Our X-Omics lab include the following instrumentation capabilities:
Ion Mobility Mass Spectrometer (MS)
Our Ion Mobility MS is composed of a nanoAcquity Ultra Performance Liquid Chromatography (UPLC) MS and a Synapt G2-S Ion Mobility MS. A reflectron time-of-flight (TOF) instrument is used for ion detection. Ion detection can be accomplished under various experimental setup to acquire mass spectra under high sensitivity and/or high mass resolving power (m/Δm50%) conditions.
For structural characterizations, ions can be fragmented via collision induced dissociation (CID) in various components of the IM-MS system.
nanoAcquity Ultra Performance Liquid Chromatography (UPLC) MS
Liquid Chromatography (LC) is an analytical technique used for separating components of a mixture dissolved in a liquid mobile phase. Separation occurs as different analytes flow through a solid stationary phase. Each of the components (depending on their functional group types, size, partitioning between the two stationary and mobile phases, ion-exchange, and absorption) can interact with the stationary phase in different manner which can cause these various components to flow through the stationary phase at different speeds and hence be separated. The different flow rates can be used to separate the two components. UPLC is one of the most efficient instruments that employs liquid chromatography (LC). The UPLC gains efficiency by utilizing columns that are packed with “packing material” smaller than 2.5 micrometers. Specifically, these columns are packed with Acquity BEH, which is a silica-polymeric mixture that has a bridging between the methyl groups in the silica for extra stability. The UPLC also allows for temperature control, automated switching between columns, a bypass channel for flow injections, and three different detectors (the photodiode, Turnable UV, and Evaporative Light Scattering). The UPLC has a flow rate range of 200 nanoliters/minute to 100 microliters/minute and can have columns with 75 micrometers to 1 millimeter diameter and 100 micrometers to 25 millimeters length which can operate under 10,000 psi. This allows the UPLC to have improved peak capacity, peak shape, and resolution (as compared to the conventional HPLC). The UPLC has applications in biomarker discovery, protein identification, and characterization, degradation studies, amongst other areas of study.
Synapt G2-S Ion Mobility MS
The Synapt G2 employs Ion Mobility and Time of Flight (TOF) Mass Spectrometry for multidimensional analyses (e.g., drift times, m/z values, relative abundances). Ion mobility MS operates by first ionizing the components of a sample (biological fluids, protein mixtures, etc) and guiding selected ions into the mobility device where a drift gas flow restricts the movement of ions as they are being accelerated electrically toward the detection region of the mass spectrometer. Ion acceleration or drift is accomplished by establishing a potential difference on the drift rings, which line the tube or drift region. As ions flow through the drift region, they collide with the inserted drift gas, which slows the ions advance though the tube. Smaller ions travel faster, as they are hit less often and vice versa. As ions reach the detection region and are accelerated toward the detector, they hit the detector and generate a cascade of electrons which can be recored to yield a spectrum based on the flight time of each ion. TOF (t) will depend on the acceleration electric field amplitude (V), flight tube length (x, or distance ions travel), and mass-to-charge ratio (m/z) and ion velocities (v) will depend on translational or kinetic energy (KE) of ions governed by KE = qV = 1/2mv^2. Using x = vt TOF of each can be calculated. In other words, since the ions vary in mass, they will travel through the flight tube at different velocities and hit the detector at different times. This data can then be collected (using an oscilloscope) and converted into m/z values. The Synapt G2-S is a ion mobility mass spectrometer that has several additions that allows it to have increased signal intensities, higher signal-to-noise ratios (S/N), and increased limits of quantitation. At its maximum data collection capacity, this instrument has an acquisition rate of 30 spectra/second. It employs stepwave ion transfer-an off-axis design, which increases the efficiency of ion transfers from the ion source. It also contains software, such as MSE Data Viewer, High Definition Imaging, and Time-Aligned Parallel Fragmentation and Electron Transfer Dissociation, which allows for visualization and interpretation of multidimensional MS data, maximization of selectivity and confidence in MALDI imaging experiments (we do not have this capability with the current setup), and faster and more complete structural identification and characterization. The Synapt G2 has applications in proteomics, biomarker discovery, pharmaceuticals, lipidomics, structural elucidations, metabolomics, polymer analysis, petroleum characterization, metabolite identification, among other x-omics research areas.
Gas Chromatography (GC)
Gas Chromatography (GC) is a technique used to separate and detect volatile substances, using an inert gas as the mobile phase and a liquid stationary phase. The components of a mixture are separated based on their interactions with mobile and stationary phases; variations in retention times (RT) within the column will allow different molecule to elute from the column at different times. Detection of these “slow” and/or “fast” eluting compounds will allow their separation and detection to yield a gas chromatogram.
