Chemoenzymatic Approach for the Preparation of Asymmetric Bi‑, Tri‑, and Tetra-Antennary <i>N</i>‑Glycans from a Common Precursor

Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric <i>N</i>-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has <i>N</i>-acetylglucosamine (GlcNAc), <i>N</i>-acetyllactosamine (LacNAc), and unnatural Galα­(1,4)-GlcNAc and Manβ­(1,4)-GlcNAc appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a substrate, and thus this structure can be uniquely extended. Next, the β-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural α-Gal and β-Man terminating antennae can sequentially be decaged by an appropriate glycosidase to liberate a terminal β-GlcNAc moiety, which can be converted into LacNAc and then elaborated by a panel of glycosyltransferases. Asymmetric bi- and triantennary glycans could be obtained by removal of a terminal β-GlcNAc moiety by treatment with β-<i>N</i>-acetylglucosaminidase and selective extension of the other arms. The power of the methodology is demonstrated by the preparation of an asymmetric tetra-antennary <i>N</i>-glycan found in human breast carcinoma tissue, which represents the most complex <i>N</i>-glycan ever synthesized. Multistage mass spectrometry of the two isomeric triantennary glycans uncovered unique fragment ions that will facilitate identification of exact structures of glycans in biological samples

Parsimonious Charge Deconvolution for Native Mass Spectrometry

Charge deconvolution infers the mass from mass over charge (<i>m</i>/<i>z</i>) measurements in electrospray ionization mass spectra. When applied over a wide input <i>m</i>/<i>z</i> or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new “parsimonious” charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously and, when focused on the <i>m</i>/<i>z</i> range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using <i>m</i>/<i>z</i> peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies