Native-MS analysis of monoclonal antibody conjugates by Fourier transform ion cyclotron resonance mass spectrometry

Antibody drug conjugates (ADCs) are an important class of therapeutic molecule currently being used to treat HER2-positive metastatic breast cancer, relapsed or refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, relapsed or refractory B-cell precursor acute lymphoblastic leukemia and acute myeloid leukemia. An ADC typically consists of a small molecule or peptide-based cytotoxic moiety covalently linked, via lysine or cysteine residues, to amonoclonal antibody (mAb) scaffold. Mass spectrometric (MS) characterization of these molecules afford highly accurate molecular weight (MW) and drug-to-antibody ratio (DAR) determination, and is typically performed using orthogonal acceleration time-of-flight (oa-ToF) analysers and more recently Orbitrap instruments. Herein we describe for the first time the use of a 15 Tesla solariX Fourier transform ion cyclotron mass spectrometer to characterize an IgG1 mAb molecule conjugated with biotin via native lysine and cysteine residues, under native-MS and solution conditions. The cysteine biotin conjugates remained fully intact, demonstrating the ability of the FT-ICR to maintain the noncovalent interactions and efficiently transmit labile protein complexes. Native-MS was acquired and is displayed in magnitude mode using a symmetric Hann apodisation function. Baseline separation is achieved on all covalent biotin additions, for each charge state, for both the lysine and cysteine biotin-conjugates. Average DAR values obtained by native-MS for the lysine conjugate are compared to those derived by denaturing reversed phase liquid chromatography using an oa-ToF MS system (1.56 ±0.02 versus 2.24 ±0.02 for a 5-molar equivalent and 3.99 ±0.09 versus 4.43 ±0.01 for a 10-molar equivalent, respectively). Increased DAR value accuracy can be obtained for the higher biotin load, when using standard ESI conditions as opposed to nanoESI native-MS conditions. Both denatured LC-MS and native-MS spectral data were deconvoluted using a parsimonious based algorithm, without the need for parameter adjustment

Baseline resolution of isomers by traveling wave ion mobility mass spectrometry: investigating the effects of polarizable drift gases and ionic charge distribution

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)We have studied the behavior of isomers and analogues by traveling wave ion mobility mass spectrometry (TWIM-MS) using drift-gases with varying masses and polarizabilities. Despite the reduced length of the cell (18cm), a pair of constitutional isomers, N-butylaniline and para-butylaniline, with theoretical collision cross-section values in helium ((He)) differing by as little as 1.2 angstrom(2) (1.5%) but possessing contrasting charge distribution, showed baseline peak-to-peak resolution (Rp-p) for their protonated molecules, using carbon dioxide (CO2), nitrous oxide (N2O) and ethene (C2H4) as the TWIM drift-gas. Near baseline Rp-p was also obtained in CO2 for a group of protonated haloanilines (para-chloroaniline, para-bromoaniline and para-iodoaniline) which display contrasting masses and theoretical (He), which differ by as much as 15.7 angstrom(2) (19.5%) but similar charge distributions. The deprotonated isomeric pair of trans-oleic acid and cis-oleic acid possessing nearly identical theoretical (He) and (N2) as well as similar charge distributions, remained unresolved. Interestingly, an inversion of drift-times were observed for the 1,3-dialkylimidazolium ions when comparing He, N-2 and N2O. Using density functional theory as a means of examining the ions electronic structure, and He and N-2-based trajectory method algorithm, we discuss the effect of the long-range charge induced dipole attractive and short-range Van der Waals forces involved in the TWIM separation in drift-gases of differing polarizabilities. We therefore propose that examining the electronic structure of the ions under investigation may potentially indicate whether the use of more polarizable drift-gases could improve separation and the overall success of TWIM-MS analysis. Copyright (c) 2013 John Wiley & Sons, Ltd.489989997Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Brazilian Science Foundation FINEPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Submicrometer Emitter ESI Tips for Native Mass Spectrometry of Membrane Proteins in Ionic and Nonionic Detergents

Native mass spectrometry (native-MS) of membrane proteins typically requires a detergent screening protocol, protein solubilization in the preferred detergent, followed by protein liberation from the micelle by collisional activation. Here, submicrometer nano-ESI emitter tips are used for native-MS of membrane proteins solubilized in both nonionic and ionic detergent solutions. With the submicrometer nano-ESI emitter tips, resolved charge-state distributions of membrane protein ions are obtained from a 150 mM NaCl, 25 mM Tris-HCl with 1.1% octyl glucoside solution. The relative abundances of NaCl and detergent cluster ions at high m /z are significantly reduced with the submicrometer emitters compared with larger nano-ESI emitters that are commonly used. This technique is beneficial for significantly decreasing the abundances (by two to three orders of magnitude compared with the larger tip size: 1.6 μm) of detergent cluster ions formed from aqueous ammonium acetate solutions containing detergents that can overlap with the membrane protein ion signal. Resolved charge-state distributions of membrane protein ions from aqueous ammonium acetate solutions containing ionic detergents were obtained with the submicrometer nano-ESI emitters; this is the first report of native-MS of membrane proteins solubilized by ionic detergents. Graphical Abstract