FAQ

FAQ’s Mass Spectrometry

Mass spectrometry (MS) makes use of the motion of ions in electric and magnetic fields in order to sort them according to their mass-to-charge ratios. Thus, MS is an analytic technique by which chemical substances are identified by the sorting of gaseous ions in electric and magnetic fields. The instruments used in these studies operate on the principle that moving ions may be deflected by electric and magnetic fields. A device that performs this operation and uses electrical means to detect the sorted ions is called a mass spectrometer.
MS provides qualitative and quantitative information about the atomic and molecular composition of inorganic and organic materials.

(https://en.wikipedia.org/wiki/Tandem_mass_spectrometry)
Mass Spectrometry – Instrumentation
Mass spectrometers consist of four basic parts; a handling system to introduce the unknown sample into the equipment; an ion source, in which a beam of particles characteristic of the sample is produced; an analyzer that separates the particles according to mass; and a detector, in which the separated ion components are collected and characterized.
The spectrometer requires a collision free path for the ions and therefore operates under vacuum or near vacuum conditions. The sample inlet system is designed for minimal loss of vacuum. The ion source creates gaseous ion fragments from the sample. There exist two kinds of ion sources; gas-phase sources and desorption sources.

Gas-phase Sources

In gas-phase sources, the sample is first volatilized before the ionization of the gaseous components takes place in various ways. The sample is vaporized outside the ion source. Examples of ionization methods are chemical ionization, electron-impact ionization and field ionization.

Chemical Ionization

In chemical ionization, a small amount of gaseous atoms is ionized by collision with ions produced by electron bombardment of the reagent gas. Some widespread reagent gases are methane, oxygen, ammonia and hydrogen.

Electron-Impact Ionization

The electron-impact ion source is the most commonly used ionization method. An electron beam, generated from a tungsten or rhenium filament, is used to ionize gas-phase atoms or molecules. Ions are formed during collision of the electron beam and sample molecules.
M + e- -> M+. + 2e-
Here M represents the analyzed molecule and M+. is its molecular ion. The positive ions are accelerated by an electric field and passed into a magnetic field. By changing the accelerating voltage, i.e. the speed of the particle, or the magnetic field strength, ions of different mass-to-charge ratio can be collected and measured.

Field Ionization

Molecules can lose an electron when placed in a very high electric field. High fields can be created in an ion source by applying a high voltage between a cathode and an anode, a field emitter. A field emitter consists of a wire covered with microscopic carbon dendrites, which greatly amplify the effective field of the carbon points. The gaseous sample from an inlet system is allowed in to the high-field area around the microtips of the emitter. Electrons from the analyte are extracted by the microtips and there is a little or no fragmentation of the ion.

Desorption Sources

In desorption sources, ions are formed from samples in a condensed phase. A major advantage of desorption ionization is that it permits the examination of non-volatile and thermally unstable molecules. Two examples of desorption source are field desorption and fast atom bombardment.

Field Desorption

Field desorption is a valuable technique for studying surface phenomena such as adsorbed species and the results of chemical reactions on surfaces. It is also a useful method for large lipophilic polar molecules. In field desorption a multi-tipped emitter similar to that employed in field ionization is used. The electrode is mounted on a probe that can be removed from the sample compartment and coated with a solution of the sample. Ionization takes place by the application of a high potential to the electrode. Sometimes it is necessary to heat the emitter with an electric current.

Fast Atom Bombardment (HCD)

In fast atom bombardment, a high-energy beam of neutral atoms, typically xenon or argon, strikes a solid sample causing desorption and ionization. This technique is used for large biological molecules that are difficult to get into the gas phase. The atomic beam is produced by accelerating ions from an ion source through a charge-exchange cell. The ions pick up an electron in collision with neutral atoms to form a beam of high energy atoms.

Mass Analyzer Designs

The purpose of the mass analyzer is to separate the ions produced in the ion source according to their different mass-to-charge ratio. The most common analyzer designs include the quadruple, magnetic sector and time-of-flight mass analyzers.

