TITLE: K-ε-GG Peptide Enrichment and Analysis by Tandem Mass Tagging-based proteomics
AUTHORS: Harper JW, Alban Ordureau

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details K-ε-GG peptide enrichment and analysis by tandem mass tagging-based proteomics.</div></div>

[STEPS]
?. [Harvest, precipitation and digestion]
Lyse cells in  of lysis buffer and pass through a 21G needle 10 times.
3 mL
?. [Harvest, precipitation and digestion]
Centrifuge suspensions at  (high speed) for  at  and collect supernatant.
Centrifuge: 13000 33
4 °C
?. [Harvest, precipitation and digestion]
Quantify protein lysate concentration and transfer  of lysates to a clean tube.
1 mg
?. [Harvest, precipitation and digestion]
Reduce lysates for  at  with  TCEP.
4 Room temperature
?. [Harvest, precipitation and digestion]
Alkylate cysteine residues with  Chloroacetamide (, ).
4 Room temperature
?. [Harvest, precipitation and digestion]
Extract protein content by methanol-chloroform precipitation and subsequent MetOH washes.
?. [Harvest, precipitation and digestion]
Add 4x volumes of MeOH and vortex.
?. [Harvest, precipitation and digestion]
Dry protein pellet down to get rid of MetOH traces.
?. [Harvest, precipitation and digestion]
Spin down at  for  at high speed.
4 Room temperature
?. [Harvest, precipitation and digestion]
Add 4x volumes of MeOH and vortex.
?. [Harvest, precipitation and digestion]
Remove both the aqueous and organic layers carefully, discard.
?. [Harvest, precipitation and digestion]
Spin down at  for  at high speed.
4 Room temperature
?. [Harvest, precipitation and digestion]
Add 3x volume of water and vortex.
?. [Harvest, precipitation and digestion]
Add 1x volume of chloroform and vortex.
?. [Harvest, precipitation and digestion]
Resuspend protein pellets in  urea,  EPPS () buffer.
?. [Harvest, precipitation and digestion]
Dilute samples to  urea with  EPPS () and digest at  for  with endoproteinase Lys-C (Wako, Japan) at a 1/200 enzyme/protein ratio.
30 °C
?. [Harvest, precipitation and digestion]
Dilute samples to  urea with  EPPS ().
?. [Harvest, precipitation and digestion]
Digest with Trypsin (1:100) o/n at .
37 °C
?. [Harvest, precipitation and digestion]
Stop digestion by acidification with formic acid (FA) 5% (v/v) (pH ~ 2).
?. [Harvest, precipitation and digestion]
Subject peptides to C18 SepPak solid-phase extraction cartridges (SPE Waters) and dry down.
?. [Harvest, precipitation and digestion]
Resuspend the desalted peptides in  IAP buffer.
1.3 mL
?. [Capture of K-ε-GG containing peptides with a-K-ε-GG Antibody]
Add the resin to a  Eppendorf tube.
One IP per sample.For one IP:   of a-K-ε-GG Antibody per   slurry (Pierce™ Protein A Plus UltraLink™ Resin, Cat. No. 53142) - see below for coupling of antibody to resin.
15 mL
?. [Capture of K-ε-GG containing peptides with a-K-ε-GG Antibody]
Wash 3x with PBS and centrifuge 1’ at .
Centrifuge: 1000 34
?. [Capture of K-ε-GG containing peptides with a-K-ε-GG Antibody]
Add the a-K-ε-GG Antibody and add enough PBS to have a total volume of  in  tube.
10 mL
15 mL
?. [Capture of K-ε-GG containing peptides with a-K-ε-GG Antibody]
Incubate O/N at  with gentle rotation.
4 °C
?. [Chemical cross-linking of K-ε-GG-specific antibody to resin]
Wash the anti-K-ε-GG Antibody coupled beads 3x with  sodium borate, pH 9.0.
3 mL
?. [Chemical cross-linking of K-ε-GG-specific antibody to resin]
Resuspend the beads in  of  DMP in  sodium borate (pH 9.0) and incubate at for  with gentle end-over-end rotator.
3 mL
4 Room temperature
?. [Chemical cross-linking of K-ε-GG-specific antibody to resin]
Stop the reaction by washing the beads 2x with  of antibody blocking buffer ( ethanolamine pH 8.0).
3 mL
?. [Chemical cross-linking of K-ε-GG-specific antibody to resin]
Resuspend in  of Antibody blocking buffer and incubate the Ab for  at  with gentle rotation.
3 mL
4 °C
?. [Chemical cross-linking of K-ε-GG-specific antibody to resin]
Wash the cross-linked antibody 3x with  IAP buffer.
3 mL
?. [Immunoprecipitation]
Add each sample to a clean  Eppendorf tube containing the cross-linked anti-K-ε-GG Antibody ( slurry of resin).
2 mL
40 µl
?. [Immunoprecipitation]
Incubate the IPs for  at  with gentle end-over-end rotation.
4 °C
?. [Immunoprecipitation]
Centrifuge each IP at  for  and remove the supernatant. Store supernatant at .
Centrifuge: 2000 34
-80 °C
?. [Immunoprecipitation]
Wash the beads 3x with  of IAP buffer followed with a wash with  PBS.
2 mL
2 mL
?. [Immunoprecipitation]
To elute K-ε-GG peptides add  of elution solution (0.15% TFA), gently tap the bottom of the tube several times and let the tube stand at for .
75 µl
-80 Room temperature
?. [Immunoprecipitation]
Repeat elution step and combine both eluates.
?. [Immunoprecipitation]
Dry down in speedvac and proceed to stage-tip.
?. [Stage TiP]
Resuspend samples in 5% FA, 5% ACN.
