Jan 14, 2022
Version 1
Acetylation of lysines on affinity-purification matrices to reduce co-digestion of bead-bound ligands V.1
- David M. Hollenstein1,
- Margarita Maurer2,
- Thomas Gossenreiter3,
- Natascha Hartl3,
- Dorothea Anrather3,
- Markus Hartl4
- 1Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104, Freiburg, Germany;
- 2CCRI, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria;
- 3Max Perutz Labs, Mass Spectrometry Facility, Vienna BioCenter, Austria;
- 4Max Perutz Labs, Mass Spectrometry Facility, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 7, Vienna, Austria
External link: https://doi.org/10.1371/journal.pgen.1010150
Protocol Citation: David M. Hollenstein, Margarita Maurer, Thomas Gossenreiter, Natascha Hartl, Dorothea Anrather, Markus Hartl 2022. Acetylation of lysines on affinity-purification matrices to reduce co-digestion of bead-bound ligands. protocols.io https://dx.doi.org/10.17504/protocols.io.b3sqqndw
Manuscript citation:
Holzer E, Rumpf-Kienzl C, Falk S, Dammermann A (2022) A modified TurboID approach identifies tissue-specific centriolar components in C. elegans. PLoS Genet 18(4): e1010150. doi: 10.1371/journal.pgen.1010150
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: January 13, 2022
Last Modified: January 14, 2022
Protocol Integer ID: 56880
Keywords: on-bead digestion, affinity-purification, proteomics, BioID, streptavidin, acetylation, TurboID, interaction proteomics, LC-MS
Abstract
In mass-spectrometry-based interaction proteomics on-bead digestion protocols are commonly applied after affinity-enrichment due to their simplicity and high efficiency. However, on-bead digestion often leads to strong background signals due to co-digestion of the bead-bound ligands such as streptavidin or antibodies. We present an effective, rapid and low-cost method to specifically reduce the peptide signals from co-digested matrix ligands. A short pre-incubation of matrix beads with Sulfo-NHS-Acetate (S-NHS-Ac) leads to acetylation of free amines on lysine side-chains of the bead-bound ligands making them resistant to Lys-C-mediated proteolysis. After binding of bait proteins to the acetylated beads we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion with trypsin. The strong reduction of interfering ligand peptides improves signal strength and data quality for the peptides of interest in liquid chromatography mass spectrometry (LC-MS).
Guidelines
This protocol has been successfully tested for:
Pierce™ Streptavidin Magnetic BeadsThermo FisherCatalog #88816 and
GFP-Trap Magnetic AgaroseChromoTekCatalog #gtma-100
Application to other affinity matrices or antibodies requires prior testing to ensure that the binding efficiency is not affected.
Please note that Rafiee et al. (https://doi.org/10.15252/msb.20199370) have described a similar approach for the modification of lysines and arginines using a different chemistry for bead modfication.
Materials
Acetylation protocol:
Buffers:
- Reaction buffer: 50 mM HEPES-NaOH, pH 7.8, 0.2 % Tween 20; you will require at least 17x the desired bead suspension volume (e.g. 340 µL for 20 µL beads, incl. 1 volume shrink).
- Quenching buffer: 50 mM Ammonium bicarbonate (=Ammonium hydrogen carbonate), 0.2% Tween 20; you will require at least 16x the desired bead suspension volume (e.g. 320 µL for 20 µL beads, incl. 1 volume shrink).
- Storage buffer: 1x PBS-T (i.e. 1x PBS, pH 7.4, 0.2 % Tween 20), 0.02 % Sodium azide; you will require at least 6x the desired bead volume (e.g. 120 µL for 20 µL beads, incl. 1 volume shrink).
Reagents:
- Pierce™ Sulfo-NHS-AcetateThermo FisherCatalog #26777 ; prepare a 100 mM stock solution in water-free DMSO, aliquot and store at -80°C; note that the reagent is labile in water.
Beads as required (see guidelines):
- Pierce™ Streptavidin Magnetic BeadsThermo FisherCatalog #88816
or
- GFP-Trap Magnetic AgaroseChromoTekCatalog #gtma-100
Further materials required:
- Magnetic rack suitable for the applied tube size.
On-bead digestion protocol:
Buffers & reagents
NOTE: Use ultra-pure water for the preparation of all solutions.
