Analytical Instrumentation : Mass spectrometry

Analytical Instrumentation Portfolios


New Mass Spectrometry Detector Uses Optically Active Membrane

UW–Madison researchers and collaborators have developed a mass spectrometry detector that is more sensitive to large molecule impacts and may provide better spatial sensitivity. The detector incorporates a thin membrane made of semiconducting materials. The membrane is optically active, converting the kinetic energy of the molecules that strike its front surface into light photons. The photons are detected and converted to an electrical signal by a photosensor.

Fast Calorie Feedback to Manage Weight and Exercise

UW–Madison researchers and others have developed a weight management test based on the breath carbon isotope ratio. The new test can be performed before and after aerobic exercise, for example, to determine if a person is burning more calories than he/she is consuming.

Breath samples can be collected in bags and analyzed via cavity ringdown spectroscopy (CRDS) or used on site. CRDS uses infrared laser absorption to measure the concentrations of 13CO2 and 12CO2 signals and determine the ratio.

The new test could be embodied in a handheld device and made accessible in health centers and clinics.

Identifying Related Peak Sets to Boost Mass Spectrometry Throughput

UW–Madison researchers have developed an algorithm for identifying related peak sets from MS1 spectra data.

First, an intensity peak is selected from the MS1 data and its peak location is identified. Based on intensity values associated with all potentially related peak locations, an intensity score is calculated. This score determines whether or not the peak locations form a related set. Related peaks may optionally be selected for MS2 processing.

Multiplexed Mass Spectrometry Quantification with Neutron-Encoded Mass Tags

UW–Madison researchers have developed customized tagging reagents for MS proteome quantification. Called neutron encoded mass tagging, or ‘NeuCode,’ the method exploits small mass differences, on the order of millidaltons (mDa), between heavy isotopes, such as nitrogen or carbon, which can be coupled with amino acids used during cell culture similarly to SILAC. Through instrument resolution settings, NeuCode labeling allows the user to control when multiplexed information is masked, limit sample complexity, and when it is revealed, produce quantitative data. Furthermore, these mDa mass differences can be incorporated into novel chemical tags that couple to peptides and enable analysis of biological samples from any source. Unlike isobaric tags, however, specialized reporter groups, linkers and charge sites are not required.

Cost-Effective Isobaric Tandem Mass Tags for High Throughput Quantitative Proteomics and Peptidomics

UW–Madison researchers have designed and synthesized novel N,N-dimethylated amino acid eight- and 16-plex isobaric MS/MS tagging reagents.

The reagents consist of a reporter group and a balancing group that are isotopically coded to provide eight compounds with equal molecular weights. The balancing group is designed to provide eight isotopic combinations. The reagents feature an amine reactive group capable of reacting with the molecule to be tagged. Compared to iTRAQ reagents, the eight-plex dimethyl leucine reagents also give rise to high intensity parent and reporter ions, offering enhanced sensitivity and dynamic range for detection and quantitation of low-abundance analytes.

Gas-Phase Purification of Peptides Reaps Accuracy in Mass Spectrometry Quantitation

UW–Madison researchers have developed a method to eliminate interference by directly segregating ions of interest from similarly massed and charged non-targets or contaminants that were unintentionally co-isolated between stages of MS/MS.

This is accomplished using samples embedded with isobaric tags. Following initial ionization, an established proton transfer reaction (PTR) is commenced, reducing the charges of ions in the gas phase by introducing even-electron anions. The populations thus diverge according to mass-to-charge ratio, with the precursors of interest able to be selected.

During subsequent analysis of the purged ions, their tags are cleaved, fragmenting into charged particles that generate data readouts. Relative abundance of the purified peptides thus can be derived with significantly improved accuracy.

Increasing Peptide and Protein Identifications with Prioritized Mass Spectrometry

UW–Madison researchers have developed a universally compatible, computationally based method in which multiple precursor ion attributes—such as mass, intensity, m/z ratio and charge state, as well as results obtained from previous scans—are used to calculate the likelihood of identification, thus prioritizing subsequent analysis.

The method comprises MS/MS analysis of a sample (or training sample) containing proteins and peptides, with one or more compounds optionally labeled with isobaric tags. Established procedure for analyte ionization and mass-to-charge separation generates precursor ions. The ions then are detected and analyzed for information related to two or more physical properties (mass, charge state, etc.) and directed for MS2 dissociation—selecting and/or allotting resources to the most identifiable ions as determined by the algorithm. Segregated and detected for mass and abundance, the fragmented ions provide further characteristic data used to identify the compounds. Additionally, the process permits novel, or first-time, identification of precursor ions during an experiment or within an ID database.

