Digestion of undepleted plasma
- Dilute 2.5µl of plasma to 1ml with 50mM triethylammonium bicarbonate (TEAB) ([protein] 0.2µg/µl taking the average [protein] of plasma as 80mg/ml).
- Take 100µl (20µg plasma protein) and reduce with 5mM dithiothreitol (DTT; 11µl of 50mM stock) for 30 mins at 56oC and alkylate with 20mM iodoacetamide (IAM; 12.5µl of 200mM stock) for 20 mins in the dark.
- Total volume 123.5µl containing 20µg of plasma protein.
- Use a 1:20 trypsin:protein ratio for digestion i.e. 1µg trypsin. Incubate at 37°C for 2 hrs, then overnight at room temperature.
Digestion of depleted plasma
This method is for plasma samples depleted of the six most abundant proteins using MARS HPLC columns (4.6mm x 100mm, protocol as recommended by Agilent Technologies). Samples should be assayed for protein content using the standard Bradford method.
- Prepare plasma sample to a protein concentration of ~2.5µg/µl.
- Take a 10µl aliquot of plasma and make up to 100µl with 50mM TEAB.
- Reduce cysteine residues in 5mM DTT (Add 11µl of a 50mM stock) and heat at 56oC for 30 min.
- Alkylate sample by treatment with 20mM iodoacetamide in the dark for 20 min at ambient temperature (Add 12µl of a 200mM stock).
- Digest plasma sample with 1µg trypsin (1:20 trypsin:protein ratio. Incubate at 37°C for 2 hrs, then overnight at room temperature.