TITLE: STAGE (STop And Go Extraction) C18 tips for Desalting and Clean Up
AUTHORS: Teesha C. Luehr

[STEPS]
?. [Preparing Solutions]
Abbreviations:ACN: acetonitrileTFA: trifluoroacetic acidFA: Formic AcidMeOH: Methanol
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1% TFA
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2-propanol
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Buffer A: 2% ACN with 0.1% TFATo make
100 ml
[1% TFA]
[ACN]
[Milli-Q water]
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Buffer B: Anywhere from 30-80% ACN with 0.1% TFAGeneral peptides are eluted with 60% ACN with 0.1% TFA (***this is also used for tip conditioning***)To make Immunopeptides are eluted with 30% with 0.1% TFATo make
100 ml
[1% TFA]
[ACN]
[Milli-Q water]
100 ml
[1% TFA]
[ACN]
[Milli-Q water]
?. [Preparing Solutions]
Mass Spectrometry Buffer A: 2% ACN with 0.1% FA
It is important to use FORMIC ACID for mass spectrometry solvents
?. [Making tips]
Prepare as many tips as necessary by one of two methods: C18 empore filter or frit + C18 powder
?. [Making tips]
The filter/frit will act to keep the powder in the P200 pipette tip. The frit has a pore size, but this is much larger than the pore size of C18 filter.
?. [Making tips]
Punch out a disk using a 17G flat-tipped syringe. Use C18 Empore filters or a SPE frit.Careful not to squish the frit as this will cause a blockageThe easiest way to do this is to line the syringe up gently on top of the frit, then hammer the syringe down (rather than twisting and manually pushing).
?. [Making tips]
Eject the frit/frit disk into a P200 pipette tip with an unfolded paper clip
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Ensure that the filter/frit is secrely wedged in the bottom of the tip.
?. [Adding Powder]
Using a multi-channel pipette resevoir, add in C18 powder and wet it with methanol to create a slurry
This step is optional if a C18 membrane was used.
?. [Adding Powder]
Using a pipette, add C18 slurry on top of the filter/frit
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Compact the slurry and remove the ethanol by forcing the methanol from the slurry. This can be done with a Luer lock syringe fitted to the tip of the pipette tip. Alternatively, this can be done by centrifuging the tips at 800g. However, when conditioning the tips it is advised to manually force the solvent through to check the tightness/loseness of the frit + slurry.
?. [Sample Preparation]
If your sample is dry, reconstitue it in Buffer A. If the sample is aqueous, acidify it with 1% TFA to a pH or up to depending on the sample concentration.
[Buffer A]
[Buffer A]
It is important to acheive a pH less than 2.5 to ensure all amino acids are in their protonated form
?. [Sample Preparation]
If the concentration of the sample is unknown, determine it by NanoDrop
10mg of C18 powder can handle ~ 200-500µg of protein from a full cell lysate
?. [Tip Wetting]
Wet the tips with
[2-propanol]
?. [Tip Conditioning]
Condition the tip with
[Buffer B (50-60% ACN with 0.1% TFA)]
?. [Tip Hydration]
Hydrate the tip with
[Buffer A]
?. [Tip Washing]
Wash the tip with
[1% TFA]
Optional: collect flow through and store at -20˚C in case of improper tip usage
Optional: If there is not time to elute and dry the sample, the peptides can be stored "dry" on the tip at 4˚C overnight or -20˚C for longer periods. This is a better option than eluting the peptides and storing in Buffer B.
?. [Sample Elution]
Elute the sample with 2X into a or a 96-well plate
[Buffer B (your selected % ACN)]
[microtube]
?. [Sample Loading]
Pass the reconstituted sample slowly through the tip
Optional: collect flow through and store at -20˚C in case of improper tip usage
?. [Drying]
Dry the sample completely in a SpeedVac and recostitute in the Mass Spec Buffer
?. [Adding Powder]
A different amount of C18 powder will be added each time as the slurry is not homogenous. Add slurry up to a desired height. 10mg of C18 powder is ~11mm above the frit.