ASMS: Agilent HRAM user meeting

Name: ASMS: Agilent HRAM user meeting
Start: 2025-06-01T11:00:00.000Z
End: 2025-06-01T15:00:00.000Z

1 - 1. June 2025

Explore the future of high-resolution accurate mass spectrometry at Agilent’s HRAM User Meeting, June 1 in Baltimore. Topics include native MS, CIU, 2DLC, and digital Q-TOF. Includes lunch and networking!

For more information click here

HRAM user meeting

Location: Baltimore Marriott Inner Harbor, 110 S Eutaw St, Baltimore, MD 21201
Room: Ballroom
Date: Sunday, 1 June 2025
Time: 1:00 pm - 5:00 pm

Register

12:00 PM - 1:00 PM Networking Lunch

*Please indicate whether or not you will be attending in the "Personal Information" form of the registration process.

1:00 PM - 1:10 PM Innovation in HRAM Software and Technology to Solve New Challenges in Characterization of Native Biomolecules, Lipids, and Small Molecules

John Sausen - Agilent Technologies, Inc.

Working with our collaborators, Agilent is making advances in Higher Order Structural Tools enabling characterization of biomolecules through the utilization of automated microdroplet flash Characterization for subunit analysis, as well leveraging ECD alternative fragmentation - increasing coverage at both the peptide and top down native intact protein level. Collaborators have further improved the selectivity of intact native proteins with Digital Quadrupole technology enabling isolation of high mass individual charge states. New Approaches from GenNext enable protein foot printing as an alternative to HDX while eliminating back exchange. Coupling these techniques with Ion Mobility and ECD with top down software further enables a more complete interrogation of proteoforms. IM with Collision Induced Unfolding CIU continues to increase in utility identifying subtle changes in HOS of biomolecules, extending beyond protein unfolding to also include CIU of oligonucleotides. The addition of an experimental static nano spray further enhances the ability to identify minor changes in native HOS This depth of characterization can be further enhanced with the addition of 2D chromatography for native top down proteoform characterization.

Enhancements in the PNNL Pre-Processing software tools now enable processing of 4D DIA IM data using QTOF software, very effective for a number of applications including looking at DIA of drift separated neutral Loss experiments of co-eluting lipids and enhancing identification of unknowns with DIA data. We can also use this capability when studying impurities - collecting DIA Ion Mobility of multiple drift resolved oligo charge states and associated fragments, or multiple drift resolved GLP-1 conformers, and then comparing the fragmentation of specific conformers in either Bioconfirm or “ExD Viewer” leveraging the drift dimension to compare fragmentation of a charge states or conformers using DIA with minimal interference.

1:10 PM - 1:50 PM Collision Induced Unfolding of Biotherapeutics: Rapid, Sensitive, Information-Rich Stability Measurements

Brandon T. Rutuolo - Associate Chair for Research and Professor, Department of Chemistry, University of Michigan

Medicines of the future will rely heavily upon our ability to quickly assess the structures and stabilities of complex macromolecular machines and evaluate the influence of large libraries of conformationally-selective small molecule binders. In addition, the ever-growing and complex array of protein and nucleic acid-based biotherapeutics similarly demands improved tools that can rapidly detect changes in therapeutic structure upon target binding, alterations in formulation, or stress. Current structural biology tools lack the throughput and sensitivity to meet these challenges. In this presentation, I will discuss recent developments surrounding collision-induced unfolding (CIU) methods that aim to bridge this technology gap. CIU uses ion mobility-mass spectrometry to measure the stability and unfolding pathways of gas-phase proteins, without the need for covalent labels or tagging, and consumes 10-100 times less sample than almost any other label-free technology. Recent developments in high-throughput CIU screening methods, their ability to track small alterations in structure over a wide array of biotherapeutics, integration with electron capture dissociation, and software developments that seek to enhance CIU information content will be discussed.

