Louis, MO, USA)

Louis, MO, USA). Boc Anhydride opportunity to gain deeper insights into their gas-phase structural differentiation. The full discrimination of IgG antibody isoforms that possess different disulfide bond numbers and even subtle disulfide bonding patterns can be achieved based on their charge-dependent gas-phase unfolding behaviors and root-mean square deviation in CIU difference spectra. Taken together, the incorporation of all charge states observed in a native ion mobility-mass spectrometry (IM-MS) experiment to CIU analysis could make this strategy sensitive to more subtle structural discrepancies, facilitating the rapid discrimination and evaluation of innovative structurally comparable biotherapeutic candidates with unexplored functions. 1.?Introduction As highly active and specific antibodies produced by the immune system, immunoglobulins (Igs) play a vital role in mediating an immunological response network.1 Recent decades have witnessed an increasing interest in developing Igs Boc Anhydride as biotherapeutics, which benefit from higher efficiency, higher specificity, and fewer side effects than conventional small-molecule-based therapeutics. Antibodies have proven to be a clinically and commercially successful drug class with regard to clinical treatment for various diseases, such as cancer, cardiovascular and neurodegenerative disorders, and other autoimmune diseases.2C4 Among them, as the most abundant of the five classes of Igs, Ig gamma (IgG) comprises three-quarters of all human antibodies in serum. To this end, all marked antibody drugs approved for clinical use are IgG-based therapeutic design templates, which have been widely exploited, including antibodyCdrug GPC4 conjugates5,6 and bispecific antibodies.7 Despite the tremendous market for IgG antibodies, their structural and compositional complexity normally compromises their pharmaceutical benefits, potentially posing challenges in the discovery and development of antibody-based therapeutics.8,9 Thus, the clinical significance of these therapeutic proteins accompanied by high structural complexity has stimulated the development of analytical methods for rapid and comprehensive characterization of therapeutic antibodies. The architecture of IgG antibodies is usually relatively conserved as a characteristic Y-shaped topology through evolution, which is composed of two heavy chains and two light chains united by disulfide bonds. The number of disulfide bonds within the hinge region and connection type between heavy and light chains contribute to four subclasses of human IgGs (utilized restricted enzymatic digestion and 193 nm ultraviolet photodissociation (UVPD) for middle-down proteomics, allowing comprehensive sequencing of large peptides ranging from 4C12 kDa for unambiguous IgG identification.21 Fornelli combined the IdeS enzyme and electron transfer dissociation (ETD) fragmentation for sequencing and characterization of IgGs.22 Mao used top-down electron capture Boc Anhydride dissociation (ECD) with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) for structural characterization of intact IgG1 with extensive sequence coverage.23 Recently, Burgt used matrix-assisted laser desorption/ionization (MALDI) Boc Anhydride FTICR MS coupled with in-source decay fragmentation for top- and middle-down characterization of antibodies.24 As a highly complementary method, native Boc Anhydride MS has recently emerged as a promising method for producing intact gas-phase protein complexes and even membrane proteins, providing valuable stoichiometry and topology information.25,26 Gratifyingly, native MS can be hyphenated with ion mobility-mass spectrometry (IM-MS), which has been widely acknowledged in studying intact protein assemblies, such as dynamics,13,27 thermal stability,28,29 protein refolding,26,30 and antibodyCdrug conjugates.31,32 Despite these successful applications, great challenges remain because the available IM-MS normally has limited spectral resolution, and therefore, fails to produce sufficient information for defining macromolecular structures in detail. Inspired by the pioneering efforts of Ruotolo’s group, these challenges motivated the development of native IM-MS-based collision-induced unfolding (CIU), which has gained rapidly increasing attention owing to its ability to rapidly interrogate subtle tertiary structure differences and chemical modifications in the protein isoforms based on their unfolding patterns and gas-phase stabilities.9,33,34 Upon stepwise collisional activation of protein assemblies, a range of partially unfolded intermediates can occur in the gas phase, which provides a multitude of comparative and diagnostic information of protein complexes detected by IM-MS.35,36 Unfortunately, only protein ions with a single charge state can be quadrupole-selected prior to CIU analysis, resulting in iterative data collection process or a loss of complementary.