Protocol
Dimethyl labelling of peptides for mass spectrometry bad proteomics
Bad on:
Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics
Boerma PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJ.
Nat Protoc. 2009;4(4):484-94. doi: 10.1038/nprot.2009.21.
On-column dimethyl labelling (1-5mg protein/sample):
Reagents
奖状制作
Acetic acid
Acetonitrile
Formaldehyde (CH2O) 37% (vol/vol)
Formaldehyde (CD2O) 20% in D2O (492620-20G Sigma)
Formaldehyde (13CD2O) 20% in D2O (596388-5.00G Sigma)
Formic acid
Sodium cyanoborohydride (NaBH3CN)
Sodium cyanoborodeuteride (NaBD3CN) (D-1797, CDN Isotopes)
Sodium dihydrogen phosphate (NaH2PO4)
Di-sodium hydrogen phosphate (Na2HPO4)
Triethylammonium bicarbonate
Equipment
Sep-Pak cartridges C18 3 cc, 200 mg (WAT054945, Waters)故事长篇
VALVE(STOPCOCK)W/NEEDLE TIP (WAT200685, Waters)
Solutions will flow through slowly by gravity alone, so a vacuum manifold is not needed. A low flow rate is critical for the columns to work. If any columns do become a bit blocked they can be placed inside a 15ml falcon tube and spun at 20-50g which will slowly force liquid through.
Preparation of solutions
Prepare formaldehyde and cyanoborohydride solutions fresh, just prior to labelling reaction
Buffer A
0.6% acetic acid
Buffer B
50% acetonitrile 0.6% acetic acid
4% Formaldehyde (CH2O)
Volume of 37% formaldehyde (CH2O) = ( #samples × 250µl)/9.25
Volume of water = ( #samples × 250µl) – volume of 37% formaldehyde
4% Formaldehyde (CD2O)
Volume of 20% formaldehyde (CD2O) = ( #samples × 250µl)/5
Volume of water = ( #samples × 250µl) – volume of 20% formaldehyde
4% Formaldehyde (13CD2O)
Volume of 20% formaldehyde (13CD2O) = ( #samples × 250µl)/5
Volume of water = ( #samples × 250µl) – volume of 20% formaldehyde
0.6M NaBH3CN
MW = 62.84g/mol
促进就业法Volume required = #light+medium samples × 250µl
0.0754g in 2ml water for 8 samples
NaBD3CN
MW = 65.86g/mol
Volume required = #heavy samples × 250µl
0.039516g in 1ml water for 4 samples
50mM sodium phosphate pH 7.5泰州城隍庙
Per sample: 1ml 50mM NaH2PO4 + 3.5ml 50mM Na2HPO4
50ml NaH2PO4 = 0.3449g in 50ml
50ml Na2HPO4 = 0.354g in 50ml
Volume of 50mM NaH2PO4 = #samples × 1ml
Volume of 50mM Na2HPO4 = #samples × 3.5ml
1.Measure amount of protein in each sample (by Bradford or BCA assay for example) and
perform digest. You will need to know protein amounts so that you can mix dimethyl
labelled peptides in the correct ratios
2.Resuspend dried peptides in 5% formic acid. 1ml for each sample. Leave peptides solubilising
for a couple of hours at RT
3.Mix prepared solutions according to the table below to make up the labelling solutions.
Carry out protocol in a fume hood:
Light Medium Heavy
4.5ml 4.5ml 4.5ml
50mM sodium
phosphate buffer
pH7.5
4% formaldehyde 250µl CH2O 250µl CD2O 250µl 13CD2O
250µl NaBH3CN 250µl NaBH3CN 250µl NaBD3CN
0.6M
cyanoborohydride
4.Set up solid pha extraction cartridges with a stopcock valve each (e picture below).
Condition each 200mg C18 column with 3ml acetonitrile
寒假的作文5. 2ml buffer A × 2
6. Load peptides onto columns
7. Wash columns with 3ml buffer A
8. Flush each column with 5 × 1ml of appropriate labelling mix (light, medium or heavy) 9. Wash eac
h column with 2ml buffer A
10. Elute peptides with 1ml buffer B into Eppendorf tubes 11. Mix labelled peptides according to the desired ratio
12. Peptides are now desalted and labelled. Dry down peptides. They can either be analyd by
LC-MS or fractionated further.
In-solution dimethyl labelling (>25µg protein/sample):
Reagents
Ammonia solution (25% vol/vol)
Formaldehyde (CH2O) 37% (vol/vol)
Formaldehyde (CD2O) 20% in D2O (492620-20G Sigma)
Formaldehyde (13CD2O) 20% in D2O (596388-5.00G Sigma)
Formic acid煎豆腐的做法
Sodium cyanoborohydride (NaBH3CN)
Sodium cyanoborodeuteride (NaBD3CN) (D-1797, CDN Isotopes)
Triethylammonium bicarbonate
Equipment
Test-tube mixer
Preparation of solutions
4% Formaldehyde (CH2O)
Volume of 37% formaldehyde (CH2O) = (#samples × 4µl)/9.25
Volume of water = (#samples × 4µl) – volume of 37% formaldehyde
4% Formaldehyde (CD2O)
Volume of 20% formaldehyde (CD2O) = (#samples × 4µl)/5
Volume of water = (#samples × 4µl) – volume of 20% formaldehyde
4% Formaldehyde (13CD2O)
Volume of 20% formaldehyde (13CD2O) = (#samples × 4µl)/5
Volume of water = (#samples × 4µl) – volume of 20% formaldehyde
0.6M NaBH3CN
MW = 62.84g/mol
Volume required = #light+medium samples × 4µl
0.6M NaBD3CN
MW = 65.86g/mol
Volume required = #heavy samples × 4µl
1% ammonia
Volume required = #samples × 16µl
领导干部讲党课5% formic acid
Volume required = #samples × 8µl
1.Measure amount of protein in each sample (by Bradford or BCA assay for example) and
perform digest. You will need to know protein amounts so that you can mix dimethyl
labelled peptides in the correct ratios
2.Resuspend dried peptides in 100 mM TEAB. 100µl for each sample. Leave peptides
solubilising for a couple of hours at RT. The pH should be between 5 and 8.5. The sample should not contain any molecules with primary amines (other than the peptides) as the will reduce labelling efficiency of peptides. Carry out the protocol in a fume hood
3.Add 4µl of 4% (vol/vol) of the appropriate formaldehyde: CH2O (light samples), CD2O
(medium samples), or 13CD2O (heavy samples)
4.Mix and centrifuge solutions
5.Add 4µl 0.6M NaBH3CN to light and medium samples and 0.6M NaBD3CN to heavy samples
6.Incubate in fume hood for 1 hour at RT, mixing periodically.新年英语怎么写
7.Add 16µl of 1% (vol/vol) ammonia solution to each tube to quench the reaction. Cool
samples on ice if necessary
8.Mix and spin centrifuge solutions
9.Add 8µl formic acid
10.Mix labelled peptides according to the desired ratio
11.Desalt peptides with C18 and analy by LC-MS