1:50 PM - 2:10 PM High-Efficiency Electron Capture Dissociation Enables Top-Down and Bottom-Up Discovery Proteomics at Chromatographic and Ion Mobility Timescales with an LC-Q-TOF

Blaine R. Roberts - Associate Professor, Emory University School of Medicine

Therapeutic monocolonal antibodies (mAbs) are one of the fastest growing classes of drugs. The silencing of unwanted binding to Fc‐gamma receptors has resulted in many different variants which have entered clinical trials. Therefore, fast structural characterization of antibodies is in need. One of the best known is “LALA” (L234A/L235A), which has shown resistance to IdeS cleavage in both solution and microdroplet reactions, limiting the analysis of subunits. Here we show for the first time, the efficient and rapid microdroplet digestion of antibodies that have mutations “LALA”, “LAGA”, and “LFLE” with FabRICATOR Xtra, an IdeS enzyme derived from Streptococcus castoreus. The digestion took place during the spray ionization process, using an Agilent jet stream (AJS) ion source in less than 1 ms, with digestion efficiency over 80%. An increased enzyme to antibody ratio was shown to increase digestion efficiency. Reduced antibody with a single chain showed a higher digestion efficiency than the intact one with two chains, leading to subunits with high ion abundance for structural identification. Our digestion is an integrated microdroplet protein digestion technique with automated sample flow injection, Agilent Jet Stream (AJS) ion source and online mass spectrometry analysis, providing a rapid and robust method for structural characterization of mAbs with mutations. In this talk, I will also present ultrafast digestion of IgG with various enzymes, including trypsin and ProAlanase, for rapid IgG characterization. We expect that our method will have a high impact on monoclonal antibody drug development.

2:10 PM - 2:30 PM Optimization of Q-TOF Instruments for Native Protein Analysis

David Russell - Professor, Department of Chemistry, Texas A&M University

Native mass spectrometry (nMS) is now widely recognized for its high information content for studies of large biomolecules, especially proteins/protein complexes. Figures of merit for nMS include sensitivity, resolution, and the capability to investigate specific reaction pathways via measurement ensemble-averaged products, including conformational analysis and ligand binding thermodynamics. Although nMS shows promise for analysis of native proteins, the large size, low charge, and number of states that proteins adopt (i.e. microstates) can make characterization of native proteins difficult. Our lab focuses on optimizing Q-TOF mass spectrometers to characterize native protein dynamics and stabilities by adapting variable-temperature electrospray ionization (vTESI), digital quadrupole (DigiQ) isolation, and electron capture dissociation (ECD) to the instruments. vTESI allows the temperature of a protein solution to be varied in the nanospray tip, which controls the distribution of protein microstates present in solution; those microstates are then “freeze-dried” with electrospray ionization before analysis. DigiQ isolation employs digital electronics to select ion populations at high m/z (m/z > 8000) and can be used to effectively select charge states of native protein complexes. ECD can be used to generate fragment ions that return relevant sequence, modification, and structure information on native proteins, which can be used to obtain information on their dynamics and stabilities. Optimization of Q-TOF-MS instruments for native investigation of proteins using these methods provides new ways to characterize the protein energy-folding landscape, and unlocks new avenues for protein research and analysis.

2:30 PM - 2:45 PM Break

2:45 PM - 3:05 PM Automated Flash Characterization of Therapeutic Antibodies with Fc-Silencing Mutations using Ultrafast Microdroplet Digestion

Hao Chen - Professor, Chemistry and Environmental Science, New Jersey Institute of Technology

3:05 PM - 3:25 PM High-Throughput Online Two-Dimensional Chromatography Strategies for Top-Down Proteomics

Ying Ge - Professor of Cell and Regenerative Biology and Chemistry, University of Wisconsin-Madison

Top-down proteomics (TDP) provides a holistic view of the proteoforms for deciphering proteoform function, uncovering disease mechanisms, and advancing precision medicine. However, the proteome complexity poses a significant challenge for TDP, requiring effective separation of proteins prior to mass spectrometry. We have recently developed an online two-dimensional liquid chromatography (2DLC) for high-throughput and automated TDP by leveraging Agilent's advanced LC-QTOF-MS platform with extensive fragmentation (MSn). Our newly developed native 2DLC method achieves comprehensive coverage of endogenous protein complexes within the chromatographic timescale, enabling high-throughput structural analysis of protein complexes and enhanced proteoform characterization. We have successfully detected 133 native proteoforms and endogenous protein complexes (up to 350 kDa) from human heart tissue in under two hours. We have also developed a unified software package, MASH Native, for analysis of native TDP data to process complex datasets, providing a “one-stop shop” for characterizing both native protein complexes and proteoforms. These advancements will significantly improve large-scale TDP studies in health and disease.

3:25 PM - 3:45 PM Advances in Digital Quadrupole Mass Spectrometry

Elizabeth Groetsema - Brian Clowers Group, Department of Chemistry, Washington State University

Quadrupole technology is a vital component of modern mass spectrometry instrumentation, given its considerable role in facilitating robust tandem MS experiments. Digital quadrupole technology differs from conventional methods by applying a high-precision square or rectangular RF waveform instead of the traditional high-voltage sinusoidal RF waveform. As a complementary approach, digital waveform technology (DWT) can augment existing instruments with new capabilities, including constant resolution scans and agile m/z transitions. Using a quadrupole time-of-flight MS platform, we illustrate the capacity of DWT to access higher Mathieu stability zones and realize narrow m/z isolation widths using low-voltage rectangular waves. In addition to narrowed isolation windows for low m/z analytes, we will provide insights into how DWT can complement existing sinusoidal workflows, and how new digital prefilter approaches suggest the need for novel quadrupole arrangements for next-generation platforms.

3:45 PM - 4:00 PM Lipid Perturbations Due to Wildfire Smoke Exposure

Haley C. Jostes - Erin Baker Group, Department of Chemistry, University of North Carolina at Chapel Hill

As global temperatures rise, wildfires have increased in duration, frequency, and intensity. Emitted smoke is a growing public health concern due to its association with numerous adverse health effects, including respiratory-related illnesses and cardiovascular diseases. Moreover, recent studies illustrated that biological responses are dependent on smoke composition, which can be highly variable and complex due to factors such as the biomass fuel type and age, and the combustion conditions. Lipids are essential regulators of cellular health, yet their involvement in wildfire smoke-induced toxicity remains to be characterized. To evaluate perturbed molecular pathways due to smoke inhalation, a lipidomic analysis was performed for mice exposed to various wildfire simulations.

Extracts were analyzed with a multidimensional platform coupling liquid chromatography, ion mobility spectrometry, collision induced dissociation, and mass spectrometry (LC-IMS-CID-MS). Data was processed using a Skyline library containing over 877 unique lipids. A total of 401 lipids were identified, and statistical comparisons between the negative control samples and each wildfire condition identified four to seven significant lipids for each exposure type (α= 0.05 and log2FC =1). Interestingly, across all exposures, the abundance of monosialodihexosylgangliosides (GM3s), the simplest type of gangliosides, increased. The enrichment of GM3s contributes to the formation of highly dynamic and specialized microdomains including caveolae and lipid rafts, which alter the regulation of cell functions. To understand the molecular pathways influenced by increased GM3 abundance, transcriptomic data of the same samples were evaluated. Initial screening of GM3-related genes revealed an increased expression of IL-6 (a pro-inflammatory mediator) across most smoke exposures. Decreased expression of a GUSB transcript (a glycosaminoglycan degradative enzyme) was also observed, which could be linked to lysosomal storage disorders.

4:00 PM - 4:10 PM Thank you, Intro to Agilent Content at ASMS, Closing Remark

John Sausen - Agilent Technologies, Inc.

Agilent Technologies