- ABC buffer: 50 mM Ammonium bicarbonate (=Ammonium hydrogen carbonate; purity ≥ 99.5%), prepare fresh (min. 300 µL per sample, depending on bead volume)
- 1 M urea (≥ 99.5%) in ABC buffer, prepare fresh, do not heat (min. 50 µL per sample, depending on bead volume used)
- DTT stock solution: 250 mM dithiothreitol (97%) in water (stock solution aliquots can be prepared before and kept frozen until use; min. 4 µL)
- IAA stock solution: 500 mM iodoacetamide (purity ≥ 99.5%) in water (prepare fresh, min. 4 µL per sample required, depending on set-up)
- Lys-C solution: 100 ng/µL lysyl-endopeptidase C (Mass spectrometry grade) in water (stock solution aliquots can be prepared before and kept frozen until use; 150 ng per sample required, depending on set-up and protein amount)
- Trypsin solution: 100 ng/µL trypsin (Mass spectrometry grade) in 1 mM HCl (stock solution aliquots can be prepared before and kept frozen until use; 150 ng per sample required, depending on set-up and protein amount)
- 10% trifluoroacetic acid (sequencing grade; TFA)
Further materials required:
- Low-protein-binding PCR tubes
- Magnetic rack for PCR tubes
Safety warnings
Please refer to the safety-data-sheets of the according chemicals, especially before handling Sodium azide and Sulfo-NHS-acetate.
Before start
This protocol consists of two parts: 1. bead acetylation, 2. on-bead digestion after the affinity enrichment experiment. The protocol for the enrichment depends on the experiment and is not part of this protocol. Please note that the preparations below only relate to part 1 (bead acetylation). Preparations necessary for on-bead digestion are mentioned in the according protocol step and in Materials.
Preparations for bead acetylation:
We recommend to prepare one large batch of beads for the same experimental series. The protocol allows the acetylation of 20 µL beads and can be simply scaled to the amounts required. Please determine and prepare the required reagents accordingly before starting the protocol.
When using magnetic beads all washing steps are performed using a magnetic rack to allow easy removal of buffer.
Using storage buffer is not necessary when the beads are used for affinity purification directly after washing. For longer storage it is highly recommended to prevent bacterial growth.
Acetylation protocol
Acetylation protocol
1h 30m
1h 30m
Wash beads 3x with 100µl Reaction buffer, using the magnetic rack.
Remove supernatant after the final wash.
10m
Add 19 µl Reaction buffer and 1 µl Sulfo-NHS-Acetate (100 mM) to obtain a final concentration of 5 mM Sulfo-NHS-Acetate.
Incubate 1h at room temperature with gentle mixing.
NOTE: for up to 20 µL bead suspension use 20 µL reaction volume. For higher amounts use the bead suspension volume as the reaction volume, keeping the Sulfo-NHS-Acetate concentration at 5 mM. Consider that the reagent is labile in water. Thus the buffer must not be prepared before but the reagent must be added from stock immediately to the buffer.
1h 10m
Discard supernatant and wash 3x with 100 µl Quenching buffer.
10m
Use directly or store in 100 µL Storage buffer at 4°C for later use.
Affinity enrichment protocol
Affinity enrichment protocol
This step is not part of the protocol and will depend on the experiment, as for example BioID, enrichment via GFP and GFP-nanobodies, etc.
NOTE: Be aware that detergents as for example Triton X100 or Tween 20, can be used during these steps but should be removed by repeated washing with detergent-free buffer. Even small amounts of detergent can strongly interfere with chromatography and mass spectrometry and thus should be avoided by all means. We usually recommend five washes with a detergent free buffer before continuing to the on-bead digest protocol. Alternatively, clean-up protocols that are capable of reliably removing detergents could be used after digestion.
On-bead digest protocol
On-bead digest protocol
1d 1h 10m
1d 1h 10m
Prepare all reagents and materials as specified in Materials.
30m
Remove remaining wash buffer from beads. Add 150 µL ABC buffer, gently mix and transfer the beads to a 0.2 mL PCR tube and discard supernatant.
NOTE: The volumes specified in the following steps work depend on initial bead volumes and thus might have to be adjusted accordingly. The volumes specified in this protocol work for up to 30 µL GFP-trap magnetic agarose or 200 µL of streptaviding magnetic beads (see Materials).
5m
Add 50 µL of Digestion buffer.
5m
Add 2 µL DTT stock solution to reach a final concentration of 10 mM. Incubate for 30 min at room temperature.
35m
Add 2 µL IAA stock solution to reach a final concentration of 20 mM. Incubate for 30 min at room temperature in the dark.
35m
Quench remaining reactive IAA by adding 1 µL DTT stock solution (half the amount used in step 8) and incubate for 10 min at room temperature in the dark.
15m
Add 1.5 µL (150 ng) Lys-C solution and digest over night at 25°C in the dark.
18h
Transfer supernatant to a new PCR tube. Add 1.5 µL (150 ng) Trypsin solution and digest at 37°C for 5h.
5h
Acidify sample by adding 3 µL 10% TFA to reach a final concentration of approximately 0.5%.
5m
Proceed to desalting using according protocols (e.g. C18 StageTips or similar).