TITLE: TMTpro HUNTER N-terminomics
AUTHORS: Edward Emmott

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for N-terminomic analysis of protease substrates. This method is an adaption of the Weng et al. 2019 Mol. Cell. Proteomics (</span><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823850" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823850/</span></a><span>) method for N-terminomics, modified to allow sample multiplexing and quantification using TMTpro reagents rather than the original implementation from the Lange lab which used dimethyl or DIA-based quantification. This method was used in Meyer et al. 2020 - </span><a href="https://www.biorxiv.org/content/10.1101/2020.09.16.297945v1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.biorxiv.org/content/10.1101/2020.09.16.297945v1</span></a><span style = ":;">.</span></div><div class = "text-block">If you use this protocol, please cite both the Meyer et al. and Weng et al. publications.</div><div class = "text-block">This adaption has advantages for the analysis of protease substrates, permitting sample multiplexing early in the protocol and allowing all samples to be processed subsequently as a single sample. This protocol has disadvantages compared to its parental protocol for analysis of native N-termini where blocking of the native N-terminus by acetylation or pyroglutamine prevents TMT labelling, and thus quantification of non-lysine-containing peptides. As neo-N-termini generated by viral proteases are not expected to possess natively-blocked N-termini (aside from a possible minority of pyroglutamine N-termini), these are not a consideration here.</div><div class = "text-block">In brief:</div><div class = "text-block">1. Cells are lysed in SDS and heated</div><div class = "text-block">2. Samples are reduced and alkylated.</div><div class = "text-block">3. Protein is precipitated on SP3 beads</div><div class = "text-block"><span>4. Protein is resolubilised and TMTpro labelling is performed at the </span><span style = "font-weight:bold;">protein level </span><span>to block N-termini and lysine residues</span></div><div class = "text-block">5. Unreacted TMTpro reagent is quenched and washed away.</div><div class = "text-block">6. Samples are pooled.</div><div class = "text-block">7. Overnight Tryptic digestion.</div><div class = "text-block">8. Unblocked N-termini generated by tryptic digestion are hydrophlabelled with undecannal.</div><div class = "text-block">9. Undecannal-labelled N-termini are depleted by passing the peptides over C18 resin in 50% ethanol, resulting in flow-through containing natively-blocked and TMTpro-labelled N-termini.</div><div class = "text-block">This protocol can be accomplished in aproximately two days.</div></div>

[STEPS]
?. [Day 2 - Sample cleanup]
Cell pellets resuspended in 200uL lysis buffer consisting of:- 1% SDS- 2x Thermo HALT protease inhibitor- 100mM HEPES, pH8- 1% NP40
?. [Day 1]
One T25 dish of ACE2-A549 cells/sample was collected by centrifugation, washed 3x with PBS, and the pellet frozen in a 5mL low-bind Eppendorf for use the next day.
?. [Day 2 - Sample cleanup]
A protein assay should be performed e.g. BCA. Normalise sample volumes to 110uL, containing 25ug of material
?. [Day 2 - Sample cleanup]
Sample heated to 95C for 5 minutes
?. [Day 2 - Sample cleanup]
Sample chilled on ice for 5 minutes
?. [Day 2 - Sample cleanup]
Sample briefly centrifuged to collect condensation
?. [Day 2 - Sample cleanup]
Benzonase added at 1 in 200 dilution and incubated at 37C for 30 minutes
?. [Day 2 - Sample cleanup]
2uL of 1M DTT added and incubated at 37C for 30 minutesNote: DTT should be made up fresh from stock
?. [Day 2 - Sample cleanup]
11.2uL of 0.5M 2-chloroacetamide added, incubated at RT in the dark for 30 minutes.Note: 2-CAA should be made up fresh from stock
?. [Day 2 - Sample cleanup]
6uL of 1M DTT added to quench CAA, incubated at RT in the dark for 20 minutes.
?. [Day 2 - Sample cleanup]
2.5uL of previously prepared SP3 beads was added to each low-bind tube.Note 1:10 protein:bead ratio with beads at 20ug/uL
?. [Day 2 - Sample cleanup]
Check volume: If you have been following the instructions above, each sample should be 141uL volume. If not, adjust volume with HPLC-grade water
?. [Day 2 - Sample cleanup]
Add 564uL 100% ethanol to initiate binding, incubate for 18 minutes at RT.
?. [Day 2 - Sample cleanup]
Incubate on magnetic stand for 5 minutes.
?. [Day 2 - Sample cleanup]
Wash beads twice with 400uL 90% ethanol.Note: do not disturb beads
?. [Day 2 - Sample cleanup]
Briefly centrifuge beads and pipette off any remaining liquid
?. [Day 2 - Sample Labelling]
Resuspend beads in:22.5uL 6M GuCL30uL 0.5M HEPES pH84.5uL TCEP to 10mM final (diluted 1 in 3.5 from 500mM stock)
?. [Day 2 - Sample Labelling]
Incubate beads at room temperature for 30 minutesDuring this incubation, prepare the TMT in the following two steps
?. [Day 2 - Sample Labelling]
Dissolve TMTpro labels in 62uL of anhydrous DMSO, mix well. The TMTpro can take a while to dissolveNote - seriously, use the GOOD anyhydrous DMSO. TMT aint cheap!
