Simultaneous Identification and Quantification of Nitrosylation Sites by Combination of Biotin Switch and ICAT Labeling - Plant Proteomics: Methods and Protocols - page 585

2. A pack of fi lter devices (Amicon Ultra-0.5 centrifugal fi lter unit
UFC503096, Amicon Ultra-4 UFC803096, and Amicon
Ultra-15 UFC903096) ( see Note 4 ).
3. HENU buffer, pH 7.7: 150 mM Hepes, 5 mM EDTA, 0.5 mM
neocuproine (208.269 g/mol), 6 M Urea (60.06 g/mol): To
prepare 250 mL of HENU buffer, mix 37.5 mL of 1 M Hepes,
pH 7.7 (from stock), with 25 mL of 0.5 M EDTA (from
stock). Add 0.026 g of neocuproine and 90.1 g of urea. Bring
the volume up to 250 mL with water. Mix and verify the pH
(7.7).
4. HEN buffer, pH 7.7: 150 mM Hepes, 5 mM EDTA, 0.5 mM
neocuproine: To prepare 250 mL of HEN buffer, mix 37.5 mL
of 1 M Hepes, pH 7.7 (from stock), with 25 mL of 0.5 M
EDTA (from stock). Add 0.026 g of neocuproine. Bring the
volume up to 250 mL with water. Mix and verify the pH (7.7).
5. A tabletop centrifuge (for 1.5 mL centrifuge tube).
6. SNO reducing solution: 0.5 M ascorbic acid (176.12 g/mol)
and 100 mM CuCl 2 2 H 2 O (170.48 g/mol). To prepare
0.5 mL of 0.5 M ascorbic acid and 0.1 M CuCl 2 2 H 2 O; weigh
44 mg of ascorbic acid and 8.6 mg of CuCl 2 . Add 5 μ L to

L of sample ( see Subheading 3.2 , step 2 ) Prepare fresh
and store at room temperature in the dark until use.
7. Acetonitrile.
8. ICAT labeling kit including buffer, labels, avidin column and
cleaving reagents (ABSCIEX 4337336).
9. Ammonium carbonate solution: To prepare 1 M ammonium
bicarbonate solution, dissolve 0.79 g of ammonium carbonate
(79.06 g/mol) in 8 mL of water. Vortex to mix and bring the
volume up to 10 mL.
10. Trypsin.
11. A vacuum concentrator.
12. An incubator.
13. A syringe-pump.
14. 5 mL syringes.

500 μ

1. C18 desalting microcolumns ( see Note 5 ).
2. Formic acid, acetonitrile as above ( see Subheading 2.3 ).
3. Binding buffer: 0.1 % trifl uoroacetic acid (vol/vol) in H 2 O.
4. Elution buffer: 50 % acetonitrile (vol/vol), 0.5 % formic acid
(vol/vol) in H 2 O.

2.4 Peptide Cleaning
with C18 Desalting
Microcolumns

2.5 Mass
Spectrometry Analysis

1. Mass spectrometer coupled to ESI-nanofl ow HPLC ( see Note 6 ).
2. Convenient software for quantitative analysis of MS spectra
and database searching ( see Note 6 ).