?. [Stage TiP]
Perform C-18 cleanup:
?. [Stage TiP]
a. Wash C-18 with  100% ACN.
1 mL
?. [Stage TiP]
g. Repeat step f.
?. [Stage TiP]
h. Elute with 2 x  75% ACN/1% FA.
500 µl
?. [Stage TiP]
f. Wash with  of 1% FA/5% ACN.
3 mL
?. [Stage TiP]
e. Collect flow through and freeze.
?. [Stage TiP]
d. Load sample (1 drop per second).
?. [Stage TiP]
c. Repeat step b.
?. [Stage TiP]
b. Equilibrate with  of 1% FA.
3 mL
?. [Stage TiP]
Dry down in speedvac.
?. [Stage TiP]
Proceed to labeling.
?. [Labeling]
Resuspend the peptide pellet in  of  EPPS (pH 8.2) containing 20% ACN.
50 µl
?. [Labeling]
Add - of the TMT reagent to each sample.
3 µl
4 µl
?. [Labeling]
Incubate for  at .
-80 Room temperature
?. [Labeling]
Stop the reaction with  of hydroxylamine 5%.
4 µl
?. [Labeling]
Combine samples, acidify (5% FA) and speed-vac to dryness (gel like consistency).
?. [Stage TiP]
Resuspend samples in 5% FA, 5% ACN.
?. [Stage TiP]
Perform C-18 cleanup:
?. [Stage TiP]
a. Wash C-18 with  100% ACN.
1 mL
?. [Stage TiP]
b. Equilibrate with  of 1% FA.
3 mL
?. [Stage TiP]
h. Elute with 2 x  75% ACN/1 % FA.
500 µl
?. [Stage TiP]
g. Repeat step f.
?. [Stage TiP]
f. Wash with  of 1% FA/5% ACN.
3 mL
?. [Stage TiP]
e. Collect flow through and freeze.
?. [Stage TiP]
d. Load sample (1 drop per second).
?. [Stage TiP]
c. Repeat step b.
?. [Stage TiP]
Dry down in speedvac.
?. [Stage TiP]
Proceed to B-pH RP fractionation.
?. [Basic-pH RP peptide fractionation kit (follow manufacturer's instructions)]
Follow manufacturer’s instructions (Thermo Cat# 84868).
Elution used: 17.5% ACN, 20% ACN, 22.5% ACN, 25% ACN, 27.5% ACN and 70% ACN.
?. [Basic-pH RP peptide fractionation kit (follow manufacturer's instructions)]
Speed vac individual samples to dryness.
?. [Basic-pH RP peptide fractionation kit (follow manufacturer's instructions)]
Proceed to stage-tip.
?. [Stage TiP]
Resuspend samples in 5% FA, 5% ACN.
?. [Stage TiP]
Perform six C18-based stage-tips (one per fraction).
?. [Stage TiP]
a. Wash C-18 with  100% ACN. Centrifuge at  for  at , discard flowthrough.
50 µl
Centrifuge: 2000 34
-80 Room temperature
?. [Stage TiP]
h. Elute with 1 x  75% ACN/% FA, in mass-spec vial. Centrifuge at  for  at .
50 µl
Centrifuge: 2000 34
-80 Room temperature
?. [Stage TiP]
g. Repeat step f.
?. [Stage TiP]
f. Wash with  of 1% FA/5% ACN. Centrifuge at  for  at , discard flowthrough.
50 µl
Centrifuge: 2000 34
-80 Room temperature
?. [Stage TiP]
e. Collect flow through and freeze.
?. [Stage TiP]
d. Load sample. Centrifuge at  for  at .
Centrifuge: 1500 34
-80 Room temperature
?. [Stage TiP]
c. Repeat step b.
?. [Stage TiP]
b. Equilibrate with  of 1% FA. Centrifuge at  for  at , discard flowthrough.
50 µl
Centrifuge: 2000 34
-80 Room temperature
?. [Stage TiP]
Dry down in speedvac.
?. [Stage TiP]
Resuspend in  5% FA, 5% ACN.
10 µl
?. [Mass spectrometry]
Inject  for each LC–MS/MS analysis using available mass spectrometer with a 120-minute online LC separation.
The analysis of K-ε-GG peptides by mass spectrometry will depend on the type of instrument/platform used. Typical instrument settings for analysis on a Thermo Fusion Lumos instrument are provided in the following section.
3 µl
?. [Mass spectrometry]
Search raw data against UniProt human protein database using any proteomic analysis software with the followingparameters: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.
?. [Mass spectrometry]
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).
?. [Instrument settings]
Seperate peptides on a  inner diameter microcapillary column packed in house with ~ of Accucore150 resin ( , 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  run at ~.
35
400
?. [Instrument settings]
For analysis, load 1/3 of each fraction onto the column.
Each analysis used the Multi-Notch MS3-based TMT method (McAlister et al., 2014). The scan sequence began with an MS1 spectrum (Orbitrap analysis; resolution 120,000 at 200 Th; mass range 400-1250 m/z; automatic gain control (AGC) target 1×106; maximum injection time 100 ms).
?. [Instrument settings]
Select precursors for MS2 analysis using a Top 4 sec method.
MS2 analysis consisted of collision-induced dissociation (quadrupole Orbitrap analysis; AGC 1×105; isolation window 0.7 Th; normalized collision energy (NCE) 35; maximum injection time 300 ms resolution was 7,500 at 200 Th)
?. [Instrument settings]
Use monoisotopic peak assignment, and exclude previously interrogated precursors using a dynamic window (120 s ± 7 ppm).
?. [Instrument settings]
As described previously, select only precursors with a charge state between 3 and 6 for downstream analysis (Rose et al., 2016).
?. [Instrument settings]
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).
?. [Instrument settings]
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).