Gas Chromatography (GC)-Fourier Transform Ion Cyclotron Resonance (FT-ICR)
Successful operation of conventional FT-ICR MS experiments involves seven specific steps or events separated in time. Hence, unlike TOF or sector type instruments whereas detectors and ionization sources are separated in space (“spatial” mass spectrometers), in FT-ICR MS ,the ionization and detection events are separated in time (i.e., a “temporal” instrument).
A typical event sequence in an FT-ICR MS experiment includes the following steps: (1) ion quenching or cleaning of the ICR cell, (2) ionization or generation of using CI, EI, ESI, MALDI, RFI, or other types of ionization mechanisms, (3) ion transfer into the ICR cell within the homogenous part of a superconducting magnet (please note that for internal ion sources this step is not necessary as ions can be generated inside the ICR cell, (4) ion trapping, (5) reaction delay (for ion-molecule reactions in kinetics studies and/or to perform ion isolation or ion fragmentation in tandem MS), (6) ion excitation using CHIRP or other types of waveforms, and (7) ion detection and subsequent Fourier transform operation followed by frequency to m/z conversion. Quenching is an ion removal process in which potential contaminants and leftover ions are ejected from the ICR cell to have a clean FT-ICR cell to start the experiment and data acquisition. Quenching is achieved by applying a positive voltage on one of the ICR trapping plates (e.g., the Quadrupole trapping plate in our instrument) and a negative voltage on the opposing trapping plate (e.g., filament trapping plate in our instruments). Positive and negative ions can be attracted to plates with opposite signs and lost. For example, positive ions and negative ions will be accelerate toward negative and positive trapping plates, respectively. Once ions hit the trapping plates, they can be neutralized and pumped away. Ions need to be removed from the FT-ICR cell to increase the signal.
Ionization is controlled by FT-ICR MS and GC eluting compounds are ionized in an external GC source. Prior to trapping ions, the ICR cell is “quenched” to remove any potential ions and contaminants from a previous run. At the same time, ions are accumulated before they are injected into the ICR cell. Accumulation refers to the collection of a sufficient number of ions for a successful FT-ICR experiment. Accumulation is achieved in the Hexapole by applying AC voltage to the six poles. The six poles create a funnel for the ions to flow through. Three of the poles have positive voltages and the other three have negative voltages. These alternating voltages allow ions to be accumulated within the hexapole assembly. Once accumulations is achieved, ions can be injected into a quadrupole assembly for ion transfer into the ICR cell. Ions flow through the Hexapole, and then through the Quadrupole to enter into the FT-ICR cell. The Quadrupole operates in a similar manner as the Hexapole, except it has four poles instead of six poles. Appropriate voltages (such as “getting”), and sometimes neutral collisions, are used to slow down the ions, restrict their “z” motion (along the magnetic field lines) and trap them within the cell. Recall that the entire ICR assembly is located inside of strong magnetic field and hence ions’ x-y motions are restricted. This combination of electric (E) and magnetic (B) fields allows for ions to a have a number of periodical motions that include trapping frequency, magnetron frequency, and ion cyclotron frequency up to this point.
Excitation is the process by which ions are excited with RF voltages in order to for the mass spectrum to be created. The FT-ICR cell is set up with two sets of parallel plates. One of the sets is an excitation plate and the other set is a detection plate. The excitation plate excites ions in the specific m/z window and starts them into a motion known as the cyclotron motion. This motion is caused by a magnet propelling the ions into a rotation in the FT-ICR cell and an electric field propelling the ions in a lateral and rotational motion. If the radius becomes too large, the ions will hit one of the six plates in the FT-ICR cell and neutralize upon impact. Various frequencies are used until all unwanted ions have neutralized in the FT-ICR cell. Isolation can also be achieved with stored waveform inverse Fourier transform (SWIFT). It essentially uses a mathematical function to turn the m/z value into a frequency that the FT-ICR can use to isolate the designated ion. Once all unwanted ions have left, the detector plate registers the induced current caused by the ions as they lose energy when the excitation plates are turned off. Fourier Transform is used to convert the included current values into a mass spectrum during the detection step.
GC/FTICR with 9.4 T Magnet
Our GC/FT-ICR is equipped with a 9.4 tesla (T) superconducting magnet (Cryomagnetics, Inc – Oak Ridge, TN). Ions within a magnetic field will experience “Lorentz force” and follow a circular motion (cyclotron motion); the frequency of this ICR motion is inversely proportional to the mass ion (m) and directly related to the number of charges (z). The ICR angular frequency (ω) of an ion within a magnetic field (B) can be calculated using ω = 2Πf = ezB/m. On the other hand, cyclotron frequencies of unknown ions can be experimentally measured and converted to m/z values to construct a mass spectrum. Higher magnetic fields are more suitable to obtain higher mass resolving power and detect larger mass ions. Currently FT-ICR MS systems equipped with magnets of up to 15 T magnetic field strength are available but lower magnetic field instruments (e.g., 12, 9.4, 7, 3, etc) are more common. Our 9.4 tesla system was one of the first modern GC/FT-ICR MS systems worldwide and it was designed and configured in-house. Currently this system is equipped with CI, EI, and RFI sources and can be used for analysis of complex sample mixtures such crude oil, petroleum products, and volatile organic compounds (VOCs).
