(1053-B) High-throughput intact protein analysis using Acoustic Ejection Mass Spectrometry (AEMS) on a quadrupole time of flight mass spectrometer
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: Proteins play a key role in almost every biochemical process. The analysis of intact protein molecules via traditional mass spectrometry allows for the characterization of these proteins. For example, accurate molecular weight determination is important to understand their role in the biophysical landscape. The need for high-throughput intact protein analysis has increased in the pharmaceutical/biopharmaceutical industries. The most common way to analyze these proteins is by peptide mapping or a “bottom-up” experiment, in which a protein is digested with an enzyme (such as trypsin) and the resulting peptides are analyzed by LC-MS to determine site-specific post-translational modifications. Throughput currently limits peptide mapping LC-MS analysis, as minutes or hours are needed to achieve the necessary characterization. An alternative approach is to analyze the intact mass of the protein first and compare the data against a known sequence, therefore allowing faster characterization and analysis time, while preserving high sensitivity and mass accuracy.
This presentation describes the use of a prototype mass spectrometer capable of Acoustic Ejection Mass Spectrometry (AEMS) for the analysis of intact proteins. The prototype system uses an alternative approach to rapidly analyze proteins and solve some challenges faced by traditional techniques. Samples are placed into well plates held by the system and acoustic energy is applied to the bottom of the well plate, causing a droplet of the sample to be ejected. The Open Port Interface (OPI) is located above the well plate and captures the ejected droplet for dilution and transport by a carrier solvent (~400 µL/min) of the diluted sample to an ESI for ionization and delivery of diluted sample ions to a high resolution mass spectrometer for analysis.
This study explores important parameters (for example, carrier solvent composition, chemical additives and varying matrices) that affect data quality of high resolution AEMS on a high-throughput scale for intact protein analysis. Modal proteins (for example, ubiquitin, lysozyme, streptavidin, c-reactive protein and NISTmAb IgG1k) were selected to benchmark the system in various concentrations and buffers with a Beckman Coulter® Biomek i7 liquid handler to create assay-ready plates. Preliminary AEMS data demonstrate the rapid analysis of compounds, as a 384-well plate can be analyzed in 10-30 minutes (1-5 seconds/sample).