Quadruple

A quadruple field is formed by four electrically conducting parallel rods. The applied voltages affect the trajectory of ions traveling down the flight path centered between the four rods. For given voltages, only ions of a certain mass-to-charge ratio are allowed to pass through the quadruple filter, while others are carried away as uncharged molecules. By varying the electrical signals to a quadruple it is possible to vary the range of the mass-to-charge ratio transmitted. This makes spectral scanning possible.

Magnetic Sector Analyzer

The magnetic sector analyzer employs a magnetic field that causes ions to travel in a circular path of 180, 90 or 60 degrees. Initially ions are accelerated through slit B into the metal analyzer tube. Ions of different mass can be scanned across the exit slit by varying the field strength of the magnet or the accelerating potential between slit A and B. The ions passing through the exit slit fall on a collector electrode, resulting in an ion current that is amplified and recorded.

Time-of-Flight Analyzer

A time-of-flight mass spectrometer uses the differences in transit time through a drift region to separate ions of different masses. The ions from the ion source are accelerated by an electric field pulse. The accelerated particles then pass into a field-free drift tube that is about a meter in length.
The essential principle of time-of-flight mass spectrometry is that all ions are accelerated to the same kinetic energy. Their velocities are inversely proportional to the square roots of their masses. The lighter ions of high velocity arrive at the detector earlier than the heavier ions of low velocity.

Ion Collection System

The ion collection system is measures the relative abundance of ion fragments of each mass. Several types of detectors are available for mass spectrometers. The detector used for most routine experiments is the electron multiplier. Another type of detector is photographic plates coated with a silver bromide emulsion, it is sensitive to energetic ions. A photographic plate can give a higher resolution than an electrical detector.

Mass Spectrometry – LC-MS

Liquid Chromatography – Mass Spectrometry (LC-MS) is a powerful analytical technique used in industries requiring very low detection limits of sometimes unknown samples, such as food analysis and pharmaceutical drug development. The efficient physical separation of chemical substances dissolved in a mobile phase, performed by liquid chromatography, is combined with the mass spectrometer being able to sort and identify the components (gaseous ions) in electric and magnetic fields according to their mass-to-charge ratios. The samples analyzed by LC-MS are often complex mixtures.

MS/MS

Tandem mass spectrometry, also known as MS/MS or MS2, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages. In a tandem mass spectrometer, ions are formed in the ion source and separated by mass-to-charge ratio in the first stage of mass spectrometry (MS1). Ions of a particular mass-to-charge ratio (precursor ions) are selected and fragment ions (product ions) are created by collision-induced dissociation, ion-molecule reaction, photodissociation, or other process. The resulting ions are then separated and detected in a second stage of mass spectrometry (MS2).

Top-Down Proteomic

Top-down proteomics is a method of protein identification that uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry analysis. Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins. The name is derived from the similar approach to DNA sequencing. Proteins are typically ionized by electrospray ionization and trapped in a Fourier transform ion cyclotron resonance (Penning trap) or quadrupole ion trap (Paul trap) mass spectrometer. Fragmentation for tandem mass spectrometry is accomplished by electron-capture dissociation or electron-transfer dissociation. Effective fractionation is critical for sample handling before mass-spectrometry-based proteomics. Proteome analysis routinely involves digesting intact proteins followed by inferred protein identification using mass spectrometry. Top-down proteomics interrogates protein structure through measurement of an intact mass followed by direct ion dissociation in the gas phase.

MRM

Selected reaction monitoring (SRM) is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection.

Maldi

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is similar in character to electrospray ionization (ESI) in that both techniques are relatively soft ways of obtaining ions of large molecules in the gas phase, though MALDI produces far fewer multiply charged ions.

MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and can then be accelerated into whichever mass spectrometer is used to analyse them.

Bottom-Up Proteomics

Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry. The proteins may first be purified by a method such as gel electrophoresis resulting in one or a few proteins in each proteolytic digest. Alternatively, the crude protein extract is digested directly, followed by one or more dimensions of separation of the peptides by liquid chromatography coupled to mass spectrometry, a technique known as shotgun proteomics. By comparing the masses of the proteolytic peptides or their tandem mass spectra with those predicted from a sequence database or annotated peptide spectral in a peptide spectral library, peptides can be identified and multiple peptide identifications assembled into a protein identification.