Biology Reference
In-Depth Information
emerged from mass spectrometry (MS)-based phosphoproteomics
strategies. To facilitate protein phosphorylation analysis, a number
of enrichment methods have been applied to separate phosphory-
lated proteins or peptides from non-phosphorylated ones. A col-
lection of metal-based affi nity methods such as immobilized metal
affi nity chromatography (IMAC), metal oxide affi nity chromatog-
raphy (MOAC), and strong cation exchange (SCX) are among the
most commonly used. These enrichment methods, in combination
with MS, now allow detection and quantifi cation of hundreds of in
vivo phosphorylation sites from complex biological samples. Today,
enrichment of phosphorylated peptides using TiO
2
upon proteoly-
sis digestion of the protein extract has become popular and is
applied most frequently. Besides enrichment strategies that target
the phosphate moiety of phosphopeptides, methods for the enrich-
ment of phosphoproteins have also been described. However,
dynamics of protein abundance in a given cell or tissue, the tran-
sient nature and low-stoichiometry of the phosphorylation event
remain major challenges in phosphoproteomics.
In this chapter we describe a cost-effi cient workfl ow for the
consecutive enrichment of phosphoproteins and peptides and their
subsequent analysis by LC/MS. We combine denatured protein
extraction with highly selective Al(OH)
3
-MOAC enrichment of
phosphoproteins [
1
,
2
], proteolytic cleavage of enriched phospho-
proteins, and TiO
2
-MOAC of phosphopeptides. In our hands, this
approach ensured reproducible enrichment of transiently modifi ed
peptides allowing direct identifi cation and site-specifi c quantifi ca-
tion of phosphorylation of many low abundant phosphoproteins
such as transcription factors, kinases, and phosphatases [
3
].
2
Materials
Prepare all solutions using ultrapure water (bi-distilled or double
distilled, deionized) and analytical/ultra HPLC grade reagents.
1. 10 % (w/v) Trichloroacetic acid (TCA) in acetone.
2. 10 % (w/v) TCA in dH
2
O.
3. Dense SDS buffer: 100 mM tris(hydroxymethyl)aminometh-
ane (Tris) pH 8.0, 30 % (w/v) sucrose, 2 % (w/v) sodium
dodecyl sulfate (SDS), 5 % (v/v)
2.1 Total Protein
Extraction
β
-mercaptoethanol.
4. Phenol, Tris-saturated pH 8.0.
5. 100 mM Ammonium acetate in methanol (MeOH).
6. Incubation buffer A (IB/A): 30 mM 2-(
N
-morpholino)-
ethanesulfonic acid (MES) pH 6.1, 0.25 % (w/v)
3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate
(CHAPS), 7 M urea, 2 M thiourea.
