Higher-order structural interrogation of antibodies using middle-down hydrogen/deuterium exchange mass spectrometry.

Although X-ray crystallography is the "gold standard" method for protein higher-order structure analysis, the challenges of antibody crystallization and the time-consuming data analysis involved make this technique unsuitable for routine structural studies of antibodies. In addition, crystallography cannot be used for the structural characterization of an antibody in solution, under conditions where antibody drugs are active. Intact antibodies are also too large and too complex for NMR. Top-down mass spectrometry coupled to hydrogen/deuterium exchange (HDX) is a powerful tool for high-resolution protein structural characterization, but its success declines rapidly as protein size increases. Here we report for the first time a new hybrid "middle-down" HDX approach that overcomes this limitation through enabling the nonspecific enzyme pepsin to perform specific restricted digestion at low pH prior to HPLC separation at subzero temperatures and online electron transfer dissociation (ETD). Three large specific peptic fragments (12 to 25 kDa) were observed from the heavy chain and light chain of a therapeutic antibody Herceptin, together with a few smaller fragments from the middle portion of the heavy chain. The average amino-acid resolutions obtained by ETD were around two residues, with single-residue resolution in many regions. This middle-down approach is also applicable to other antibodies. It provided HDX information on the entire light chain, and 95.3% of the heavy chain, representing 96.8% of the entire antibody (150 kDa). The structural effects of glycosylation on Herceptin were determined at close-to-single residue level by this method.

Fast Comparative Structural Characterization of Intact Therapeutic Antibodies Using Hydrogen-Deuterium Exchange and Electron Transfer Dissociation.

Higher-order structural characterization plays an important role in many stages of therapeutic antibody production. Herein, we report a new top-down mass spectrometry approach for characterizing the higher-order structure of intact antibodies, by combining hydrogen/deuterium exchange (HDX), subzero temperature chromatography, and electron transfer dissociation on the Orbitrap mass spectrometer. Individual IgG domain-level deuteration information was obtained for 6 IgG domains on Herceptin (HER), which included the antigen binding sites. This is the first time that top-down HDX has been applied to an intact protein as large as 150 kDa, which has never been done before on any instrument. Ligand-binding induced structural differences in HER were determined to be located only on the variable region of the light chain. Global glycosylation profile of antibodies and HDX property of the glycoforms were also determined by accurate intact mass measurements. Although the presence of disulfide bonds prevent the current approach from being able to obtain amino acid level structural information within the disulfide-linked regions, the advantages such as minimal sample manipulation, fast workflow, very low level of back exchange, and simple data analysis, make it well-suited for fast comparative structural evaluation of intact antibodies.

Hydroxyflavones as a new family of matrices for MALDI tissue imaging.

The discovery of new matrices that are suitable for in situ analysis of low molecular-weight compounds by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important technological aspect of tissue imaging. In this work, ten natural flavonoid compounds, including flavone and nine of its mono- or polyhydroxyl-substituted analogues (3-hydroxyflavone, 5-hydroxyflavone, 3,7-dihydroxyflavone, chrysin, 7,3',4'-trihydroxyflavone, fisetin, luteolin, quercetin, and morin) were evaluated as potential MALDI matrices for the profiling and imaging of endogenous lipids in mouse liver, using a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer with a 355-nm Nd:YAG UV laser, in the positive ion mode. When an electronic sprayer was used for matrix coating and with a high-pH (0.1-0.5% ammonia hydroxide) matrix solvent, eight of the ten compounds, all of which had at least one OH group at the C3 or C5 position of the flavone structure, enabled the successful detection of 77 to 161 phospholipids and other lipids. The best results were observed with two penta-OH flavones (i.e., quercertin and morin). Taking quercetin as an example, this matrix showed characteristics superior to those of commonly used MALDI matrices, such as DHB (2,5-dihydroxybenzoic acid), CHCA (α-cyano-4-hydroxycinnamic acid), and 2-mercaptobenzothiazole (2-MBT). These characteristics were: µm-sized matrix crystals, uniform matrix coating, low volatility in the high vacuum (~10(-7) mbar) source, good chemical stability, low yield of matrix-related ions, low matrix consumption, low power threshold for laser desorption/ionization, and improved safety of handling. The use of quercetin led to improved lipid imaging, with 212 lipids being successfully imaged from rat brain in a single experiment and with asymmetric distributions of some lipids in left and right brain hippocampus being observed for the first time.