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.
Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan.
Chan-Hua
CH
Chen
Jing-Xiang
JX
Hong
Chun-Sheng
CS
Wu
Shu-Hui
SH
Chen
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D045744:Cell Line, Tumor; D004587:Electrophoresis, Agar Gel; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006046:Gold / Q000737:chemistry*; D006801:Humans; D055767:Immobilized Proteins; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D047468:Immunoprecipitation / Q000379:methods*; D053768:Metal Nanoparticles / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; C034284:IgG Fc-binding protein, Streptococcus; D055767:Immobilized Proteins; D007141:Immunoglobulin Fc Fragments; D010446:Peptide Fragments; D010750:Phosphoproteins; D006046:Gold