Electrospray Ionization (ESI)-FTICR MS
In this instrument, ions that are introduced into the FT-ICR MS are created externally and by electrospray ionization (ESI). Essentially, the analyte is added to an appropriate liquid solvent (e.g., water:ethanol:acetic acid mixture in 49.95%:49.95%:0.1% ratio) to prepare it for ionization. Analyte solution is generally passed through a capillary that an electric potential (high voltage such as 2000 volts) is applied to. The potential difference between the capillary needle analyte solution and grounded electrode initiates the ESI process. Ions with multiple charge can be formed where analyte and the solution sprays out in a funnel called the “Taylor cone”. As the droplets of the Taylor Cone are heated, the droplets can begin to evaporate and become smaller, thus allowing the charges to become closer. Once the Rayleigh limit, a limit where the repulsion force between two like charges is greater than the surface tension of the droplet, is reached, a Coulombic explosion occurs, and charged particles are released as a result. The FT-ICR connected to the ESI operates similarly to the GC/FTICR.
Quadrupole
The Quadrupole is a mass separator composed of four rods. It separates ions based on their masses. The four rods have both DC and AC (radio frequency) voltages. Two of the poles are positive and the other two rods are negative at any given time. The AC voltage causes the rods to alternate between positive and negative voltages. The DC voltage keeps the ions in the center of the quadrupole, and the AC voltage causes the ions to move through the quadruple. The AC voltage affects the ions motion in the x-direction and is known as the high-pass mass filter, because light ions are moving faster through the quadrupole and, once they come into an oppositely charged rod’s electric field, it will be pulled to the rod and neutralized or eliminated. The DC voltage affects the ions motion in the y-direction and is known as the low-pass mass filter, because the high mass ions are moving slower and will gradually be energized by the DC to the point of collision with a pole and neutralization. A combination of different RF and DC values can be used to isolate ions of a very specific mass.
Triple Quadrupole Mass Spectrometer Center
This instrument is kindly provided by Dr. Kevin Chambliss and is used cooperatively to conduct intergroup collaborative projects. Current Chambliss-Solouki group projects include instrument and method developments and design of novel ionization sources.
Triple Quadrupole Mass Spectrometer
The triple quadrupole MS is a form of tandem mass spectrometer, which is a mass spectrometer composed of multiple mass spectrometers lined up either in series of either space or time. In this case, three quadrupoles (Qs) are connected in space. The first quadrupole (Q1) acts as a mass selector or isolator, the second (Q2) can function as a collision cell for fragmentation by means of collision induced dissociation (CID), and the third (Q3) acts as a mass analyzer. This set up increases sensitivity and efficiency.
Post-Column Cryogenic Trap
A home-built cryogenic trap is used for the focusing of volatile compounds and results in improved resolution and detection limits of mass analyzers like the quadrupole MS. Essentially, the cryogenic trap is a trap that is held at 150 degees lower then the boiling point of the analyte of interest, and the separated analyses are pumped through the trap. Low-boiling compounds (such as the He carrier gas) will elute through the trap at a fast rate, while high boiling compounds (e.g., organic compounds) will stick to the cold trap. The trap is then heated resistively and at a fast rate to elute the high-boiling compounds. The cryogenic trap assembly consists of a capacitive discharge unit for resistive heating of the cryogenic trap element. The cryogenic trap element is made out of sulfinert stainless steel tubing and immersed in liquid nitrogen. The capacitors within the discharge unit are electrically charged with a DC power supply and discharged with the flash heating the cryogenic trapping element. Desorbed species are detected with a mass spectrometer or other types of conventional detectors such flame ionization detector (FID) to construct a gas chromatogram.
Sonicator, Refrigerated Centrifuge, and Vortex (From Left to Right)
The Sonicator is a device used in cleaning our instruments and fragile mass spectrometry parts, without damaging them. Essentially, the sonicator works by supplying an ultrasonic wave to the solution for cleaning purposes . This wave causes the solution to vibrate and create bubbles; the transducer creates a ultrasonic wave that expands the bubbles in the solution. This creates a vacuum that causes the bubbles to implode, which adds energy to the solution. This added energy agitates any unwanted debris on an unclean part and causes the contaminants to fall off into the cleaning solution.
The centrifuge contains a rotor that rotates an object in a circular path and causes a force perpendicular to the motion. This force causes the objects in the centrifuge to separate based on their masses. Lighter masses are forced to the cap and the heavier masses are forced to the bottom of the centrifuge tube. The vortex is used to mix a solution and suspend objects such as peptides in a solvent. The vortex functions by a rotor causing a circular motion. When a tube is pressed against the rubber piece that produces the circular motion, it creates a vortex in the solution, which mixes the solution and everything dissolved in it.
