Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry
March 1st, 2020
Neutral SO2 clusters of low kinetic energy (< 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.
Desorption ionization induced by neutral SO2 clusters which we call in short DINeC, is a soft and efficient desorption ionization method. We use it among others for mass spectrometry of biomolecules, such as peptides and proteins. As DINeC, desorption molecules intact from the sample surfaces, subtle chemical changes in these molecules are easily detected in the mass spectra.
The process is illustrated in the following simulation. The incoming clusters shatters through in-cluster surface impact. The surface isotope dipeptide is dissolved in one of the cluster fragments and desorpted by this process.
In the experiment, the beam of SO2 clusters is produced via supersonic expansion from a pulsed nozzle. The molecules that are dissolved and ionized during cluster surface impact are transferred into the ion trap for mass spectrometry. Typical mass spectra only show peaks at the MO set values of the intact molecule.
Demonstrating the procedure will be Karolin Bomhardt, and Pascal Schneider, two PhD students in my laboratory. For standard samples, cut the substrates from silicon wafers with thickness of approximately 0.5 to one millimeters in pieces of one by one square centimeter. Clean the silicon substrates in an ultrasound bath of ethanol and acetone for 15 minutes each.
Dry the substrates in a stream of dry nitrogen gas. Then, drop cast five to 30 microliters of the sample solution on the substrate. Depending on vapor pressure of the solvent, let the sample dry under ambient conditions or in a desiccator until all solvent has been evaporated and a dry film has been formed.
Consider the simplest preparation scheme. As an example, for the investigation of highlighter ink, just draw a dot on the substrate surface. Mount the samples on the sample holder with sticky tape or clamps tightened by screws.
Additionally mount a reference sample such as a micrometer thick film of Angiotensin II on the sample holder. Then, press Vent load lock to vent the load lock system. Wait for five minutes, or until atmospheric pressure has been reached.
Open the load lock and mount the sample holder. Close the load lock and pump down the load lock chamber to a pressure below two times 10 to the negative five millibar. Open the valve to the DINeC chamber and transfer the sample holder with the transfer rod to the main manipulator.
Attach the sample holder to the manipulator. Retract the transfer rod and close the valve between the load lock and the DINeC chamber. Open the valve between the gas cylinder and the nozzle.
Adjust the pressure of the sulfur dioxide and helium gas mixture at the outline of the gas mixing system to 15 bar. Set the position of the manipulator to the reference sample's position. For measuring cationic mass spectra, set the sample and grid bias to plus 40 volts, and plus seven volts respectively.
To drive the pulsed nozzle and the ion trap mass spectrometer, set the external function generator to two Hertz. With the delay generator, set the time delay between the clear trap signal from the ion trap and the trigger signal for the pulsed nozzle to five milliseconds. In the Bruker control software, in the Mode page of the main dialogue window, select Enhanced Resolution for Scan mode.
Type in M over Z 50 to 3000 for range, type in 0.1 milliseconds for accumulation time, type in 10 cycles for average, and for polarity, select positive for the measurement of cationic mass spectra and negative for the measurement of anionic spectra. Once a pressure below three times 10 to the negative six millibar has been reached in the DINeC chamber, start the measurement. Set the M over Z value to the respective mass in order to follow the time dependent chromatogram signal.
Press Operate in the control software. Start recording the measurements by pressing the Record button. Measure test spectrum from a reference sample, such as Angiotensin II for about 300 seconds.
Optimize signal intensity by adjustment of the time delay between the clear trap signal and the signal triggering the pulsed nozzle. With the time delay optimized, move the manipulator to the position of the sample to be measured. Click on the Record button to acquire mass spectra over the time span of interest.
After measurement is finished, load the respective data set in the Data Analysis program as shown here for the reference measurement. Select the time span of interest in the chromatogram with the right button of the mouse. The average spectrum is displayed in a separate window.
Click on Export as ASCII file to export the spectrum as a data file for further processing in a program of choice. The DINeC mass spectrum from an Angiotensin II sample changed with time as the sample was heated to about 140 degrees Celsius. Once the temperature reached the final value, the peak of the attacked protinated molecule at M over Z equalling 1047 dropped and new peaks were observed.
The process is summarized in these three graphs. The additional peaks at M over Z equaling 932, 1012, and 1029 were well observed. Analysis of the reaction kinetics demonstrates that the entity with M over Z equaling 1029 is an intermediate which was further decomposed into smaller fragments as the intensity first increased and then decreased.
The most important features of DINeC are the soft nature of the desorption process, the efficiency, and the high surface sensitivity. They all come together with the low requirements with respect to sample preparation. DINeC can thus be applied to a wide variety of different samples ranging from organic bark materials to complex surface adsorbates in the sub-monolayer region.
In all cases, full chemical information can be obtained in real time.
Overview
This study presents a novel desorption ionization method using neutral SO2 clusters, termed DINeC, for mass spectrometry analysis of biomolecules. The technique allows for real-time observation of chemical changes in peptides and proteins without extensive sample preparation.