?. [Day 2 - Sample Labelling]
Add the 57uL TMTpro/DMSO to each 57uL sample. Mix well by pipetting. Note - it is good practice to randomise the allocation of TMT/TMTpro labels to samples.
?. [Day 2 - Sample Labelling]
Remove the TMTpro from the -80C, and allow it to equilibrate to room temperature before opening
?. [Day 2 - Sample Labelling]
Incubate at RT in the dark for 1.5h.
?. [Day 2 - Sample Labelling]
Add 13uL of 1M ethanolamide, mix well and incubate for 45 min to quench unreacted TMT labels.
?. [Day 2 - Sample Labelling]
Combine samples into a single tube. For a full 16plex TMTpro experiment this will give a volume of 16 * 133uL = 2128uL, containing ~400ug total protein
?. [Day 2 - Post-label cleanup]
To ease sample handling. Divide sample into 6 * 354uL aliquots in 2mL tubes
?. [Day 2 - Post-label cleanup]
Add 13uL of SP3 beads (20ug/ul) per aliquot
?. [Day 2 - Post-label cleanup]
1468uL 100% ethanol added
?. [Day 2 - Post-label cleanup]
Incubate beads at RT off the magnetic stand for 15 minutes.
?. [Day 2 - Post-label cleanup]
Incubate beads on stand for 5 minutes (or until clear)
?. [Day 2 - Sample cleanup]
Beads should have collected at the side of the tube, and the supernatent should be clear. Remove supernatent
?. [Day 2 - Post-label cleanup]
Remove supernatant.
?. [Day 2 - Post-label cleanup]
Wash beads twice 600uL 90% ethanol.Note: do not disturb beads
?. [Day 2 - Post-label cleanup]
Briefly centrifuge beads and pipette off any remaining liquid
?. [Day 2 - Overnight digest]
Recombine the Six aliquots by resuspending in 400uLof 200mM HEPES, pH 8
?. [Day 2 - Overnight digest]
Add 20ug Trypsin Gold in 10uL of 200mM HEPES pH8.
?. [Day 2 - Overnight digest]
Incubate sample overnight at 37C. Note - minimum 13h
?. [Day 3 - Sample cleanup]
Place sample onto magnetic rack for 5 minutes.
?. [Day 3 - Sample cleanup]
Retain 10% of the sample (40uL) to assess protein labelling and protein abundance (unenriched sample)
?. [Day 3 - Sample cleanup]
Remove sample from magnetic rack.
?. [Day 3 - Sample cleanup]
Add 240uL 100% ethanol resuspending the beads Note: should take to 40% ethanol
?. [Day 3 - Sample cleanup]
Tap the tube and sonicate for 30s to mix.
?. [Day 3 - Sample cleanup]
Add 18uL undecanalNote: this is ~97% undecanal, and assumes the protocol has 400ug total protein (-10%) in the sample
?. [Day 3 - Sample cleanup]
Tap the tube and sonicate for 30s to mix.
?. [Day 3 - Sample cleanup]
Add 18.5uL of 1M sodium cyanoborohydrideNote: Gives 30mM finalNote: make 1M sodium cyanoborohydride fresh
?. [Day 3 - Sample cleanup]
Tap the tube and sonicate for 30s to mix.
?. [Day 3 - Sample cleanup]
Confirm pH7-8 by spotting 1uL of sample onto pH paper
?. [Day 3 - Sample cleanup]
Incubate at 37C for 1h.
?. [Day 3 - Sample cleanup]
Sonicate in waterbath for 15s to mix
?. [Day 3 - Sample cleanup]
Bind tube to magnetic stand foor 1 minute.
?. [Day 3 - Sample cleanup]
Transfer the supernatent to a fresh tube
?. [Day 3 - enrichment]
Acidify supernatant with 5% TFA in 40% ethanol to pH3-4.
?. [Day 3 - enrichment]
Adjust final volume to 1600uL with 1% TFA in 40% ethanol. Set tube to one side.
?. [Day 3 - enrichment]
Confirm pH3-4 by spotting 1uL onto filter paper
?. [Day 3 - enrichment]
Add 400uL methanol to each of 4 macrospin columns to condition them. Centrifuge 2m, 300g
?. [Day 3 - enrichment]
Add 400uL 0.1% TFA in 40% ethanol to each of the 4 macrospin columns. Centrifuge 2m, 300g
?. [Day 3 - enrichment]
Repeat previous step
?. [Day 3 - enrichment]
Place each macrospin column in a fresh 2mL collection tube
?. [Day 3 - enrichment]
Load 400uL of the acidified sample to each of the 4 macrospin columns. Centrifuge 2m, 300g.
?. [Day 3 - enrichment]
Discard the macrospin column, retain the flow-through liquid in the 2mL collection tube. This contains your N-terminally enriched peptides.
?. [Day 3 - post-enrichment cleanup]
Dry sample on speed-vac, resuspend in 0.1% TFA.
?. [Day 3 - post-enrichment cleanup]
Sample should be desalted by standard methods (e.g. macrospin column). N-terminal identifications can be improved by further sample fractionation after desalting, for example basic reverse phase.