In-Depth Analysis of Protein Phosphorylation by Multidimensional Ion Exchange Chromatography and Mass Spectrometry - LC-MS/MS in Proteomics: Methods and Applications - page 113

2.4.SCXHPLC

1. SCX mobile phase A: 0.1% formic acid, 25% ACN.
2. SCX mobile phase B: 300 mM ammonium acetate,
25% ACN.
3. Polysulphoethyl A (Poly LC) SCX column 4.6 mm ×

100 mm (PolyLC, Columbia, MD, USA).
4. HPLC system with fraction collector (1200 series; Agilent,
Wokingham, UK).

2.5.Sequential
Elutionfrom
ImmobilisedMetal
IonAffinity
Chromatography
(SIMAC)

1. Ni(III)-coated Sepharose high-performance beads (GE

2.5.1. ImmobilisedMetal
IonAffinity
Chromatography

Healthcare, Little Chalfont, UK).
2. IMAC chelating solution: 200 mM EDTA.
3. IMAC charging solution: 100 mM Fe(III)Cl 3 .
4. IMAC loading/washing solution: 50% ACN, 0.1% TFA.
5. IMAC elution solutions: (A) 20% ACN, 1% TFA; (B)
NH 4 OH (pH 11.3) (prepare by mixing 940 μ lofwater

20 ◦ C); (C) and 60 μ l of 25% NH 4 OH; can be stored at −

NH 4 OH (pH 11.3) prepared in 50% ACN.
6. 100% Formic acid (FA).

1. TiO 2 beads (Hichrom Ltd, Theale, UK).
2. 100% ACN.
3. 4 M Urea.
4. 1% Sodium dodecyl sulphate (SDS).
5. TiO 2 loading buffer: 80% ACN, 5% TFA, 1 M glycolic acid.
6. TiO 2 washing solutions: (A) 80% ACN, 5% TFA; (B)
10% ACN.
7. TiO 2 elution buffers: (A) NH 4 OH (pH 11.3) (prepare as
indicated in 2.5.1 step 5 ); (B) 30% ACN.
8. 100% FA.

2.5.2.TitaniumDioxide
(TiO 2 )Chromatography

2.6.MSIdentification

Peptide separation is performed by nanoflow reverse-phase
chromatography using a C18 column in a high-pressure liq-
uid chromatography (HPLC) system. In our example, we use
a nanoflow ultrahigh-pressure liquid chromatograph (UPLC;
Acquity, Waters/Micromass) connected on line to a quadrupole