Key Study Components
Area of Science
- Mass Spectrometry
- Biomolecular Analysis
- Surface Chemistry
Background
- DINeC is a soft desorption ionization method.
- It enables the analysis of complex surface molecules.
- Real-time monitoring of reactions is possible.
- Minimal sample preparation is required.
Purpose of Study
- To demonstrate the effectiveness of DINeC for mass spectrometry.
- To analyze subtle chemical changes in biomolecules.
- To provide a detailed protocol for sample preparation and measurement.
Methods Used
- Production of SO2 clusters via supersonic expansion.
- Desorption of molecules through cluster surface impact.
- Transfer of ionized molecules into an ion trap mass spectrometer.
- Real-time data acquisition and analysis of mass spectra.
Main Results
- Mass spectra show intact molecular peaks.
- New peaks emerge as temperature increases during analysis.
- Reaction kinetics reveal intermediate species formation.
- DINeC demonstrates high surface sensitivity and efficiency.
Conclusions
- DINeC is a versatile method applicable to various samples.
- It provides comprehensive chemical information in real time.
- The method's low sample preparation requirements enhance its utility.
Frequently Asked Questions
Executive Industry Relevance
DINeC enables soft, matrix-free desorption/ionization of complex biomolecules from surfaces, supporting real-time monitoring of chemical modifications without extensive sample preparation. This capability enhances target validation and mechanistic de-risking in early discovery by providing quantitative, fragmentation-free mass spectra of peptides and proteins at sub-monolayer sensitivity. The method’s compatibility with ion trap MSn allows for detailed structural elucidation, improving predictive confidence in lead identification and preclinical workflows.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by detecting intact molecular ions and subtle chemical changes in peptides and proteins.
- Operational Value: Supports biological de-risking through soft desorption that preserves labile modifications, reducing false negatives in target engagement studies.
- Predictive Value: Provides quantitative surface coverage data down to 0.1% of a monolayer, aiding in portfolio triage based on target abundance and modification status.
Screening & Assay Development
- Scientific Value: Delivers matrix-free, quantitative mass spectra that enable reliable detection of surface-bound analytes without ionization suppression.
- Operational Value: Ensures assay standardization and reproducibility via consistent cluster beam generation and ion trap transfer, minimizing run-to-run variability.
- Scalability: Facilitates platform reuse across diverse sample types, from organic extracts to engineered surfaces, supporting high-throughput screening readiness.
Translational & Preclinical Research
- Scientific Value: Enables real-time observation of surface reactions such as H/D exchange or thermal decomposition, allowing kinetic analysis of biomolecular stability.
- Translational Continuity: Bridges discovery and preclinical workflows by providing continuous chemical information from surface adsorbates to bulk molecules under identical analytical conditions.
- Risk-Adjusted Decisions: Supports go/no-go criteria by identifying degradation intermediates and reaction pathways that may impact drug candidate stability or efficacy.
Pipeline & Workflow Integration
DINeC integrates into the discovery continuum from early target validation through lead identification to preclinical assessment, particularly for studies requiring surface-sensitive, real-time monitoring of biomolecular interactions or modifications.
- Discovery Biology: Supports hypothesis testing and pathway clarification by detecting post-translational modifications and ligand-induced conformational changes on surfaces.
- Screening: Enables assay readiness through reproducible desorption of intact molecules, providing quantitative outputs for hit confirmation and structure-activity relationship mapping.
- Analytics: Delivers high-resolution mass spectra and MSn capabilities that allow teams to compare molecular states, modification sites, and fragmentation patterns across conditions.
- Translational Research: Connects to preclinical validation by monitoring time-dependent chemical changes in peptides and proteins under controlled thermal or chemical stress.
- Enterprise Reuse: Functions as a reusable analytical capability across departments, reducing redundant method development and enabling cross-project data consistency.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by preserving molecular integrity and enabling detection of labile species that may be lost in harsher ionization methods.
- Operational Value: Enhances reproducibility and scalability via standardized cluster beam parameters and minimal sample handling, reducing technical variance.
- Strategic Value: Improves capital efficiency by eliminating matrix requirements and enabling direct analysis of native samples, lowering consumable costs and preparation time.
- Portfolio Impact: Informs risk-adjusted advancement decisions through real-time kinetic data on surface reactions, supporting early identification of liabilities.
Implementation Considerations
- Requires expertise in cluster beam generation, ion trap operation, and surface science principles for optimal setup and data interpretation.
- Needs access to a pulsed nozzle system, sulfur dioxide and helium gas handling infrastructure, and a compatible ion trap mass spectrometer with MSn capabilities.
- Demands cross-team standardization of sample mounting, substrate cleaning, and drying protocols to ensure surface homogeneity and reproducible desorption efficiency.
- Involves adaptation considerations when transitioning from model peptides to complex native samples or heterogeneous surfaces, necessitating surface coverage optimization.
- Includes practical limitations such as the need for ultra-high vacuum conditions and potential cluster fragmentation effects at higher impact energies, which must be controlled to maintain soft desorption.
