STUDY A
Untagged (no label) tube sample: a digested yeast protein sample
Following trypsinization, the digested sample was desalted and dried on a SpeedVac. A total of 50 ug of digested yeast protein was dispatched to each participant. The most important point is that all participants should inject 200 ng of sample in each LC-MS/MS analysis.
In the following, we provide an example on making 10 aliquots:
- Spin the tube before opening it. (STRONGLY RECOMMENDED)
- Resuspend the peptide sample with 100 uL of 10% acetonitrile and vortex for 15 sec.
- Spin down and make 10 aliquots of 10 uL each. Dry them on a Speedvac and store at -80ºC until further use.
Then, we prepare samples (from aliquots) at 50 ng/uL so as injections of 4 uL will contain 200 ng:
- Resuspend an aliquot (5ug) in 100 uL of 2% ACN, 0.1% FA.
- Inject 4 uL of the sample (=200 ng)
STUDY B
Green-tagged tube sample: In study B, participants should perform at least 6 in-solution digestions throughout the whole study. Ideally, an aliquot will be digested once a month and its product analyzed together with the sample from study A (once a week or once every two weeks). Upon receiving the samples, we recommend the participant to prepare 10 to 15 aliquots and store at -80ºC until further use (there is no need to quantify the sample proteins). To prepare the required aliquots, proteins should be first resuspended in an appropriate buffer. Although participants are free to choose their own digestion protocol, trypsin must be the enzyme of choice.
Important. There is a small visible pellet in the protein tubes. You should spin the tube before opening it.
Despite the relative freedom in terms of digestion protocol in Study B, participants should follow the same guidelines of Study A when it comes down to the LC-MS/MS analysis. Following the example provided above in Study A, if you make 10-micrograms aliquots of the yeast protein sample and digest one of these aliquots, then you should make it in a final volume of 200 uL of 2% ACN, 0.1% FA. Finally, 4 microliters of such sample should be injected for LC-MS/MS analysis; that is, inject 200ng of digested protein sample.
Important. The LC-MS/MS instrument method used with this sample should be the same method used with the sample of Study A. |