Enabling prioritized, probability-focused mass spectrometry, the innovative software achieves increased sample identification with little or no time increase and requiring no additional hardware.

Mass Spectrometry Data Acquisition Method Enables More Reliable Large-Scale Protein Quantitation

UW–Madison researchers have developed a platform for analyte quantitation that prevents the acquisition of mass spectra that will not result in usable data due to interference by modifying the data acquisition software. During the automated precursor selection process, candidate precursors with significant interference are rejected until a suitable replacement is found. This method enables increased quantitation accuracy while maintaining high levels of throughput.

Specifically, a distribution of precursor ions from an analyte is analyzed using mass spectrometry to identify a precursor peak in the ion mass spectrometry data corresponding to a precursor ion. The data allows determination of the amount of interference within a preselected range about the precursor peak. Then, an adjusted range of ions may be selected for analysis such that the amount of interference is less than a selected value.

Parallel Measurements by Fluorescence and Mass Spectrometry for Absolute Quantification of Proteins

UW–Madison researchers have developed an improved method for MS-based quantification using an integrated electrospray emitter and fluorescence detector. Solution-phase measurements are employed to overcome the limitations in standard quantification techniques using measured intrinsic fluorescence to quantify selected amino acids. The inventors have developed a label-free technique that exploits the property of intrinsic fluorescence exhibited by tryptophan-containing peptides and proteins as a means to provide both relative and absolute quantification of proteins and peptides in complex mixtures identified through tandem mass spectrometry.

Integration of the emitter and detector permits quantification on a single stream of analyte while placement of the fluorescence detector immediately before the electrospray emitter provides improved correlation between the measurements and reduction of chromatographic dead volume. A continuous capillary may be used to provide not only the electrospray emitter, but also the liquid chromatography column. This combination allows effective analysis of extremely small amounts of material at nanoliter flow rates and a significant reduction in dead volume, which may cause loss of chromatographic resolution and sensitivity.

Microwave Cavity Detector for Highly Sensitive Mass Spectrometry Detection of Large Molecules

UW-Madison researchers have developed a detector for mass spectrometry that employs a tuned microwave cavity to increase the sensitivity of time measurements. The detector can be used in mass spectrometers that provide a source of ionized molecules, which are accelerated in an electric field before reaching the detector. The design includes a cavity of conductive material providing an electromagnetically tuned cavity, which includes an opening positioned to receive the accelerated molecules. An antenna communicates with the cavity to receive an electrical signal caused by electromagnetic resonance of the cavity. Detection electronics receive this electrical signal, which marks the arrival of the ionized molecule in the cavity and allows for time of flight measurement. With proper configuration of the frequency of resonance in the cavity, its modes and its quality factor, time resolutions on the order of one nanosecond are possible. 

Labeling Peptides with Basic Functional Groups to Improve Mass Spectrometric Analysis

UW–Madison researchers have developed methods of labeling proteins and peptides with tertiary amines or other functional groups to enhance ion fragmentation and improve identification of target molecules. Labeling the proteins or peptides allows them to acquire more charges during electrospray ionization mass spectrometry. The more highly charged ions fragment more extensively during ETD. This leads to more sequence information and thus improved protein identification. It also helps to locate the sites of post-translational modifications (PTMs). 

The methods utilize a chemical labeling strategy that reacts a tagging reagent with one or more carboxylic acid groups of the target molecule. The tagging reagent comprises a binding group that can react with the target peptide and a functional group that can improve the fragmenting characteristics of the target. The functional group may be a tertiary amine or other functional group having high gas-phase basicity. Alternatively, the functional group may comprise a protected amine, phosphonium group or sulfonium group. The tagging reagent also may contain one or more stable isotopes, allowing a single mass spectrometric analysis of labeled peptides or proteins to provide precise relative quantification as well as improved protein identification.

Use of Nanomaterials to Enrich Phosphopeptides for Mass Spectrometry-Based Proteomics

UW–Madison researchers have developed a method and materials for isolating, purifying and enriching the concentration of compounds containing phosphate groups, including phosphorylated peptides and proteins. The mesoporous nanomaterials are made from transition metal oxides, which selectively and reversibly bind phosphorylated compounds. These materials are relatively easy to prepare and have nanopore structures attractive for enrichment due to large surface area, high flow-through capacity, chemical stability and robustness.

In practice, the enrichment materials are contacted with a sample containing a mixture of phosphorylated and non-phosphorylated compounds. When contacted with the mesoporous enrichment material, the phosphorylated compounds reversibly bind to the surface while the remainder of the solution passes through the column. Once separated, the phosphorylated compounds are then removed from the mesoporous metal oxide enrichment material via controlled release.

