10. Tris–Glycine buffer 5Â (pH 8.3): dissolve in 700 mL of H 2 O
(deionized): 15.1 g Tris base; 94 g glycine; 50 mL of 10% SDS.
After solid is dissolved, adjust the volume to 1 L with H 2 O.
11. Coomassie Brilliant Blue G-250.
12. Acetic acid, 10% made with deionized (Milli Q) water.
13. Sodium dodecyl sulfate (SDS), 10% made with deionized
water.
14. Laemmli Sample buffer: 60 mМ Tris–HCl pH 6.8, 2% SDS,
10%
glycerol,
5%
2-mercaptoethanol,
0.01%
bromophenol blue.
15. 2Â Laemmli sample buffer (10 mL): mix 2.4 mL of 0.5 M
Tris–HCl pH 6.8, 400 mg SDS, 2 mL glycerol, 1 mL
2-mercaptoethanol, 5 mg bromophenol blue and bring volume to 10 mL with deionized water.
2.4 In-Gel Trypsin
Digestion
1. Carbon steel sterilized scalpels.
2. 100% Acetonitrile (ACN).
3. 50% Acetonitrile in the 50 mМ NH 4 HCO 3 made with deionized water.
4. 55 mМ iodoacetamide (IAA) in the 100 mМ NH 4 HCO 3
made with deionized water.
5. 98–100% Formic acid (FA).
6. Trypsin Gold (mass spectrometry grade).
2.5 Tandem Mass
Spectrometric
Analysis Combined
with Nano-Liquid
Chromatography
1. Microcentrifuge/vortex.
2. Eppendorf Centrifuge.
3. Mass spectrometer Q-Exactive/TripleTof.
4. Nanospray ion source Flex.
5. Nanochromatograph Ultimate 3000 RSLCnano system.
6. Mass spectrometer control and data analysis Xcalibur 3.0
software.
7. Chromatographic device control Chromeleon Xpress software.
8. Autosampler vials.
9. Autosampler Snap-It Caps and Septa.
10. Clear glass or polypropylene inserts for HPLC and GC autosampler vials.
11. HPLC Columns Acclaim PepMap, (15 cm  50 μm, C 18 ,
3 μm, 100 A and 2 cm  75 μm C 18 , 3 μm, 100 A).
12. Coated Fused-silicaPicoTip Emitter SilicaTip.
13. Acetonitrile, LC-MS grade.
14. Methanol, LC-MS grade.
194
Julia Bespyatykh et al.
(deionized): 15.1 g Tris base; 94 g glycine; 50 mL of 10% SDS.
After solid is dissolved, adjust the volume to 1 L with H 2 O.
11. Coomassie Brilliant Blue G-250.
12. Acetic acid, 10% made with deionized (Milli Q) water.
13. Sodium dodecyl sulfate (SDS), 10% made with deionized
water.
14. Laemmli Sample buffer: 60 mМ Tris–HCl pH 6.8, 2% SDS,
10%
glycerol,
5%
2-mercaptoethanol,
0.01%
bromophenol blue.
15. 2Â Laemmli sample buffer (10 mL): mix 2.4 mL of 0.5 M
Tris–HCl pH 6.8, 400 mg SDS, 2 mL glycerol, 1 mL
2-mercaptoethanol, 5 mg bromophenol blue and bring volume to 10 mL with deionized water.
2.4 In-Gel Trypsin
Digestion
1. Carbon steel sterilized scalpels.
2. 100% Acetonitrile (ACN).
3. 50% Acetonitrile in the 50 mМ NH 4 HCO 3 made with deionized water.
4. 55 mМ iodoacetamide (IAA) in the 100 mМ NH 4 HCO 3
made with deionized water.
5. 98–100% Formic acid (FA).
6. Trypsin Gold (mass spectrometry grade).
2.5 Tandem Mass
Spectrometric
Analysis Combined
with Nano-Liquid
Chromatography
1. Microcentrifuge/vortex.
2. Eppendorf Centrifuge.
3. Mass spectrometer Q-Exactive/TripleTof.
4. Nanospray ion source Flex.
5. Nanochromatograph Ultimate 3000 RSLCnano system.
6. Mass spectrometer control and data analysis Xcalibur 3.0
software.
7. Chromatographic device control Chromeleon Xpress software.
8. Autosampler vials.
9. Autosampler Snap-It Caps and Septa.
10. Clear glass or polypropylene inserts for HPLC and GC autosampler vials.
11. HPLC Columns Acclaim PepMap, (15 cm  50 μm, C 18 ,
3 μm, 100 A and 2 cm  75 μm C 18 , 3 μm, 100 A).
12. Coated Fused-silicaPicoTip Emitter SilicaTip.
13. Acetonitrile, LC-MS grade.
14. Methanol, LC-MS grade.
194
Julia Bespyatykh et al.