, and pool the peptides.