Quantification of Protein Kinase Activities by LC-MS - LC-MS/MS in Proteomics: Methods and Applications - page 324

surement of PI3K-dependent kinase enzymatic activity by mass
spectrometry ( 11 ) . This approach allows quantitative analysis of
the PI3K/Akt axis with great sensitivity, precision and specificity,
representing an advantage when compared with alternative meth-
ods based on radioactivity, immunochemistry or fluorescence
( 12 , 13 ) . In this chapter we will provide a thorough description
of this new strategy, which allows for accurate measurement of
the PI3K signalling pathway. Furthermore, this approach can in
principle be applied to measure the activity of any kinase-driven
signalling pathway for which specific peptide substrates are avail-
able, making this method suitable for multiplexing kinase mea-
surements.

2. Materials

2.1.CellCulture
andLysis

1. Human acute monocytic leukaemia cell line (P31/FUJ) as
example of a cancer cell line showing PI3K/Akt signalling
pathway activation. P31/FUJ cell line can be obtained from
the Health Science Research Resources Bank, Japan ( see
Note 1 ).
2. Phosphate-buffered saline (PBS): Prepare 10X stock with
1.37 M NaCl, 27 mM KCl, 100 mM Na 2 HPO 4 ,18mM
KH 2 PO 4 (adjust to pH 7.4 with HCl if necessary) and auto-
clave before storage at room temperature. Prepare working
solution by dilution of one part with nine parts water ( see
Note 2 ).
3. Roswell Park Memorial Institute (RPMI) medium
(Gibco/BRL, Bethesda, MD) supplemented with 10%
foetal bovine serum (FBS, HyClone, Ogden, UT),
100 units/ml penicillin and 100 μ g/ml streptomycin

(Invitrogen, Carlsbad, CA).
4. Example of compound to be tested: Wortmannin
(Calbiochem, San Diego, CA). Prepare a stock solu-
tion of 10 mM in DMSO and store small aliquots at

− 20ºC

( see Note 3 ).
5. Triton buffer for cell lysis: 50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 1 mM EDTA, 1% (w/v) Triton X-100,
0.5mMNaF,1mMNa 3 VO 4 , 0.05 TIU/ml aprotinin,
10 μ M leupeptin, 0.7 μ g/ml pepstatin A, 10 μ g/ml TLCK,

1 mM DTT, 1 mM PMSF, 1 M okadaic acid. Prepare the

stock lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mMNaCl,
1 mM EDTA, 1% (w/v) Triton X-100) and store in aliquots
at

μ

20ºC. The day of the experiment supplement 1 ml of
stock lysis buffer with 1

−

μ l 0.5 N NaF, 10 μ l 100 mM

Na 3 VO 4 ,10 μ l 5TIU/ml aprotinin, 1 μ l 10 mM leupeptin,

1 μ l 0.7 mg/ml pepstatin A, 1 μ l 10 mg/ml TLCK, 1 μ l