Protein Protocols Protein Protocols

Mass Spectrometry of Protein Phosphorylation 611
2. C 18 reverse-phase packing material: For preparing the capillary columns, Vydac C 18
material (5 µm particle size), removed from old Vydac 218TP51 columns (Vydac,
Hesperia, CA), was used.
3. Chemicals: Trifluoroacetic acid (TFA) and acetonitrile were from Pierce (Rockford, IL)
and from J. T. Baker (Phillipsburg, NJ), respectively. Ba(OH) 2 was purchased from Merck
(Darmstadt, Germany). Dithiothreitol (DTT) and 10× times concentrated dephosphorylation
buffer (0.5 M Tris-HCl, pH 8.5; 1 mM EDTA) were from Roche Diagnostics
(Mannheim, Germany). Iodoacetamide was from Fluka Chemie AG (Buchs, Switzerland).
4. Enzymes: Enzymes were obtained from the following suppliers: modified trypsin:
Promega (Madison, WI), endoproteinase LysC (Achromobacter protease): Wako Pure
Chemical Industries (Osaka, Japan), endoproteinase GluC, sequencing grade, and calf
intestinal alkaline phosphatase (1000 U/mL): Roche Diagnostics (Mannheim, Germany).
3. Methods
3.1. Enzymatic Digestions of Phosphoproteins
To efficiently trace sites of phosphorylation in proteins, it is important to obtain an
essentially complete proteolysis of the protein of interest (see Note 1). This is best
achieved if the protein is fully denatured by reduction and alkylation with DTT and
iodoacetamide. For reduction, the protein is dissolved in 10 µL of 100 mM Tris-HCl,
pH 8.0 and 8 M urea (freshly prepared); 1.5 µL of 75 mM DTT (dissolved in water) is
added and the protein is reduced for 1–2 h at 37°C. Alkylation is achieved by adding
1 µL of 625 mM iodoacetamide and incubating for 15 min at room temperature.
At this point the protein can be digested with the endoproteinase LysC. The residual
iodoacetamide and urea (approx 6 M) do not appreciably inhibit the enzymatic activity.
The enzyme-to-substrate ratio is kept between 1:50 and 1:20 (w/w). Incubation with
endoproteinase LysC is carried out at 37°C for 1 h. The reaction is stopped by the
addition of 10% TFA to a final concentration of 0.5% TFA.
For trypsin digestion, the urea concentration has to be lowered to 2 M by dilution
with 100 mM Tris-HCl, pH 8.0, whereas endoproteinase GluC was found to be severely
inhibited even by 2 M urea. When using this enzyme, it is best to remove urea completely.
This can be achieved by reverse-phase chromatography or, if the protein fails
to chromatograph with acceptable yields, by acetone precipitation. Digestion with
trypsin or endoproteinase GluC is carried out at an enzyme-to-substrate ratio of 1:50 to
1:20 (w/w) for 2 h at 37°C.
3.2. Enzymatic Dephosphorylation with Alkaline Phosphatase
Dephosphorylation is achieved by dissolving the phosphoprotein or the enzymatic
digest in 10 µL of 100 mM Tris-HCl, pH 8.0. To this, 1 µL of 10× concentrated dephosphorylation
buffer (0.5 M Tris-HCl, pH 8.5, 1 mM EDTA) is added, followed by 1 µL
of calf intestinal alkaline phosphatase (CIP), and dephosphorylation is allowed to proceed
at 37°C for 15 min. The solution is acidified with 3 µL of 1% TFA and analyzed
by liquid chromatography.
3.3. β-Elimination with Ba(OH) 2
A dilute alkali solution is prepared from 150 mM Tris-HCl, pH 8.0, to which solid
Ba(OH) 2 is added in excess of saturation (0.16 M at 20°C) (19). Peptides resulting from
proteolytic digests are dissolved in 10 µL of Ba(OH) 2 solution and incubated at 37°C