QExactive Sedio sample protocol

Steps to follow before starting a sample sequence:

Calibrate the instrument once a month. For calibration, follow the steps in the Wiki page for proteomics under QExactive calibration.
Once samples are at the lab or confirmed arrival within 1 hour, start reconditioning the columns (run preconditioning method at 300 ul/min then reconditioning method at 500 ul/min in the sequence). The column compartment will heat to 45C and the solvent will be changed to 5%B.
Run 2 blank runs injecting solvent A.
Run two BSA (2ug) to QC the instrument, check peak shape and run protein ID for check of mass calibration and signal intensity, as well as column max pressure.

General notes on samples:

Minimum safe autosampler injection needle height is 2 mm. Samples in plates must have liquid levels higher than 2 mm to avoid injecting air.
Match the plate wells to the volume injected. Less than 12 ul spare volume in standard Sedio 96 well plates is not enough.
Low volume samples should be run in inserts in vials.
Sample order should be randomized prior to entering into plate or numbered vials.
Processing blank should be included as a sample.

Sample submission by user:

Submit FBS form for metabolite analysis.
Fill out number of samples, solvent, and desired method.
List QC samples and frequency of QC runs. For example, QC in plate position A1, run every 10 samples.
Create excel sheet with 2 columns 1) sample name and 2) plate position. Submit excel sheet with FBS form submission.
Put the samples in the fridge next to the door on the shelf with racks labeled Samples to be run. Put PF number on the sample vials or tray, do not cover barcode.

Samples for a new project or new student:

Before running all the samples in a project, request a few samples at a certain resuspension volume to see if the signal is sufficient. If not, reduce or increase the resuspension volume.
Once the resuspension volume is determined, use that volume to resuspend the rest of the samples and filter it.
Samples should be in 10%MeOH and 90%water during the injection.
If they are in 90% MeOH and 10% water, dilute it 8x times with water to bring the final MeOH % to 10.
Take 6ul of sample and pipette into the insert; inject 4ul of the sample into system. If signal is in e9 TIC, sample is sufficient concentration.
Always start a new sequence with blank runs for both the columns.
The samples use the top5 method.