Biomedical Engineering Reference
In-Depth Information
2. Materials
1. Triethylammonium bicarbonate (TEAB) supplied as 1 M
stock.
2. Sodium dodecyl sulphate (SDS). Dissolved at 0.1% (w/v) in
1 M TEAB and remainder discarded after use.
3. iTRAQ
R
2.1.Sample
Preparationand
iTRAQLabelling
reagent labelling kit (AB Sciex, Warrington, UK).
20
◦
C. Extremely susceptible to hydrolysis.
4. Tris-(2-carboxyethyl)phosphine (TCEP). 50 mM stock pre-
pared in water and stored at
Store at
−
20
◦
C.
See
Note 1
.
5. Methylmethanethiosulphate (MMTS; Perbio Scientific,
Cramlington, UK). 200 mM stock prepared in isopropanol
and stored at
−
20
◦
C.
6. Sequencing grade-modified trypsin.
7. Lysis buffer: 1 M TEAB with up to 0.1% SDS.
−
2.2.SampleAnalysis
by2D-Liquid
Chromatographyand
TandemMass
Spectrometry
1. Strong cation exchange chromatography (SCX) buffer A
(SCX-A): 10 mM KH
2
PO
4
(Sigma), 20% (v/v) acetonitrile
(Fisher Scientific, Loughborough, UK), pH 2.7, made with
HPLC-grade water (Rathburn Chemicals, Waterburn, UK).
Lifetime 2-3 days.
2. Strong cation exchange chromatography (SCX) buffer B
(SCX-B): 10 mM KH
2
PO
4
, 20% (v/v) acetonitrile, 1 M KCl
(Sigma), pH 2.7, made with HPLC-grade water. Lifetime
2-3 days.
3. Strong cation exchange chromatography column: (10
×
m beads, 200 Å
pore size; Hichrom Ltd., Reading, Berks, UK).
4. Reverse-phase chromatography (RP) buffer A (RP-A): 2%
(v/v) acetonitrile, 0.1% (v/v) formic acid, made with
HPLC-grade water. Lifetime 2-3 days.
5. Reverse-phase chromatography (RP) buffer B (RP-B): 80%
(v/v) acetonitrile, 0.1% (v/v) formic acid, made with
HPLC-grade water. Lifetime 2-3 days.
6. Reverse-phase
2.1 cm PolyLC Polysulfoethyl A column, 5
μ
chromatography
trap column: Acclaim
PepMap C18 (5
μ
m, 100 Å) in a 300
μ
m inner diameter
×
5 mm column (LC Packings/Dionex, Camberley, UK).
7. Reverse-phase chromatography analytical column: Acclaim
C18 PepMap100 (3
μ
μ
m, 100 Å)
in a 75
m
inner diameter
×
15 cm column (LC Packings/Dionex,
Camberley, UK).
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