Shu-Hua Chen

Fabrication of Oriented Antibody-Conjugated Magnetic Nanoprobes and Their Immunoaffinity Application

In an attempt to fabricate highly active immunoprobes for serum biomarker detection, we report a simple and effective method for site-specific and self-oriented immobilization of antibodies on magnetic nanoparticles (MNPs). Through boronate formation, the carbohydrate moiety within the constant domain, Fc, of the antibody can be specifically and covalently linked to a boronic acid-functionalized MNP (BA@MNP) without hindering the antigen binding domain, Fab. The performance was evaluated by immunoaffinity extraction of multiple serum antigens. Compared with the random immobilization of antibody on a MNP, the antibody self-oriented immunoprobe provides long-term stability (>2 months) and 5-fold extraction efficiency. It also provides 5-fold improved sensitivity at a low nM range (0.4 nM), presumably through enhanced antibody@MNP activity. In addition, false-positive detections arising from nonspecific binding can be completely minimized by effective surface protection using concentration-dependent dextran blocking. Compared with conventional antibody site-specific immobilization through protein G, this new BA-mediated covalent antibody immobilization provides interference-free extraction resulting from noncovalent immobilization of antibody by protein G. The new immunoassay was applied in comparative profiling of serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP) in human serum. Our triple immunoassay revealed a distinct pattern among normal patients, patients with cancer, and patients with cardiovascular disease. Using the previously reported quantization capability of the MALDI MS readout, we expect that this site-specific immunonanoprobe-based immunoassay can be highly active, rapid, and accurate in nanodiagnosis.

Multiplexed Immunoassay: Quantitation and Profiling of Serum Biomarkers Using Magnetic Nanoprobes and MALDI-TOF MS

Taking advantage of efficient affinity extraction by surface-functionalized magnetic nanoparticles (MNPs) and accurate MALDI-TOF MS readout, we present a multiplexed immunoassay for simultaneous enrichment and quantitation of multiple disease-associated antigens, serum amyloid A (SAA), C-reactive protein (CRP), and serum amyloid P (SAP) from human serum. To obtain reproducible MALDI signal response with direct on-MNP detection, the seed-layer method improved homogeneity of the cocrystallization of MNPs and captured antigens. Our methodology demonstrated good quantitation linearity of targeted analytes (R(2) approximately 0.97) with reduced signal variation (RSD < 10%). The lower limit of quantitation is in the nanogram level with overall assay precision (intraday, 7.0%; interday, 11.3%) and accuracy (intraday, 6.3%; interday, 17.5%) including steps of nanoprobe extraction and MALDI-TOF MS analysis. This triplexed immunoassay showed overexpression of SAA and CRP in patients with cardiac catheterization or gastric cancer (P < 0.05), consistent with single-analyte ELISA and previous studies. Compared to the determination of disease onset by single protein quantitation, our multiplexed immunoassay revealed a distinct triplexed pattern in the control group, patients with gastric cancer, and cardiac catheterization. On the basis of the advantages of flexibility in nanoprobe preparation, high specificity and sensitivity, and rapid screening by MALDI-TOF MS, this platform may provide a new methodology for disease diagnosis.

Carbohydrate-encapsulated gold nanoparticles for rapid target- protein identification and binding- epitope mapping