These methods and materials are highly versatile and can be used for highly efficient enrichment, purification and effective analysis of phosphorylated compounds in a variety of biological environments. They also are highly complementary to both bottom-up and top-down mass spectrometric-based protein identification methods and can be used to effectively apply these methods in the study of proteomics.

Improved Molecule Mass Detection Using Electron Field Emission from Kinetically Impacted Membranes

A UW-Madison researcher has developed an active detector and method for sensing molecules based on the generation of electrons through field emission (FE) and/or secondary electron emission (SEE).  This device is capable of detecting large molecules at higher temperatures than previous devices.

The detector is made of a semiconductor membrane, such as silicon or silicon nitride, with an “external” and “internal” surface.  The external surface contacts the desired molecules, and the internal surface is made of a thin electron emitting layer.  Kinetic energy from the molecules is absorbed through the external surface to the internal surface via vibrational quanta, which cause electrons to be emitted from the internal surface.  The emitted electrons then can be detected by an electron detector. 

Any material that emits electrons via FE or SEE can be used on the internal surface, including highly doped semiconductors and doped diamond materials.  The electron emitting layer can be electrically biased to enhance FE or SEE.

Space and Time Variant Electric Field Enhancement Improves Accuracy of Mass Spectroscopy for Large Compounds

UW-Madison researchers have developed a mass spectroscopy technique that uses a dynamic electric field to increase the differences in particle velocity.  By making the electric field variable with respect to space and time, particles of different masses experience different charges, allowing for greater differences in velocity.  Thus smaller acceleration voltages, drift regions and detector sizes are required.

The electric field gives heavier ion particles less kinetic energy than the lighter particles.  This spreads the particle velocities out and enhances the spectrograms of heavier mass species.  A computer program then finds the amount of energy gained by each particle and, with the particle velocity, calculates the particle masses.  The ability to control the electric field strength in such a way allows for the magnification of the spectrogram axes as well.  The user can zoom in on the different peaks and even focus the peaks to reduce their widths.  This dramatically increases the versatility of mass spectroscopy.

Real-Time Tandem Mass Spectral Data Analysis for Protein Sequence Identification

UW-Madison researchers have developed a technique that allows for real-time identification of unknown peptides using mass spectral analysis to identify and characterize proteins during a tandem mass spectral analysis. 

With sufficient MS resolution, the z-type product ions of the ETD technique can be uniquely identified, in turn allowing for immediate identification of c-type product ions.  Because of the labeling of both product ions, other spectra peaks due to noise can be eliminated and the overall spectra quality can be determined.  The masses of the z- and c-type product ions are compared against a computer database to identify one or more “putative chemical compositions,” which are the amino acid sequences of the peptide.  The putative chemical compositions then are confirmed by comparison to peptide amino acid sequences in a database or via de novo analysis using a computational model without extrinsic comparison.  The results from either of these methods can be used to identify and characterize the protein from which the peptide was derived.  This process is incorporated into an algorithm that can make automated decisions to determine the best course of action for the mass spectral analysis.

Ionizable Isotopic Labeling Reagents for Relative Quantification by Mass Spectrometry

UW-Madison researchers have developed small, inexpensive ionizable tags for non-targeted relative quantification of a variety of biological metabolites, peptides and proteins by liquid chromatography-mass spectrometry (LC-MS). The tags differ in their isotopic composition and react quantitatively with amines or carboxylic acid groups, offering a powerful approach to relative quantification of multiple analytes between two samples by electrospray ionization-mass spectrometry (ESI-MS).

One sample is labeled with an isotopically-light reagent and the other sample is labeled with an isotopically-heavy reagent, which yields a characteristic mass shift in the spectra. The relative intensity for each peak pair allows for the precise determination of the relative concentration of multiple analytes between samples. In addition, these reagents enhance the ionizability of the analytes in positive-ion mode MS, resulting in lower detection limits.

Faster Gas-Phase Protein Sequencing

UW–Madison researchers have developed a sequencing method that combines the time-tested de novo capability of Edman degradation chemistry with the speed and sensitivity of mass spectrometry.

In the new process, a protein or polypeptide molecule is introduced into a mass spectrometer and allowed to accumulate in the linear ion trap. Edman degradation reactions then are conducted in gas phase (within the linear ion trap) by introducing chemical reagents. After each cycle the product ions are selectively transmitted through the mass spectrometer to determine their mass-to-charge ratios. The chemical identities of the amino acid residues are determined based on the mass spectra thus generated.

