Prince:Yeast FASP

• Template File provides easy calculation of volumes and masses for Solution preparation

FASP Template

Cell Lysis

• Incubate yeast cells at 96°C for 10 minutes
• Add 10% SDS to yeast cells 1:1 (v/v) for a 5% total SDS concentration. Incubate at 96°C for 10 minutes
• Add DTT to .1 M concentration
• Sonify samples at 50% amplitude for 1 minute 8 times with a 30 second delay between sonications
• Centrifuge samples at 16000g to remove cell lysates

Sample Processing

• Dilute supernatant with UA buffer to 15ml. Load into Vivaspin 15R 10k filters. Centrifuge at 3000g for 50 minutes
• Wash sample with 10 ml of UA buffer and centrifuge for 40 minutes at 3000g (twice)
• Add 2 ml UA-IAA, mix well, incubate at room temperature in the dark for 20 minutes. Centrifuge at 3000g for 30 minutes
• Wash with 10 ml UA buffer and centrifuge 3000g 40 minutes (twice)
• Wash with 10 ml ABC buffer and centrifuge at 3000g for at least an hour and half (twice)
• Protein on small portion of sample assay if desired
• Add trypsin (proteomic grade) to the sample in the ratio (1:40) (make sure at least 1 ml of ABC is in the filter during trypsin addition)
• Pulse sample filters to 1000g
• Incubate sample filters at 37 °C in a rotating incubator for 18 hours
• Clean the collection portion filter tube very well with distilled water. After washing rinse with mass spec water
• Centrifuge at 3000g for 30 minutes
• Rinse with 1 ml ABC. Centrifuge 30 minutes at 3000g
• The flow through contains the peptides
• Acidify with .1% Formic Acid unless fractionating with SCX or SAX

Strong Cation Exchange

• Wash SCX column with 1 ml methanol
• Wash SCX column with 1 ml .1 M HCl
• Load SCX column with slightly acidified sample (collect flow through, collect following washes)
• Wash SCX column with 1 ml .1 M HCl
• Wash SCX column with 1 ml .1 M HCl in MeOH
• Elute SCX column with .5 ml 5% NH4OH in MeOH (Twice) combine elutions
• Add 400 ul of H2O to fractions containing MeOH
• Speed vac fractions to 400 ul
• Peptide Assay (you may have to add 200 ul H2O to fill the cuvettes)
• Concentrate sample to about 2 ug of peptides/ul using speed vac (you may combine low abundance fractions)