TITLE: Tandem Mass Tag (TMT) 10-plex Labeling of Yeast Peptides
AUTHORS: Rebecca E. Hardman, Jeffrey Lewis

[STEPS]
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Thaw frozen cells (collected in 50-ml conical tubes) at room temperature, making sure not to leave cells thawed longer than necessary. Centrifuge at max speed (4,696 rcf) in a swinging bucket rotor for 2 minutes. Carefully remove remaining media by pipetting. Suspend in 1 ml water and transfer to 1.5-ml microfuge tube.
For any samples you need to split, add more water. For example, if one sample is 100 OD units, resuspend in 2 ml water and transfer 1 ml to each of two tubes to split the sample in half.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Centrifuge at 10,000 rcf for 1 min, then carefully remove the water by pipetting.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Repeat centrifugation at 10,000 rcf for 1 min, and again carefully remove the remaining water by pipetting.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Resuspend cells in 150 μl 6M  guanidine hydrochoride 100 mM Tris-HCl pH 8 solution.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Boil samples for 5 min in 100°C heat block (make sure the water has not evaporated). Rest samples for 5 min by removing the tubes from the heat block and placing in a rack at room temperature.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Boil sample again for 5 min.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Precipitate proteins by adding methanol to a final concentration of 90%. Briefly vortex.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Centrifuge samples at 9,000 rcf for 5 min. Decant the supernatant by carefully pouring and then then pipetting off as much residual liquid as possible.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Air dry pellet for 5 min in a Biosafety Cabinet.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Resuspend pellet in 1ml 8M urea - 100 mM Tris solution.
Peptide concentration should be
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Transfer samples to Oak Ridge tubes.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Dilute urea to 2M with 100 mM Tris-HCl pH 8.0.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Quantitate with Bradford Assay, no dilution of samples is necessary.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Add tris(2-carboxyethyl)phosphine (TCEP) to 2.5 mM and chloroacetamide to 10 mM.
Make 100x stock by dissolving 0.36g TCEP and 0.47g chloroacetamide in 5ml of 100mM Tris-HCl pH 8. Requires mild heat (37°C) to fully dissolve. Store at -20°C.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Add trypsin at a ratio of 1:50 (1 mg trypsin per 50 mg protein) and incubate overnight at 25°C on a rotating mixer.
Prepare trypsin stock fresh just before use at 1mg/ml dissolved in 50 mM acetic acid.
?. [DAY 2: DESALTING AND LYOPHILIZATION OF PEPTIDES]
Quench digestion by adding trifluoroacetic acid (TFA) to 0.6% final concentration.
100% TFA should be stored at 4°C. TFA is toxic and should only be opened in a chemical hood.
?. [DAY 2: DESALTING AND LYOPHILIZATION OF PEPTIDES]
Check that pH is
?. [DAY 2: DESALTING AND LYOPHILIZATION OF PEPTIDES]
Centrifuge 9,000 rcf for 5 min in large floor centrifuge.
Pour off supernatant immediately after samples stop spinning. If you are not ready for them, spin a little longer, but don’t let pellets sit in buffer. They will quickly begin to re-dissolve and break apart.
?. [DAY 2: DESALTING AND LYOPHILIZATION OF PEPTIDES]
Desalt peptides (see protocol Peptide Desalting with a Vacuum Manifold), and freeze samples in liquid nitrogen.
?. [DAY 2: DESALTING AND LYOPHILIZATION OF PEPTIDES]
Dry down or lyophilize peptides to remove acetonitrile.
Due to the acetonitrile in the samples, lyophilization must be done in a lyophilizer capable of maintining -70°C or lower.
?. [DAY 3: TMT LABELING]
Suspend peptides in 200 mM tetraethylammonium bromide (TEAB) to ~8 μg/μl.
Use concentrations calculated previously by Bradford Assay to determine how many total mg protein went into trypsin digest.
Before resuspending peptides, make sure there is liquid nitrogen available. If you cannot acquire liquid nitrogen, store lyophilized peptides at -80°C.
