Sep 18, 2020
TMTpro HUNTER N-terminomics
This protocol is a draft, published without a DOI.
- Edward Emmott1
- 1University of Liverpool
- Emmott Lab
External link: http://emmottlab.org
Protocol Citation: Edward Emmott 2020. TMTpro HUNTER N-terminomics. protocols.io https://protocols.io/view/tmtpro-hunter-n-terminomics-bi44kgyw
Manuscript citation:
https://www.biorxiv.org/content/10.1101/2020.09.16.297945v1
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 works for us (please tell us if bits are unclear)
Created: July 29, 2020
Last Modified: September 18, 2020
Protocol Integer ID: 39804
Keywords: Proteomics, Mass Spectrometry, N-terminomics, Protease,
Disclaimer
It works for me (TM).
Abstract
Protocol for N-terminomic analysis of protease substrates. This method is an adaption of the Weng et al. 2019 Mol. Cell. Proteomics (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823850/) 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 - https://www.biorxiv.org/content/10.1101/2020.09.16.297945v1.
If you use this protocol, please cite both the Meyer et al. and Weng et al. publications.
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.
In brief:
1. Cells are lysed in SDS and heated
2. Samples are reduced and alkylated.
3. Protein is precipitated on SP3 beads
4. Protein is resolubilised and TMTpro labelling is performed at the protein level to block N-termini and lysine residues
5. Unreacted TMTpro reagent is quenched and washed away.
6. Samples are pooled.
7. Overnight Tryptic digestion.
8. Unblocked N-termini generated by tryptic digestion are hydrophlabelled with undecannal.
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.
This protocol can be accomplished in aproximately two days.
Day 1
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
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
Sample heated to 95C for 5 minutes
5m
Sample chilled on ice for 5 minutes
5m
Sample briefly centrifuged to collect condensation
Benzonase added at 1 in 200 dilution and incubated at 37C for 30 minutes
30m
A protein assay should be performed e.g. BCA. Normalise sample volumes to 110uL, containing 25ug of material
2uL of 1M DTT added and incubated at 37C for 30 minutes
Note: DTT should be made up fresh from stock
30m
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
30m
6uL of 1M DTT added to quench CAA, incubated at RT in the dark for 20 minutes.
20m
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
Check volume: If you have been following the instructions above, each sample should be 141uL volume. If not, adjust volume with HPLC-grade water
Add 564uL 100% ethanol to initiate binding, incubate for 18 minutes at RT.
18m
Incubate on magnetic stand for 5 minutes.
5m
Beads should have collected at the side of the tube, and the supernatent should be clear. Remove supernatent
Wash beads twice with 400uL 90% ethanol.
Note: do not disturb beads
Briefly centrifuge beads and pipette off any remaining liquid
Day 2 - Sample Labelling
Day 2 - Sample Labelling
Resuspend beads in:
22.5uL 6M GuCL
30uL 0.5M HEPES pH8
4.5uL TCEP to 10mM final (diluted 1 in 3.5 from 500mM stock)
Incubate beads at room temperature for 30 minutes
During this incubation, prepare the TMT in the following two steps
Remove the TMTpro from the -80C, and allow it to equilibrate to room temperature before opening
10m
Dissolve TMTpro labels in 62uL of anhydrous DMSO, mix well. The TMTpro can take a while to dissolve
Note - seriously, use the GOOD anyhydrous DMSO. TMT aint cheap!
40m
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.
5m
Incubate at RT in the dark for 1.5h.
1h 30m
Add 13uL of 1M ethanolamide, mix well and incubate for 45 min to quench unreacted TMT labels.
45m
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
Day 2 - Post-label cleanup
To ease sample handling. Divide sample into 6 * 354uL aliquots in 2mL tubes
Add 13uL of SP3 beads (20ug/ul) per aliquot
1468uL 100% ethanol added
Incubate beads at RT off the magnetic stand for 15 minutes.
15m
Incubate beads on stand for 5 minutes (or until clear)
5m
Remove supernatant.
Wash beads twice 600uL 90% ethanol.
Note: do not disturb beads
Briefly centrifuge beads and pipette off any remaining liquid
Day 2 - Overnight digest
Day 2 - Overnight digest
13h
13h
Recombine the Six aliquots by resuspending in 400uLof 200mM HEPES, pH 8
Add 20ug Trypsin Gold in 10uL of 200mM HEPES pH8.
Incubate sample overnight at 37C.
Note - minimum 13h
13h
Day 3 - Sample cleanup
Day 3 - Sample cleanup
1h 6m 45s
1h 6m 45s
Place sample onto magnetic rack for 5 minutes.
5m
Retain 10% of the sample (40uL) to assess protein labelling and protein abundance (unenriched sample)
Remove sample from magnetic rack.
Add 240uL 100% ethanol resuspending the beads
Note: should take to 40% ethanol
Tap the tube and sonicate for 30s to mix.
30s
Add 18uL undecanal
Note: this is ~97% undecanal, and assumes the protocol has 400ug total protein (-10%) in the sample
Tap the tube and sonicate for 30s to mix.
30s
Add 18.5uL of 1M sodium cyanoborohydride
Note: Gives 30mM final
Note: make 1M sodium cyanoborohydride fresh
Tap the tube and sonicate for 30s to mix.
30s
Confirm pH7-8 by spotting 1uL of sample onto pH paper
Incubate at 37C for 1h.
1h
Sonicate in waterbath for 15s to mix
15s
Bind tube to magnetic stand foor 1 minute.
Transfer the supernatent to a fresh tube
Day 3 - enrichment
Day 3 - enrichment
6m
6m
Acidify supernatant with 5% TFA in 40% ethanol to pH3-4.
Confirm pH3-4 by spotting 1uL onto filter paper
Adjust final volume to 1600uL with 1% TFA in 40% ethanol. Set tube to one side.
Add 400uL methanol to each of 4 macrospin columns to condition them. Centrifuge 2m, 300g
2m
Add 400uL 0.1% TFA in 40% ethanol to each of the 4 macrospin columns. Centrifuge 2m, 300g
2m
Repeat previous step
2m
Place each macrospin column in a fresh 2mL collection tube
Load 400uL of the acidified sample to each of the 4 macrospin columns. Centrifuge 2m, 300g.
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
Day 3 - post-enrichment cleanup
Dry sample on speed-vac, resuspend in 0.1% TFA.
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.