The interactions of cell-surface glycoproteins and glycolipids play important roles in cell–cell communication, proliferation, and differentiation. Combinations of saccharides, orientations of glycosidic bonds, and branching patterns of linkages allow complex carbohydrates to have a vast diversity of structures for molecular recognition. Thus, studies of carbohydrate-related interactions might provide new insights into their biological roles and reveal new possibilities for drug development. 4] Disclosure of the carbohydrate-recognition sites by X-ray crystallography and NMR spectroscopy has been a challenge due to the difficulty of cocrystallization of targeting proteins and carbohydrates. At present, most of the binding-epitope analysis methodologies are time-consuming as they screen sets of overlapping peptides spanning a known protein sequence. 7] The advent of an efficient, sensitive, general strategy to identify new carbohydrate-binding lectins and map epitopes is awaited to unravel the complexities of carbohydrate recognition. Recent developments in mass spectrometry have greatly expanded the possibility of characterizing unknown proteins, including mapping of protein glycosylation sites. Despite the advantages, the simultaneous characterization of the hundreds to thousands of proteins present in a complex medium still remains a challenge. However, when mass spectrometry is combined with a biologically active probe to rapidly and specifically target proteins of interest, this targeted proteomic approach can accelerate research for class-specific proteins or biomarkers. Recently, metal nanoparticles have been used in biological separation and promise to be superior to microbeads. Furthermore, biomolecule-conjugated gold nanoparticles (AuNPs) are the most popular probes because of their readily assembling with thiolated molecules, their large area/ volume ratio for investigating three-dimensional interactions, and their ease of separation by centrifugation. 13] However, the use of functionalized nanoparticles as probes combined with mass spectrometry for carbohydrate–protein recognition studies has not been explored. We report here a new approach of using carbohydrate-encapsulated AuNP (c-AuNP) as an affinity probe for the efficient separation and enrichment of target proteins, and then protein identification and epitope mapping by MALDI-TOF MS. The analytical scheme of the approach, nanoprobe-based affinity mass spectrometry (NBAMS), is illustrated in Scheme 1. Unlike other mass spectrometry-based affinity capture approaches that make use of agarose beads or biochips, the core component of our scheme is a nanosized biologically active affinity probe. Target proteins can be affinity captured from a mixture by the nanoprobe and directly analyzed on-probe by MALDI-TOF MS. Most significantly, once target proteins have been captured, on-probe digestion followed by removal of unbound peptides allows rapid mapping of carbohydrate-recognition peptide sequences in the proteins. To demonstrate the general applicability of the NBAMS technique in tackling carbohydrate–protein interactions, proof-ofprinciple was performed for the specific capture and identification of the galactophilic lectin Pseudomonas aeruginosa lectin I (PA-IL) by using c-AuNP. The medium-range affinity (Ka~3.4 10 m ) of monomeric d-galactose for PA-IL was enhanced by assembling sugars on nanoparticles. The resulting multivalent interactions between c-AuNP and PA-IL facilitated highly specific and stable surface affinity separation. To probe the subtle variations in the carbohydrate-binding domain of PA-IL, two carbohydrates—galactose and P antigen (Gala1! [a] Dr. Y.-J. Chen, Y.-W. Chang, H.-K. Liao, C.-Y. Chang, M.-D. Jan, Dr. C.-C. Lin Institute of Chemistry and Genomic Research Center Academia Sinica Sec. 2 Academia Road, Taipei, 115 (Taiwan) Fax: (+ 886) 2-2783-1237 E-mail : yjchen@chem.sinica.edu.tw cclin@chem.sinica.edu.tw [b] S.-H. Chen, Y.-Y. Chien, Dr. K.-T. Wang Department of Chemistry, National Taiwan University Taipei 115 (Taiwan) Supporting information for this article is available on the WWW under http ://www.chembiochem.org or from the author.

Mass spectrometric analysis of additives in polymer extracts by desorption chemical ionization and collisional induced dissociation with B/E linked scanning

Abstract Desorption chemical ionization in combination with collisional induced dissociation and B/E linked scan proved to be a viable method for identifying additives in polyethylene extracts by mass spectrometry. In comparison with fast atom bombardment (FAB), much less fragmentation was observed with desorption chemical ionization (DCI) using ammonia as reagent gas. Furthermore, most likely due to the “matrix effect”, not all the additives in the extracts were detected by FAB. The softness and the lack of matrix effect make ammonia DCI a better ionization technique than FAB for the analysis of additives directly from the extracts. Most likely due to higher collision energy, product ion mass spectra acquired with a double focusing mass spectrometer provided more structural information than the spectra obtained with a triple quadrupole mass spectrometer. Besides contamination from the seals of the supercritical fluid extractor, high and low density polyethylenes extracted with supercritical carbon dioxide produced similar results as with toluene.

Exploring the expression bar code of SAA variants for gastric cancer detection

We reported an integrated platform to explore serum protein variant pattern in cancer and its utility as a new class of biomarker panel for diagnosis. On the model study of serum amyloid A (SAA), we employed nanoprobe‐based affinity mass spectrometry for enrichment, identification and quantitation of SAA variants from serum of 105 gastric cancer patients in comparison with 54 gastritis patients, 54 controls, and 120 patients from other cancer. The result revealed surprisingly heterogeneous and most comprehensive SAA bar code to date, which comprises 24 SAA variants including SAA1‐ and SAA2‐encoded products, polymorphic isoforms, N‐terminal–truncated forms, and three novel SAA oxidized isotypes, in which the variant‐specific peptide sequence were also confirmed by LC‐MS/MS. A diagnostic model was developed for dimension reduction and computational classification of the 24 SAA‐variant bar code, providing good discrimination (AUC = 0.85 ± 3.2E−3) for differentiating gastric cancer group from gastritis and normal groups (sensitivity, 0.76; specificity, 0.81) and was validated with external validation cohort (sensitivity, 0.71; specificity, 0.74). Our platform not only shed light on the occurrence and modification extent of under‐represented serum protein variants in cancer, but also suggested a new concept of diagnostic platform by serum protein variant profile.