TITLE: Acetylation of lysines on affinity-purification matrices to reduce co-digestion of bead-bound ligands
AUTHORS: David M. Hollenstein, Margarita Maurer, Thomas Gossenreiter, Natascha Hartl, Dorothea Anrather, Markus  Hartl

[DESCRIPTION]
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). 


[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. 


[GUIDELINES]
This protocol has been successfully tested for: 
   and 
  
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.


[STEPS]
SECTION: Acetylation protocol
1. Wash beads 3x with 100µl Reaction buffer, using the magnetic rack. 
Remove supernatant after the final wash.
SECTION: Acetylation protocol
2. 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.
SECTION: Acetylation protocol
3. Discard supernatant and wash 3x with 100 µl Quenching buffer.
SECTION: Acetylation protocol
4. Use directly or store in 100 µL Storage buffer at 4°C for later use. 
SECTION: On-bead digest protocol
7. 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).
SECTION: Affinity enrichment protocol
5. 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.
SECTION: On-bead digest protocol
8. 
Add 50 µL of Digestion buffer. 
SECTION: On-bead digest protocol
9. Add 2 µL DTT stock solution to reach a final concentration of 10 mM. Incubate for 30 min at room temperature.
SECTION: On-bead digest protocol
10. Add 2 µL IAA stock solution to reach a final concentration of 20 mM. Incubate for 30 min at room temperature in the dark.
SECTION: On-bead digest protocol
11. 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. 
SECTION: On-bead digest protocol
12. Add 1.5 µL (150 ng) Lys-C solution and digest over night at 25°C in the dark.
SECTION: On-bead digest protocol
13. Transfer supernatant to a new PCR tube. Add 1.5 µL (150 ng) Trypsin solution and digest at 37°C for 5h.
SECTION: On-bead digest protocol
14. Acidify sample by adding 3 µL 10% TFA to reach a final concentration of approximately 0.5%. 
SECTION: On-bead digest protocol
15. Proceed to desalting using according protocols (e.g. C18 StageTips or similar). 
SECTION: On-bead digest protocol
6. Prepare all reagents and materials as specified in Materials.