Improved Sensor Enables Sensitive Detection of Large Molecules in Mass Spectrometry

UW-Madison researchers have developed a new type of sensor for use in a mass spectrometer. Because this sensor does not require the molecule of interest to generate a secondary electron for detection, it avoids the size limit and is capable of detecting larger molecules with more sensitivity than currently available technology.

This device consists of a thin membrane with nano-electromechanical systems (NEMS) pillar resonators distributed in an array fashion on its inner surface. The membrane is set in motion to cause the pillar resonators to vibrate. The vibrational state of these pillars controls the spatial distribution of the electrons that are emitted. Then the molecule of interest interacts with the receiving surface of the membrane, changing the resonance frequency of at least one pillar and altering the electron emission. To sense the molecule, this change can be detected using a fluorescent film or other method.

Electrospray Ionization Ion Source with Tunable Charge Reduction

The researchers have improved the physical layout of that device by moving the corona discharge and its associated electromagnetic fields outside the charge reduction chamber, making it easier to collect the ions in the mass spectrometer for analysis. This device configuration also provides independent control over the conditions and processes involved in analyte and reagent ion formation, avoids perturbations in the trajectories of analyte ions and charged droplets caused by operation of the reagent ion source, and allows use of a wide range of reagent ion sources.

Deposition of Samples and Sample Matrix for Enhanced Sensitivity of MALDI Mass Spectrometry

UW-Madison researchers have developed a method of using an ultrasonically actuated microplotter to deposit both the sample and the overlying matrix, resulting in a MALDI target composed of small, homogeneous sample spots. See WARF reference number P01201US for more information about the microplotter itself. This technique results in smaller sample spots than can be obtained with current methods, leading to better mass spectrometry readings. Researchers in both university and industrial settings could use this method for high throughput, high-sensitivity MALDI analyses.

Inductive Detection for Mass Spectrometry

UW-Madison researchers have developed an enhanced electrospray ionization, time-of-flight mass spectrometer for the analysis of large or complex biomolecules. This device combines a single droplet ion source with multiple, “on-axis”, non-destructive, inductive ion detectors. The implementation of on-axis ion trajectories throughout the mass analyzer, along with multiple detectors, allows pre-acceleration ion velocities to be determined. A second set of ion detectors detects post-acceleration velocities, providing time-of-flight information while reducing ion detection losses of larger, slower-moving biomolecules. The detectors can be configured to simultaneously analyze charge states and mass-to-charge ratios, enabling the determination of absolute mass.

Droplet Ion Source for Mass Spectrometry

UW-Madison researchers have now developed a new TOF mass spectrometer that uses single charged droplets as ion sources and focuses these droplets, through use of an aerodynamic lens, onto the center axis of the mass analyzer. Discrete ion droplets are produced by ESI through the use of a novel piezoelectric dispenser (see WARF reference number P01294US). The combination of these advances promises to make mass spectrometry suitable to a wide range of previously unattainable applications, including DNA sequencing, protein identification, and quantification of relative protein expression levels.

Piezoelectric Charged Droplet Source for Mass Spectrometry

UW-Madison researchers have developed a new nanotechnology source that generates single packets of ions for mass spectrometric analysis. A sample solution held at a selected electric potential is ejected from a narrow exit aperture by constriction of a cylindrical piezoelectric element.

Unlike conventional nanoelectrospray sources that are continuous, this source dispenses discrete volumes (droplets) of electrically charged solutions as small as 10 picoliters. It also allows adjustable control of droplet exit time, ion formation time, droplet number, repetition rate and electric charge state of the droplet. When coupled to an orthogonal time-of-flight mass spectrometer, the source provides detection sensitivity in the attomole range.

New Method of Charge Reduction in Electrospray Mass Spectrometry

UW-Madison researchers have developed an improved means to reduce and control the charge states of ions generated by electrospray ionization. They previously developed a technique for reducing the charge state of ions by using a polonium alpha particle source (see WARF reference number P99352US). The technology featured here improves upon the previous one by using a corona discharge to reduce ion charge state distributions. It also allows users to produce gas-phase ions of positive or negative polarity, and which carry either a fixed charge or possess a charge state distribution that varies selectively over time.

Reducing the Charges of Ions Generated by Electrospray Ionization for Mass Spectrometry

UW-Madison researchers have developed a device and method to reduce the charge state of ions produced by electrospray ionization, making it possible to analyze large molecules, such as DNA, by mass spectrometry. In this method, ESI is used to convert macromolecules into ions carrying multiple charges. Next, these ions undergo reactions with bipolar ions generated from a polonium alpha particle source. This charge reduction process creates ions carrying mostly single charges, greatly simplifying data analysis once the ions are analyzed by mass spectrometry.