K-ε-GG Peptide Enrichment and Analysis by Tandem Mass Tagging-based proteomics
Harper JW, Harper JW
Abstract
This protocol details K-ε-GG peptide enrichment and analysis by tandem mass tagging-based proteomics.
Attachments
Steps
Harvest, precipitation and digestion
Lyse cells in 3mL of lysis buffer and pass through a 21G needle 10 times.
Centrifuge suspensions at 13000rpm,0h 0m 0s (high speed) for 0h 10m 0s at 4°C and collect supernatant.
Quantify protein lysate concentration and transfer 1mg of lysates to a clean tube.
Reduce lysates for 0h 20m 0s at 4Room temperature with 5millimolar (mM) TCEP.
Alkylate cysteine residues with 20millimolar (mM) Chloroacetamide (4Room temperature, 0h 30m 0s).
Extract protein content by methanol-chloroform precipitation and subsequent MetOH washes.
Add 4x volumes of MeOH and vortex.
Add 1x volume of chloroform and vortex.
Add 3x volume of water and vortex.
Spin down at 4Room temperature for 0h 5m 0s at high speed.
Remove both the aqueous and organic layers carefully, discard.
Add 4x volumes of MeOH and vortex.
Spin down at 4Room temperature for 0h 5m 0s at high speed.
Dry protein pellet down to get rid of MetOH traces.
Resuspend protein pellets in 8Molarity (M) urea, 50millimolar (mM) EPPS (8.5) buffer.
Dilute samples to 4Molarity (M) urea with 50millimolar (mM) EPPS (8.5) and digest at 30°C for 2h 0m 0s with endoproteinase Lys-C (Wako, Japan) at a 1/200 enzyme/protein ratio.
Dilute samples to 1Molarity (M) urea with 50millimolar (mM) EPPS (8.5).
Digest with Trypsin (1:100) o/n at 37°C.
Stop digestion by acidification with formic acid (FA) 5% (v/v) (pH ~ 2).
Subject peptides to C18 SepPak solid-phase extraction cartridges (SPE Waters) and dry down.
Resuspend the desalted peptides in 1.3mL IAP buffer.
Capture of K-ε-GG containing peptides with a-K-ε-GG Antibody
15mL Eppendorf tube. Wash 3x with PBS and centrifuge 1’ at 1000x g,0h 0m 0s.
Add the a-K-ε-GG Antibody and add enough PBS to have a total volume of 10mL in 15mL tube.
Incubate O/N at 4°C with gentle rotation.
Chemical cross-linking of K-ε-GG-specific antibody to resin
Wash the anti-K-ε-GG Antibody coupled beads 3x with 3mL 100millimolar (mM) sodium borate, pH 9.0.
Resuspend the beads in 3mL of 20millimolar (mM) DMP in 100millimolar (mM) sodium borate (pH 9.0) and incubate at 4Room temperaturefor 0h 30m 0s with gentle end-over-end rotator.
Stop the reaction by washing the beads 2x with 3mL of antibody blocking buffer (200millimolar (mM) ethanolamine pH 8.0).
Resuspend in 3mL of Antibody blocking buffer and incubate the Ab for 2h 0m 0s at 4°C with gentle rotation.
Wash the cross-linked antibody 3x with 3mL IAP buffer.
Immunoprecipitation
Add each sample to a clean 2mL Eppendorf tube containing the cross-linked anti-K-ε-GG Antibody (40µL slurry of resin).
Incubate the IPs for 2h 0m 0s at 4°C with gentle end-over-end rotation.
Centrifuge each IP at 2000x g,0h 0m 0s for 0h 2m 0s and remove the supernatant. Store supernatant at -80°C.
Wash the beads 3x with 2mL of IAP buffer followed with a wash with 2mL PBS.
To elute K-ε-GG peptides add 75µL of elution solution (0.15% TFA), gently tap the bottom of the tube several times and let the tube stand at -80Room temperaturefor 0h 5m 0s.
Repeat elution step and combine both eluates.
Dry down in speedvac and proceed to stage-tip.
Stage TiP
Resuspend samples in 5% FA, 5% ACN.
Perform C-18 cleanup:
a. Wash C-18 with 1mL 100% ACN.
b. Equilibrate with 3mL of 1% FA.
c. Repeat step b.
d. Load sample (1 drop per second).
e. Collect flow through and freeze.
f. Wash with 3mL of 1% FA/5% ACN.
g. Repeat step f.
h. Elute with 2 x 500µL 75% ACN/1% FA.
