N-glycosylation of proteins is now routinely characterized and monitored because of its significance to the detection of disease states and the manufacturing of biopharmaceuticals. At the same time, hydrophilic interaction chromatography (HILIC) has emerged as a powerful technology for N-glycan profiling. Sample preparation techniques for N-glycan HILIC analyses have however tended to be laborious or require compromises in sensitivity. To address these shortcomings, we have developed an N-glycan labeling reagent that provides enhanced fluorescence response and MS sensitivity for glycan detection and have also simplified the process of preparing a sample for analysis. The developed labeling reagent rapidly reacts with glycosylamines upon their release from glycoproteins. Within a 5 min reaction, enzymatically released N-glycans are labeled with this reagent comprised of an NHS-carbamate reactive group, a quinoline fluorophore, and a tertiary amine for enhancing ESI+ MS ionization. To further expedite the released N-glycan sample preparation, rapid tagging has been integrated with a fast PNGase F deglycosylation procedure that achieves complete deglycosylation of a diverse set of glycoproteins in approximately 10 min. Moreover, a technique for HILIC-SPE of the labeled glycans has been developed to provide quantitative recovery and facilitate immediate HILIC analysis of the prepared samples. The described approach makes it possible to quickly prepare N-glycan samples and to incorporate the use of a fluorescence and MS sensitivity enhancing labeling reagent. In demonstration of these new capabilities, we have combined the developed sample preparation techniques with UHPLC HILIC chromatography and high sensitivity mass spectrometry to thoroughly detail the N-glycan profile of a monoclonal antibody.

A conformationally flexible, generation-2,3 poly(aryl ether) dendrimer favors quantitative cascade fluorescence resonance energy transfer without the appearance of undesired chromophore self-quenching interactions such as excimer formation.

The synthesis and photophysical characterization of a generation 1 dendrimer containing four coumarin 2 laser dyes at the periphery and a perylenebis(dicarboximide) derivative at the core are described. It was found that 99% of the UV light absorbed by the peripheral coumarin 2 donor chromophores is transferred through fluorescence resonance energy transfer (FRET) to the core acceptor. Emission from the core via FRET is observed in the near-infrared with a 6.2-fold amplification relative to direct excitation.

A novel fluorescence resonance energy transfer (FRET) system containing a two-photon absorbing dye and a nile red chromophore has been synthesized. Upon two-photon excitation by laser at 815 nm this molecule displays efficient energy transfer from the two-photon absorbing dye to the nile red moiety, with an 8-fold increase in emission compared to the model compound. Similarly, single-photon excitation of the two-photon absorbing moiety at 405 nm results in >99% energy-transfer efficiency, along with a 3.4-fold increase in nile red emission compared to direct excitation of the nile red chromophore at 540 nm. This system provides an effective way to use IR radiation to excite molecules that, by themselves, have little or no two-photon absorption.