This repository holds the methods we use for automated magnetic bead based protein desalting, digestion, global phosphopeptide enrichment and tyrosine phosphopeptide enrichment.
We have discovered improvements and additional insights for our rapid robotic protocols and will likely discover more over time.
We will post updates to our protocols here. Please feel free to reach out with questions.
Schematic for methods contained in this repository: 
This is our automated protein desalting and digestion protocol based on the SP3 methodology from Hughes et al., 2019. Breifly, protein extracts (e.g. cell lysates) are aggregated onto carboxylated magnetic beads. Proteins are washed, digested and eluted from beads. You can directly measure total proteome from the output of this method, or proceed to phosphopeptide enrichments.
Significant updates:
- beads are mixed with organic prior to addition to protein lysate
- we have a tested overnight procedure which minimizes evaporation
KingFisher method files to download*:
- basic protocol: 5 hr, 250 ug protein input
- more protein input: 5hr, 1 mg protein input
- overnight digest, 250 ug protein input Deep well plates and holding pins at meniscus minimizes evaporation. Adjust cycles of digest step to modulate total duration.
*premix in method name refers to mixing beads with ethanol then into protein in the same plate before starting KingFisher. The first method iteration had beads in a separate plate to be added by robot to protein + ethanol mix, but we found this dampened yield.
R2-P2 is the 96-well automated protocol for global phosphopeptide enrichment.
Significant updates:
- larger peptide binding volume to mitigate peptide binding intereferences from residual salts.
- larger elution volume to increase robustness (note: acidification volume must be scaled up accordingly, e.g. 60 uL acidification buffer for 100 uL elution)
- added troubleshooting section to word doc to supplement excellent tips in original publication for common issues.
- One new tip is to start resuspension of dried peptides in just 0.1% trifluoroacetic in water, then add acetonitrile and remaining TFA. Delayed addition of organic improves resuspension success.
- We still highly recommend the optional pass through a c8 filter prior to MS measurement to avoid carryover of magentic beads.
KingFisher method files to download:
Phosphotyrosine peptide enrichment is two step procedure where pY peptides are first enriched, then desalted and further purified simultaneously with R2P2. The step 1 method uses NHS-conjugated Src SH2 superbinder beads or Cell Signaling antibody magnetic beads. The step 1 method uses CUBE Bio iron(III) magnetic beads.
Significant updates:
- pending; none yet
KingFisher method files to download:
Judit Villen, jvillen@uw.edu
biorender.com for help with schematic