diDO-IPTL protocol

Prepare dried MS-grade trypsin, 2ug per sample to be labeled. Note that separate aliquots of dried trypsin must be used for ¹⁶O- and ¹⁸O-labeling.

Prepare two separate N-methylmorpholine (NMM) and acetic acid O-isotope exchange buffers with ¹⁶O and ¹⁸O (>98%) enriched water. Volume ratio of buffer components is 48.5 H₂O : 1 NMM : 0.5 acetic acid (pH 7.4). The total volume of buffer to prepare depends on how many peptide samples are to be labeled. The protocol below is for 40µL of O-isotope exchange buffer per sample (~10-20 µg of peptide). For samples with small amounts of peptide, the concentration can be raised by adding only 20µL buffer per sample and halving the volumes of all subsequent reagent additions.

Redissolve dried trypsin aliquots in their respective O-isotope exchange buffers and transfer 40µl to each tube of dried peptide sample. KEEP TRACK of which tubes received which buffer.

Parafilm the tops of the tubes and incubate at 37ºC overnight.

After overnight incubation, add 2µl of 11.3M monochloroacetic acid (prepared in LC-MS grade water) to each peptide tube to lower pH down to 2.6.

Prepare 16% CH₂O and CD₂O formaldehyde solutions, at least 2µl per sample, diluting stocks with LC-MS grade water if necessary.

Prepare 4.8M NaBH₃CN in LC-MS water. Tare 1.5mL tube, add a small amount of NaBH₃CN powder in fume hood, weigh, dissolve.

Add 2µL of CH₂O to each ¹⁸O tube and 2µL of CD₂O to each ¹⁶O tube.

Add 2µL 4.8M NaBH₃CN to each tube.

Mix well and incubate at 45ºC for 1 hr. While waiting, prepare 5M ammonium formate in LC-MS water.

Add 2µL 5M ammonium formate to each tube and mix well.

Add 8µL of formic acid to each tube and mix well. Total sample volume is ~56µl. ¹⁶O- and ¹⁸O-labeled samples are ready to be mixed and analyzed by LC-MS.