PRTC: We know what those peptides can be

This is the last step in the lab before we go on the mass spectrometer! PRTC, or Peptide Retention Time Calibration, is a mix of known peptides and known quantities that we can use to ensure the mass spectrometer is functioning. It also provides a reference for our sample analysis.

Diluting PRTC:

The entire preparation step requires pipetting small volumes of liquids. To reduce the capacity for human error, we diluted the 0.5 pmol/µL stock solution of PRTC so we would not have to pipet any volume less than 1 µL.

• Pipetted 20 µL of 0.5 pmol/µL stock PRTC into a clean, labelled centrifuge tube
• Added 30 µL of Final Solvent (3% acetonitrile + 0.1% formic acid) to PRTC
• Vortexed gently to thoroughly mix contents

Our resulting mixture was a 50 µL 0.2 pmol/µL PRTC solution.

Preparing samples:

Our goal is to extract 3 µL of our prepared sample for each mass spectrometer replicate, which means we need 6 µL of sample total. To prepare for this, our total volume of sample and solvents will be 15 µL.

• Removed samples from -80ºC
• Placed in wet ice bath and allow to thaw
• Labelled glass autosampler vials, one for each sample. These vials are the ones we will put in the mass spectrometer
• Labelled new centrifuge tubes, one for each sample
• To each newly labelled sample centrifuge tube, we added the following reagents
• 3.13 µL of Final Solvent
• 10 µL of sample removed from -80ºC
• 1.88 µL of 0.2 pmol/µL PRTC
• Because we pipetted such small quantities of PRTC, we ensured the pipet tip was touching the bottom of the centrifuge tube so all liquid was transferred
• Vortexed gently to thoroughly mix contents
• Centrifuged liquid down to the bottom of the centrfuge tube

After we prepared the samples with PRTC, we gave everything to Emma, who put the samples in her lab’s -80ºC. When we are ready to use the mass spectrometer, we will transfer the contents of the centrifuge tubes to the labelled glass autosampler vials.