Bottom-up and Top-down Disulfide Bond Mapping of Beta-lactoglobulin on a Q-TOF with the Capability to Perform both CID and ECD

Characterization of disulfide bonds is of high importance for the structural elucidation of proteins. Disulfide bonds are an extremely common post-translational modifications in proteins that aid in the stabilization of the native conformation of proteins and maintaining their biological functions and activities. Improper disulfide bonding or scrambling can be extremely harmful, altering the protein’s physiochemical stability and biological properties. To understand the folding process of proteins it is critical to characterize and determine the arrangement of disulfide bonds.

Conventional bottom-up approaches, disulfide bonds are reduced and alkylated prior to enzymatic digestion with loss of disulfide bond connectivity. Digestion under non-reducing conditions generates disulfide-linked peptides for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). With top-down MS, the presence of post-translation modifications can be determined by the intact protein mass spectrum and modification sites can be further determined by MS/MS. Collision-induced dissociation (CID) has reduced dissociation of the protein backbone inside a disulfide loop, while electron capture dissociation (ECD) preferentially cleaves disulfide bonds.

Here we present a bottom-up and top-down disulfide bond analysis of beta-lactoglobulin with a quadrupole time-of-flight instrument capable of both CID and ECD.

Presenter: Rebecca Glaskin, Ph.D. (LC/MS Application Scientist, Agilent Technologies, Inc.)

Rebecca Glaskin is an LC/MS Application Scientist at Agilent with a focus on BioPharma applications supporting the LC-Q/TOF and IM-QTOF platforms. Prior to joining Agilent, Rebecca received her Ph.D. in analytical chemistry from Indiana University in the lab of Professor David Clemmer. While there she designed and constructed home-built instruments, pushing the limits of the mobility resolution that can be obtained with a circular drift tube for the separation of biomolecules (peptides, proteins, carbohydrates, and metabolites). While there, she also studied hydrogen/deuterium exchange of proteins in the gas-phase as a function of time and pressure. Rebecca then went to Boston University as a Postdoctoral Associate in the lab of Professor Catherine Costello to develop a database containing collision cross section values for glycans, peptides, and glycopeptides utilizing Agilent Technologies 6560 IM-QTOF. This database can be used to determine how the collision cross section is altered with the addition of individual saccharide units. The trendlines obtained from this database will be used to predict collision cross sections for glycopeptides based on the conformation and structure of the specific glycoform.