This experiment will be done using the ventricular CSF aliquoted by the Hoofnagle lab

4 replicates will be prepped over 3 days (3/19, 3/22 and 3/24) and run the day (or within 2 days) after they are prepped

MATERIALS:

• Pooled ventricular CSF
• Each prep day, thaw 4 aliquots
• BCA reagent
• 2,744 ul of reagent A
• 56 ul of reagent B
• 1M iodoacetamide (IAA)
• 1M DL-dithiothreitol (DTT)
• 2% SDS
• Dilute 20% SDS with Tris pH 8.5
• 100 ul 20% SDS in 900 ul Tris
• Resyn Biosciences MagResyn Hydroxyl particles
• 100% Acetonitrile (ACN)
• 70% Ethanol
• 95% Acetonitrile
• 100 ng/ul enolase
• 120 ul of 2% SDS into 40 ul aliquot (Freezer 2)
• 50 mM ammonium bicarbonate (ABC)
• Pierce Porcine trypsin; two 20-ug vials
• Add 100 ul of 50 mM ABC to each vial to get working concentration of 0.2 ug/ul
• HPLC grade 100% formic acid
• 500 fmol Pierce Retention Time Calibrant
• Dilute 5 pmol PRTC (stock) to 500 fmol

METHODS:

Protein Assay

Thaw 4 aliquots of ventricular CSF

Perform BCA assay on the 4 aliquots to determine protein concentration

200 ul of BCA reagent into each well of the assay plate

23.4 ul of 2% SDS into the 4 sample wells

Add 25 ul of each standard to the wells

Add 1.6 ul of sample to the wells

Pipette up and down to mix and heat for 30 min at 37 C

Use Varioskan flash to obtain photometric reading of the plates

Sample preparation

Based on the concentrations, pull 25 ug and add to plate

Add 8 ul of 100 ng/ul enolase

Protein denaturation

To each well add the same volume of 2% SDS to each sample, that was pulled from the aliquot (based on ex. Above, 54.5 ul of 2% SDS)

Pipette up and down gently

Reduction and alkylation

Volumes of reducing and alkylating agents will be calculated after the sample and buffer volumes are determined (above)

Add (vol in table below) ul of 1M DTT to each well, pipette up and down gently

Heat the plate at 95C for 10 min

Allow to cool

Add(vol in table below) ul of 1M IAA to each well

Pipette up and down gently

Incubate at room temperature in the dark for 30 minutes

Protein precipitation

Add 12.5 ul of the Hydroxyl beads (at their stock concentration of 20 ug/ul) to each well

Add 297.5 ul of 100% ACN to each well (70% final concentration) to precipitate proteins

Allow to sit for 10 minutes at room temperature

NOTE: You cannot freeze the samples at this point--the hydroxyl beads will rupture when frozen.

Wash and digestion plate preparation

Wash plates (deep well):

• Washes 1-3; 1 mL 95% ACN
• Washes 4-5; 1 mL EtOH

Digestion/elution plate (deep well):

• 125 ul 50 mM ABC + 25 ul of 0.1 ug/ul Pierce Porcine trypsin

Kingfisher automated clean-up and digestion

Turn on Kingfisher and bring up adapted Resyn protocol (see attached)

Add plates to instrument as indicated by the program

Hit "run"

The automated clean-up and digestion will take about 90 minutes.

Quenching the digestion

Add 5.4 ul of 100% formic acid to quench

Freeze (-80C) or dry down in speed vacuum

In my case, I transferred the samples from the 96 well plate and into 8 individual 1.5 mL tubes (NOT lo-bind)

Preparing samples for mass spec analysis

Prepped on EvoTips using the standard protocol

Completing the 3x4 and analysis

Repeat this two more times

Samples are to be analyzed separately on the mass spec (initiation of analysis should occur the day after prep completion)

Each analysis batch will include, using both the 44 and 88 min gradient methods

• A 6 injection chromatogram library consisting of a pool of the 4 samples
• 4 quant injections