Gold Nanoparticle-Based Immuno Dual Probes for Targeting Proteomics

Immunoprecipitation combined with mass spectrometry (MS)
is a promising technique for targeting proteomics in characterizing
submicrograms of target protein and interacting proteins in living
cells. This method, however, is limited by interference arising from
nonspecific binding. We report a novel gold nanoparticle (AuNP)-based
immuno probe approach for immunoprecipitation. By cross-linking the
antibody Fc domain to protein G covalently modified on AuNPs, the
probe was fabricated and characterized to have 60 protein G and 30
immunoglobins per AuNP. We used human immunoglobin against the target
and mouse immunoglobin with the same isotype (IgG) to fabricate the
target and preclear probe, respectively, and termed it as the dual
probe approach. Our results showed that the preclear probe (AuNP-IgG)
and the target probe (AuNP-anti-ERα) share a similar panel of
nonspecific binders but dramatic different specificity toward the
target. Thus, using the dual probe method, we showed major nonspecific
binders in the cell lysate could be largely removed without sacrificing
the target protein. Compared to the conventional agarose gel-chromatography,
the AuNP-based probe exhibited less nonspecific interference and higher
recovery yield for ERα. Moreover, the AuNP-based probe is more
inert than the agarose gel under harsh conditions and does not induce
dissociation of the cross-linked IgG that could interfere with target
identification. Using AuNP-based dual probes, ERα was shown
to be purified from MCF-7 cells with minimum nonspecific binding.
Moreover, the identity and phosphorylation sites on the C-terminus
of the purified ERα could be positively confirmed by MS using
only 1 mg of cellular protein.