?. [DAY 3: TMT LABELING]
Quantitate with Pierce Quantitative Colorimetric Peptide Assay according to manufacturer's instructions.
Generally need to dilute samples 1:50 to be in linear range of assay.
?. [DAY 3: TMT LABELING]
Dilute peptides to 5 μg/μl with 200mM TEAB.
?. [DAY 3: TMT LABELING]
Prepare TMT vials
?. Transfer 100 μl of samples to appropriate TMT tubes (=500μg of peptide per tube) and incubate for 1hr at room temperature.
RECORD WHICH SAMPLE IS LABELED WITH WHICH TMT REAGENT
?. Add 8 μl of 5% hydroxylamine and incubate for 15 min to quench reaction.
?. Transfer all 150 μl samples to a single 1.7 ml tube and pipette up and down to mix.
?. Desalt peptides (see protocol Peptide Desalting with a Vacuum Manifold), freeze samples in liquid nitrogen, and lyophilize. Samples can be stored at -80°C until ready for MS.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
E.g. add 1.35 ml of 100% methanol to 150 μl of sample = 90%.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
To make guanidine hydrochoride–Tris solution, add 2.87g guanidine hydrochoride to 5 ml 100mM Tris-HCl pH 8. Always make fresh just before thawing cells (see Guidelines & Warnings)
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Make urea – Tris solution by dissolving 4.8g urea in 10ml 100mM Tris-HCl pH 8.	Make solution fresh before thawing cells.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Resuspend by gently stirring with pipette. Pipette up and down once chunks are small. Pipette SLOWLY, trying not to introduce bubbles. Probe sonicate if necessary. The solution will be cloudy but should not have visible chunks. Because the pellets are difficult to resuspend, it works best to pipette buffer into all tubes to start the dissolution, and then work to resuspend each sample by pipetting.
If samples over 50 OD units were split in step 1, they can be re-combined here by resuspending all of the split samples serially using the same 1 ml of urea – Tris buffer.
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Add 3ml Tris to 1ml sample, then add an extra 0.5 ml tris to make sure urea is below 2M (or it will interfere with Bradford Assay).
?. [DAY 1: CELL LYSIS and TRYPSIN DIGEST]
Add 10μl 100x stock per 1ml sample.
?. [DAY 2: DESALTING AND LYOPHILIZATION OF PEPTIDES]
Pipette 10 μl of sample onto pH indicator strips to test pH
?. [DAY 2: DESALTING AND LYOPHILIZATION OF PEPTIDES]
If pH is > 2.0, add more TFA until pH is
If pH is >2.0, protein will not bind to desalting column
?. [DAY 3: TMT LABELING]
To make 200 mM TEAB, dilute from1 M stock solution using ultrapure water.
?. [DAY 3: TMT LABELING]
To resupend lyophilized peptides:Tap tubes to make sure powder is knocked off kimwipe lids before removing them.Add the TEAB. Powder should go right into solution – can do a very quick pulse on the vortex if necessary.Centrifuge at max speed in table top centrifuge for 2 min to make sure any peptides stuck to sides of tube go                 into liquid and to collect the liquid in the bottom of the tube.
?. [DAY 3: TMT LABELING]
Pull one set of TMT vials out of the -20°C freezer and equilibrate to room temperature.
?. [DAY 3: TMT LABELING]
Do a quick spin in the centrifuge to collect TMT reagents at bottom of tubes before opening.
?. [DAY 3: TMT LABELING]
Suspend each 0.8 mg TMT vial with 50μl anhydrous acetonitrile (pipette acetonitrile in chemical hood).
?. [DAY 3: TMT LABELING]
Incubate for 5 minutes at room temp with occasional vortexing. Briefly centrifuge to collect sample at bottom of tube before opening.
?. Keep remaining sample as unlabeled sample control. Store at -80°C
?. Transfer 10 μl of pooled sample to a separate tube and freeze at -80°C. Analyze this sample for peptide labeling efficiency.