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Materials
Denritic nanotechnologies
Generation 5.0 1,12-diaminodecane core polyamidoamine (PAMAM) dendrimer 10.2% in MeOH (DNT-128)
Protea Biosciences
Proteatip TiO2 10-200uL pipette tips with Reconstitution and wash 1 solution, wash 2 solution and elution solution
Sigma-Aldrich
ß-casein from bovine milk - min. 90% (22086)
Trypsin TPCK treated from bovine pancreas (93630)
Synthetic mono- and tetraphosphopeptide isolated from bovine ß-casein 0.1mg/ml (P9615)
Triethyl Ammonium Bicarbonate (TEAB) buffer, 1.0 M pH 8.5 (T7408-100ML)
(2-(N-Morphino)ethanesulfonic acid) (MES) (M3671-50G)
α-cyano-4-hydroxycinnamic acid (CHCA) (C-2020)
2,5-dihydroxybenzioc acid (DHBA)
Fisher Scientific
Analytical grade Methanol
HPLC grade acetonitrile
Sodium Chloride (S/3120/63)
Fluka
Trifluoroacetic Acid (TFA)
Acetyl chloride (CH-9470)
(N-(3-Dmethylaminopropyl)-N'-ethylcarbodiimide) (EDC) (39391)
Millipore
P10 Zip tips (Millipore)
Ultrafree-MC PBCC Biomax membrane, 5 kDa centrifugal filter unit (UFC3BCC00)
BDH Chemicals
Phosphorus pentoxide (P2O5) (29527)
Other
Imidazole
Hydrochloric acid (HCL) 1.0 M
Ammonium bicarbonate (NH4HCO3) 500 / 50 mM
Sodium hydroxide (NaOH) 500 mM
Salmonella typhimurium (SL1344) protein lysate provided by Xiao Li and the Southampton Centre for Proteomic Research (CPR)
Method
3 samples of varying complexity were used, all of which were used for both the dendrimer conugation and TiO2 enrichment methods.
Note that it is useful to take small monitoring aliquots between steps for MALDI analysis to verfiy that each step has worked. Use of zip tips for these is recommended.
1) Sample preparation
Preparation of synthetic peptides
Pipette 5uL each of the mono- and tetraphosphopeptides into a 500uL eppendorf tube.
Lyophilise in vacuo (Univap) to dryness, then dessicate overnight under vacuum with P2O5.
Preparation of ß-casein
Prepare 1mg/ml ß-casein in 50mM NH4HCO3 buffer (pH ~7.8).
Prepare 1mg/ml trypsin in 50mM NH4HCO3 buffer.
Add trypsin to ß-casein to give a final protein to enzyme ratio of 100:1 and leave at 37 degrees celsius overnight
Separate 1ml into 100uL aliquots and lyophilise in vacuo to dryness.
Keep remaining 1ml to spike complex protein mix with.
Preparation of Salmonelle protein lysate
S.typhimurium was grown in Luria Broth (LB) to mid-log phase, then harvested by centrifugation. The cell pellet was then lysed using sonication, and the cell debris was thrown down using centrifugation and the supernatant snap frozen as 20mg/ml solution.
Add 1mg/ml ß-casein digest from previous step to salmonella protein lysate (SPL) to give a ß-casein to SPL ratio of 2:1.
Prepare trypsin to 1mg/ml in 100mM NH4HCO3.
Add trypsin solution to ß-casein / SPL mixture to give a trypsin to SPL ratio of 1:25 and leave overnight at 37 degrees celsius.
Separate 1ml into 100uL aliquots and lyophilise in vacuo to dryness.
2) Dendrimer conjugation
Methyl esterification
Add anhydrous methanol to a 15ml falcon tube embedded in dry ice (low temperature is important) and add acetyl chloride dropwise to avoid violent overheating and spitting, to a final concentration of 167uL acetly chloride per ml of methanol.
Add 500uL per mg of dried peptide, using no less than 100uL per sample.
Incubate at 12 degrees celsius for 120 minutes.
Optional: pH to ~5.5 using TEAB
Lyophilise in vacuo to dryness.
Dendrimer preparation
Prepare dendrimers aliquots to give the following ratios by dry weight:
Mono- and tetra phosphopeptides to dendrimer - 1:800
ß-casein to dendrimer - 8:1
For ß-casein / SPL complex mixture - 3:1
Add dendrimer solution to 100mM MES to give a dendrimer to MES ratio of 1:5
Adjust pH to ~5.5 using 1M HCl
Lyophilise in vacuo to dryness
Reaction solution
Prepare a solution of MES at 39 mg/ml, Imidazole at 6.8 mg/ml and EDC at 17.7 uL/ml.
Adjust the pH to ~5.5 using 1M HCl
Dendrimer conjugation
Add reaction solution to dried dendrimers to give a 0.2mg/uL dendrimer concentration - vortex to solubilise (may take some time).
Add dendrimers in reaction solution to dried peptides to give the ratios described in the dendrimer preparation step.
Vortex and set to shake vigorously for 20-25 hours.
Have set this as a public entry in case anyone knows of someone not on my FL who might help.
Cheers!