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(New page: ==Overview== In gel digest of proteins samples ==Materials== '''Ammonium Bicarbonate''' (AmBic): Dissolve 0.79g in 100ml milliQ water (or 0.1975 in 25ml), filter and store for 1 month. ...)
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In gel digest of proteins samples
Ammonium Bicarbonate (AmBic): Dissolve 0.79g in 100ml milliQ water (or 0.1975 in 25ml), filter and store for 1 month.
Dithiothreitol (DTT, 154.2g/mol): Make fresh 45mM solution by adding 7.5mg to 1ml milliQ water. Always make fresh.
Iodoacetamide(IA, 185g/mol): Make fresh 100mM solution by adding 18.5mg to 1ml. Always make fresh.
Trifluoroacetic Acid (TFA)-10 x 1ml ampules Pierce cat.no. 28904: open 1ml ampule and mix 50:50 with milliQ water for 50% stock solution; then dilute 1:5 in milliQ:
Modified Trypsin -Promega cat. no. V5111: Dissolve one pellet in the vial with 200ul of the supplied suspension solution to make a 0.1mg/ml working stock solution. Aliquot 10 X 20ul volume and store at -20°C. Other proteases may need to be treated differently prior to use
Formic Acid 0.1% add used to resuspend sample for analysis on the LC-MS-MS (LTQ).
REAGENTS: Prepared on the day of processing.
50mM ammonium bicarbonate/50% acetonitrile: combine equal volumes of 100mM AmBic and 100% acetonitrile.
25mM ammonium bicarbonate: 250ul AmBic with 750ul of milliQ water.
0.1% TFA: Dilute 10ul 10% TFA into 990 milliQ water.
60% Acetonitrile/0.1% TFA: 600ul acetonitrile, 390ul milliQ water, and 10ul TFA
For Gel Bands or gel plugs that were not reduced and alkylated prior to 2D-DIGE fractionation:
- Equilibrate band or plugs in 50mM AmBic for 15mins. If bands or plugs are frozen thaw plugs in 25-50mM AmBic for 15mins. Repeat this step once or two total washes.
- Reduction and Alkylation: This step can be skipped for sample extracted from 2D-DIGE analysis as the samples are reduced an alkylated prior to fractionation. For 2D-DIGE plugs begin with step 3. Reduction – add 150ul of 50mM AmBic to the sample. Add 10ul 45mM DTT and incubate at 50°C for 15min. Alkylation – add 10ul of 100mM iodoacetamide and incubate in the dark for 15min. This results in carbamidomethylation of Cysteine residues (net addition of 57 daltons to each Cysteine residues).
For Gel Plugs reduced and alkylated prior to 2D-DIGE - START HERE:
- Equilbrate and Dehydrate: Remove liquid and replace with 100ul 50% acetonitrile/50mM AmBic and incubate 15min. Repeat this once to further remove residual stain (Coomassie, Deep Purple, etc). Replace liquid with 50-100ul 100% acetonitrile for 10min or until the gel slice turns white. Remove liquid and desiccate for 5min in vacuum centrifuge to dehydrated gel slices or plugs. Gel slice or plugs can be stored for months at -20°C
- Protease Addition: Prepare protease – for trypsin - resuspend 20ul aliquot (0.1ug/ul) 1:10 by adding 180ul 25mM AmBic to 20ul stock. Reswell the dehydrated gel slices in 10-15ul of diluted (0.01ug/ul) modified trypsin. It is important to add just enough to cover the gel slice most of which should be absorbed within 20min.
- Overlay: After all protease solution has entered the gel, add additional 25mM AmBic (without trypsin) until the gel slice is covered.
- Protease Digestion: For trypsin and AspN – digest overnight (12-18hrs) at 37°C. For Chymotrypsin – digest 4hrs at room temperature and do not exceed 4hrs or temperature higher than 25°C for the digest.
- Peptide extraction: Remove the supernatant, which contains some of the peptides that have diffused from the gel, to a new tube. Extract peptide from the gel by adding 15-25ul 60% acetonitrile/0.1% TFA and incubating 15min. Remove and combine with supernatant. Repeat the extraction one additional time and pipette up and down to ensure the pooled supernatant is mixed.
- Dry down the samples in pooled supernatant in a vacuum centrifuge to dryness. Do not over-dry as this may comprise peptide detection.
- Reconstitution and MS analysis: Dissolve dried peptides in 10-30ul of 0.1% formic Acid. Samples can be directly analyzed by LC-MS. More abundant samples can be analyzed by MALDI-TOF MS. Chemical contaminants that suppress ionization such as salt and detergent can be removed by ZipTipC18 (Millipore cat.no. ZTC18S096) ZipTip instructions: IMPORTANT RETAIN ALL FRACTIONS TO ENSURE PEPTIDES ARE NOT LOST.
- equilibrate the tip in 60% acetonitril/0.1%TFA 2X with 10ul
- wash the tip in 0.1% TFA 2X with 10ul/
- load upto 10ul of sample with repetitive pipetting (up and down 5X)
- wash tip using 0.1% TFA 2X 10ul
- elute into 2ul 60% acetonitrile/0.1% TFA with repetitive pipetting (be sure to place 2ul 60% acetonitrile/0.1% TFA in a clean tube where the washes and elution will be stored)
- For MALDI-TOF MS apply 0.4ul peptide mixture to a MALDI target and overlay with 0.4ul a-cyano-4-hydroxycinnamic acid matrix (10mg/ml in 60% acetonitrile).
Keratins and other human proteins common contaminants in all laboratories and therefore samples should be processed to limit this exposure.
Relevant papers and books
- Jeremy Myers