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.