Apr 23, 2025
Version 2
On bead digest prior to mass spec V.2
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2025. On bead digest prior to mass spec. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrkdy3lmk/v2Version created by Elias Adriaenssens
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
Created: April 09, 2025
Last Modified: April 23, 2025
Protocol Integer ID: 141122
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol was generated from an internal electronic lab notebook and contains experiment specific notes, for example, sample-specific volumes and incubation times. Due to the automatic generation process it might also contain errors and should be interpreted cautiously. An updated protocols will still be uploaded.
Materials
- urea
- ammonium bicarbonate
- LysC (mass spectrometry grade, FUJIFILM Wako chemicals)
- trypsin (Trypsin Gold, Promega)
- dithiothreitol
- iodoacetamide
Safety warnings
This protocol was generated from an internal electronic lab notebook and contains experiment specific notes, for example, sample-specific volumes and incubation times. Due to the automatic generation process it might also contain errors and should be interpreted cautiously. An updated protocols will still be uploaded.
On bead digest 1M urea LysC/Trypsin 90 min prior to Red/Alk
On bead digest 1M urea LysC/Trypsin 90 min prior to Red/Alk
2h 49m
2h 49m
Add 50 µL of ABC and transfer beads to new 0.2 mL PCR tube and remove buffer Axygen.
Add appropriate volume of 2 Molarity (M) urea 50 millimolar (mM) ABC. Here we added 30 µL .
Add 150 ng LysC/Trypsin (1:1 Mix) and incubate 01:30:00 at Room temperature . Here we added1.5 µL .
1h 30m
Transfer supernatant to new 0.2 µL PCR tube. Here, the total volume becomes:31.5 µL .
Rinse beads with 30 µL 1 Molarity (M) urea 50 millimolar (mM) ABC, combine* 30 µL , sum:61.5 µL .
Note
Optional: 2nd transfer to get rid of all beads.
Reduction: 10 millimolar (mM) DTT (aliquot 0.25 Molarity (M) ) 00:30:00 Room temperature . Here we added 2.4 µL .
30m
Alkylation: 20 Molarity (M) IAA (500mM stock = 0.092 µL ) 00:30:00 Room temperature in the dark. Here, we added2.4 µL .
30m
Quench IAA with half amount of DTT used in step 3 and incubate for 00:10:00 at Room temperature . We added 1.2 µL .
10m
Dilute to 1 Molarity (M) urea with 50 millimolar (mM) ABC. We added 30 µL , which gave rise to a total sum of:97.5 µL .
Add 150 ng LysC/Trypsin (1:1 Mix) and incubate at 37 °C Overnight . For us here, this meant 1.5 µL .
Acidify sample to final concentration of 0.5% TFA (from 10%). For us this was 4.5 µL .
Desalt: Load the peptides on a C18 STAGEtip.
Note
3-5 plugs depending on protein amount, 3 plugs for max. 20µg.
Equilibrate:
100 µL 100% MeOH, spin 2000 rpm, 00:03:00 .
3m
100 µL 80% ACN, 0.1% TFA, spin 2000 rpm, 00:03:00 .
3m
100 µL 0.1% TFA, spin 2000 rpm, 00:03:00 .
3m
Load sample: spin 1700 rpm .
Wash: 100 µL 0.1% TFA -> keep flow through (FT) and wash together.
Elute: 2x30 µL 60% ACN, 0.1% TFA in PCR tube.
Speedvac to remove ACN and take up sample in 20 µL 0.1% TFA 2% ACN.
Protocol references
https://doi.org/10.1038/nprot.2007.261