MS sample preparation using Stage Tips_Gel Plugs
Day 1
Cut gel plugs, chop each one into 1mm2 -piece (about 25 pieces per gel plug)
Add 200ul 2:1 100 mM AmBic : 100% ACN
Change the destain (2:1 100 mM AmBic : 100% ACN) as needed
Destain as needed (overnight okay)
Day 2
Wash 10 min each: water, acetonitrile, water, acetonitrile, 50mM AmBic
Add 3.125ng/ul trypsin to cover (~40ul per tube or as needed)
Digest overnight Day 3 (70ul of each sol’n per tube, do not vortex)
Remove digest solution to washed 0.65 mL tube and speedvac
Extract from gel pieces with 70% ACN, 1.7% formic acid, 0.1% TFA, 37C, 1 hour
Remove extractant, combine with dried digest solution (it is OK if not dried completely) and speedvac
Extract with 0.1 M TEAB (Triethylammonium bicarbonate), 37C, 1 hour
Remove extractant, combine with dried digest solution and speedvac
Extract with acetonitrile, 37C, 1 hour
Remove extractant, combine with dried digest solution and speedvac
Dry digest/extractant completely (this can go overnight) Day 4
Resuspend the dried peptides in 0.1% TFA, 5% formic acid (100ul 0.1% TFA + 5ul 100% formic acid)
Purify on Stage Tips by centrifugation
Tip Wet: 50ul 100% Methanol, 4000rpm (~1500 rcf.), 1’ Don’t let the tip dry completely; let the liquid go below the bottom line on the p200 tip; spin longer if needed (this is true for most wash and binding steps)
Tip Wash: 150ul 0.1% TFA, 4000rpm, 2’
Sample binding: load sample on the tip; 4000rpm, 2’
Sample Wash: 150ul 0.1% TFA, 4000rpm, 2’
Sample Wash: 150ul 0.5% HAc, 4000rpm, 2’ This is the last wash step before elution; spin harder and let all liquid pass through
Elution 1: 50ul 40% ACN / 0.5% HAc Use clean collecting tube (pre-washed with 100% ACN) Let everything pass through
Elution 2: 50ul 80% ACN / 0.5% HAc Let everything pass through Collect E1 and E2 in the same tube
Put the tube containing E1+E2 in the speed vac and dry completely