Nov 12, 2023

Public workspaceProteomics :On-bead/in-solution digestion and phosphopeptide enrichment

DOI
dx.doi.org/10.17504/protocols.io.rm7vzx195gx1/v1
  • 1University of California, San Diego
Leonardo A Parra-Rivas
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzx195gx1/v1
Protocol Citation: Leonardo A Parra-Rivas 2023. Proteomics :On-bead/in-solution digestion and phosphopeptide enrichment. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx195gx1/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: November 12, 2023
Last Modified: November 12, 2023
Protocol Integer ID: 90826
Abstract
Proteomics :On-bead/in-solution digestion and phosphopeptide enrichment
Protein digestion of GST samples was performed following published protocols
CITATION
Soucek S, Zeng Y, Bellur DL, Bergkessel M, Morris KJ, Deng Q, Duong D, Seyfried NT, Guthrie C, Staley JP, Fasken MB, Corbett AH (2016). The Evolutionarily-conserved Polyadenosine RNA Binding Protein, Nab2, Cooperates with Splicing Machinery to Regulate the Fate of pre-mRNA..
LINK
https://doi.org/10.1128/MCB.00402-16

Digestion buffer (50 mM NH4HCO3) was added to the sample, followed by treatment with 1 mM dithiothreitol (DTT, Thermo Scientific Cat#R0861) at room temperature for 30 min. Subsequently, 5 mM iodoacetamide (IAA, Sigma Cat#I1149) was added to the mixture and incubated at room temperature for 30 min in the dark.
Proteins were digested with 2 µg of lysyl endopeptidase (Wako, Cat# 125-05061) at room temperature (overnight).
Further digestion was performed with 2 µg trypsin (Promega, Cat#VA9000) at room temperature (overnight). In contrast, for phosphopeptide enrichment, proteins were digested overnight at room temperature with 1 µg of Asp-N (Promega, Cat# VA1160) for samples in solution or 5 µg of Asp-N for the samples on-beads.
The clean peptides were concentrated using a high-select Fe-NTA phosphopeptide enrichment kit (Thermo Scientific Cat#A32992) All resulting peptides were desalted and dried using an HLB column (Waters, Cat#186002034) and vacuum.
Citations
Step 1
Soucek S, Zeng Y, Bellur DL, Bergkessel M, Morris KJ, Deng Q, Duong D, Seyfried NT, Guthrie C, Staley JP, Fasken MB, Corbett AH. The Evolutionarily-conserved Polyadenosine RNA Binding Protein, Nab2, Cooperates with Splicing Machinery to Regulate the Fate of pre-mRNA.
https://doi.org/10.1128/MCB.00402-16