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286 Tchaga
proteins is variable. However, phosphohistidine is estimated to be 10- to 100-
fold more abundant than phosphotyrosine, but less abundant than phosphoserine
and phosphothreonine (8).
The only currently viable method for enrichment of the complete phosphoprotein
complement is immobilized metal ion affinity chromatography (IMAC)
with hard metal ions.
IMAC was introduced by Porath and coworkers (9) in 1975 under the name
of metal chelate affinity chromatography. This short publication reported for the
first time the use of immobilized zinc and copper metal ions for the fractionation
of proteins from human serum.
The classical system cited by most scientists in the IMAC field is that of
Pearson (10), who postulated that metal ions can be divided into three categories
according to their preferential reactivity with nucleophiles: hard, intermediate,
and soft. To the group of hard metal ions belong Fe 3+ ,Ca 2+ , and Al 3+ all of
which have a preference for oxygen.
Hundreds of papers have been published since, describing the use of immobilized
hard metal ions in group separations of phosphorylated proteins, and the
future of this particular application field looks very bright indeed (11–24). These
adsorbents are also finding broad application for enrichment of phosphorylated
peptides (25–29).
In this chapter, an outline is presented of a typical experimental protocol that
ensures reproducible and quantitative enrichment of all phosphorylated proteins
with exposed phosphorylated side chains.
When attempting to enrich the phosphorylated proteins from any given
biological sample, one needs to take into consideration the following issues:
1. Phosphorylation–dephosphorylation processes are generally quick processes.
Important consideration must therefore be given to the time for extraction, loading
of the sample and the initial washes (30). Speedy removal of phosphatases is
important as the presence of phosphatase inhibitors such as sodium ortho-vanadate,
might be undesirable during the chromatography.
2. In general, gaining an as complete as possible enrichment is more important
than obtaining a higher purification factor that results in losses of phosphorylated
proteins in the non-adsorbed fraction (see Table 1 and Fig. 1 for typical yields of
phosphorylated proteins from different sources). It is clear, therefore, that further
reduction of complexity has to occur after this first step (before one would be
able to identify and quantify the individual phosphorylated proteins from the total
proteome).
3. Selective and complete enrichment of the total phosphorylated proteome is impossible
under native conditions. A simple example is the formation of homodimeric
and heterodimeric Stat protein complexes upon their phosphorylation and
transport to the nucleus (31,32). In this case, a phosphorylated side chain of
tyrosine is involved in the formation of the Stat protein dimers. Accordingly, this