Electron Fragmentation-Based Workflows for Characterizing Proteoforms with Agilent Q-ToFs

Through a ten-year collaborative effort with Agilent, we have developed a flexible, cost-effective option that offers superior electron fragmentation of larger peptides and even proteins for the 6545-6560 family of Q-TOF’s. The ExD cell preserves labile post-translational modifications, differentiate isobaric leucine from isoleucine as well as isoaspartate from aspartate, and can cleave disulfide bonds in cysteine knot proteins. These new capabilities have created challenges in how to collect, process and interpret the far richer data sets produced by ExD fragmentation.

To meet these needs, we have developed new processing software to maximize the quality of data collected for efficient processing by a flexible open source solution based on openMS/KNIME. The processing software also produces optimized mzml date files with appropriate metadata for the electron-based fragmentation to be efficiently analyzed by third party vendors such as Protein Metrics. With these new data-processing workflows, ExD makes possible far more complete characterization of proteins and glycoproteins with unprecedented accuracy, speed and simplicity.

For Research Use Only. Not for use in diagnostic procedures.

Presenter: Joseph S. Beckman, PhD (CEO of e-MSion, Inc., e-MSion Inc.)

Joe is a Distinguished Professor of Biochemistry and Biophyics at Oregon State University and a cofounder of e-MSion, Inc. Joe was an undergraduate at the University of Colorado, a Captain in the US Army Medical Service Core and received his Ph.D. from Duke University. He became a Professor in the Departments of Anesthesiology and Biochemistry at the University of Alabama at Birmingham, before moving in 2001 to Oregon State as the Ava Helen Pauling Endowed Professor. He discovered the oxidant peroxynitrite (ONOO-) formed from superoxide and nitric oxide and nitrotyrosine, a common oxidative protein modification in pathology. He has shown protein nitration is a major pathological process mediating pathological processes as diverse as Amyotrophic Lateral Sclerosis and acute lung injury with respiratory viral infections. Shortly, after arriving at Oregon State, Joe began collaborating with Valery Voinov and Doug Barofsky to improve the mapping of oxidative protein modifications, which led to the development of electron-based fragmentation technology being commercialized at e-MSion in collaboration with Agilent Technologies for the past decade. The technology development has been supported by six SBIR awards from NIH. Joe enjoys skiing and white water rafting in the Pacific Northwest in between writing grant applications.

Presenter: Rebecca Glaskin, PhD (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.

Presenter: Cody Schwarzer (Field System Specialist, Agilent Technologies, Inc.)

Cody Schwarzer is a Field System Specialist, responsible for implementation and consulting on biopharma applications with QTOF, Ion Mobility, and ExD technology, focusing on data analysis for emerging technologies.