Dry down in speedvac.
Proceed to labeling.
Labeling
Resuspend the peptide pellet in 50µL of 200millimolar (mM) EPPS (pH 8.2) containing 20% ACN.
Add 3µL-4µL of the TMT reagent to each sample.
Incubate for 1h 0m 0s at -80Room temperature.
Stop the reaction with 4µL of hydroxylamine 5%.
Combine samples, acidify (5% FA) and speed-vac to dryness (gel like consistency).
Resuspend samples in 5% FA, 5% ACN.
Perform C-18 cleanup:
a. Wash C-18 with 1mL 100% ACN.
b. Equilibrate with 3mL of 1% FA.
c. Repeat step b.
d. Load sample (1 drop per second).
e. Collect flow through and freeze.
f. Wash with 3mL of 1% FA/5% ACN.
g. Repeat step f.
h. Elute with 2 x 500µL 75% ACN/1 % FA.
Dry down in speedvac.
Proceed to B-pH RP fractionation.
Basic-pH RP peptide fractionation kit (follow manufacturer's instructions)
Follow manufacturer’s instructions (Thermo Cat# 84868).
Speed vac individual samples to dryness.
Proceed to stage-tip.
Resuspend samples in 5% FA, 5% ACN.
Perform six C18-based stage-tips (one per fraction).
a. Wash C-18 with 50µL 100% ACN. Centrifuge at 2000x g,0h 0m 0s for 0h 2m 0s at -80Room temperature, discard flowthrough.
b. Equilibrate with 50µL of 1% FA. Centrifuge at 2000x g,0h 0m 0s for 0h 2m 0s at -80Room temperature, discard flowthrough.
c. Repeat step b.
d. Load sample. Centrifuge at 1500x g,0h 0m 0s for 0h 4m 0s at -80Room temperature.
e. Collect flow through and freeze.
f. Wash with 50µL of 1% FA/5% ACN. Centrifuge at 2000x g,0h 0m 0s for 0h 2m 0s at -80Room temperature, discard flowthrough.
g. Repeat step f.
h. Elute with 1 x 50µL 75% ACN/% FA, in mass-spec vial. Centrifuge at 2000x g,0h 0m 0s for 0h 2m 0s at -80Room temperature.
Dry down in speedvac.
Resuspend in 10µL 5% FA, 5% ACN.
Mass spectrometry
3µL for each LC–MS/MS analysis using available mass spectrometer with a 120-minute online LC separation. Search raw data against UniProt human protein database using any proteomic analysis software with the following
parameters:
- Up to 3 missed cleavages allowed for trypsin/LysC digestion.
- Carbamidomethyl (C), TMT (N-term peptide and K) set as a fixed modification.
- Oxidation (M) and di-glycine (K) set as variable modifications.
Extract signal to noise intensity values of each TMT reporter and identified proteins, and further calculate the ratio of each condition to the control sample’s intensity.
Instrument settings
Collect mass spectrometry data using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to a Proxeon EASY-nLC1200 liquid chromatography (LC) pump (Thermo Fisher Scientific).
Seperate peptides on a 100micromolar (µM) inner diameter microcapillary column packed in house with ~35cm of Accucore150 resin (2.6micromolar (µM) , 150 Å, ThermoFisher Scientific, San Jose, CA) with a gradient consisting of 3%–26% (0-100 min), 26-32% (100-110min) (ACN, 0.1% FA) over a total 2h 0m 0s run at ~400nL/min.
For analysis, load 1/3 of each fraction onto the column.
Select precursors for MS2 analysis using a Top 4 sec method.
Use monoisotopic peak assignment, and exclude previously interrogated precursors using a dynamic window (120 s ± 7 ppm).
As described previously, select only precursors with a charge state between 3 and 6 for downstream analysis (Rose et al., 2016).
Following acquisition of each MS2 spectrum, collect a synchronous-precursor-selection (SPS) MS3 scan on the top 10 most intense ions in the MS2 spectrum (McAlister et al., 2014).
Fragment MS3 precursors by high energy collision-induced dissociation (HCD) and analyze using the Orbitrap (NCE 65; AGC 2×105; maximum injection time 500 ms, resolution was 50,000 at 200 Th).
