Mapping of Phosphorylation Sites by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 127

variants thereof will detect different, partially overlapping parts
of the phosphoproteome ( 7 ) , meaning that no method can give
a complete overview, but rather a distinct subset of the biologi-
cal state. The last section of the chapter deals with some general
aspects of the mass spectrometric acquisition of phosphopeptide
samples plus a few directional hints to the evaluation and data
analysis.

2. Materials

1. Sample/wash/equilibration solution: 30% acetonitrile 2.1. IMAC

250 mM acetic acid solution.
2. Optional: methanolic HCl: Prepare methanolic HCl by
adding 160 μ L of acetyl chloride to 1 mL of anhydrous

methanol.
3. Phosphate buffer A: Prepare 50 mM phosphate buffer (pH
8.9) by dissolving 0.0063 g monosodium phosphate mono-
hydrate and 1.3 g disodium phosphate heptahydrate in 100
mL water ( see Note 2 ).
4. Phosphate buffer B: Prepare 100 mM phosphate buffer (pH
8.9) by dissolving 0.0126 g monosodium phosphate mono-
hydrate and 2.66 g disodium phosphate heptahydrate in 100
mL water.
5. 1 M Hydrochloric acid (HCl) solution.
6. PHOS-Select TM gel (Sigma-Aldrich).
7. Mobicol spin column (MoBiTec, Göttingen, Germany).
8. C18 (ultra) microspin columns (Harvard Apparatus Ltd,
Edenbridge, United Kingdom).

1. Sample-/resin-/wash solution: saturated phthalic acid solu-
tion: Dissolve 125 mg of phthalic acid in 1 mL of 80% ace-
tonitrile and 3.5% trifluoroacetic acid (TFA). Some phthalic
acid will not dissolve.
2. Wash solution 1: 0.1% TFA solution in water.
3. Wash solution 2: 80% acetonitrile (ACN), 0.1% TFA in
water.
4. Ammonium hydroxide solution: 0.3 M ammonium hydrox-
ide (NH 4 OH) solution.
5. Titanium dioxide resin (GL Science, Saitama, Japan).
6. Mobicol spin column (MoBiTec, Göttingen, Germany).

2.2.TiO 2

For the isolation of phosphopeptides using the phosphoramidite
chemistry (PAC) method, a number of chemicals are required.

2.3.PAC