Glycobiology: A spoonful of sugar : Article : Nature
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& Full TextM. WORMALD, P. RUDDA molecular model of a prostate-specific antigen with tumour-associated glycosylation (in green).Sugars occur in a variety of forms and locations throughout the human body. From those that are attached to proteins during glycosylation to the carbohydrates that decorate the surfaces of cells lining the lungs and digestive tract, the range of possible sugar conformations and glycoforms is tremendous. As a result, analysing carbohydrates is a tricky business for anyone interested in glycobiology.The term glycobiology was coined in 1988 by biochemist Raymond Dwek at the University of Oxford, UK. Dwek used the phrase simply to emphasize the importance of relating sugars back to basic biology rather than just isolating and examining them outside of their biological context. Instead, he named a field that is thriving in its own right.Today glycobiology is intertwined with fields such as immunology, virology, reproductive biology and drug discovery. "More people are starting to realize that sugars are not just there for protecting surfaces from proteolysis, but they have some functional role to play," says Ian Wilson, a structural biologist at the Scripps Research Institute in La Jolla, California.P. RUDDPauline Rudd is advancing high-throughput glycan analysis.But even as more researchers accept the importance of sugars in basic biology, many glycobiologists worry that the barrier to entry into their field remains too high, potentially delaying or hampering discovery and innovation. "The technical difficulty is so great now that many scientists are turned away," says Peter Seeberger, a chemist at the Swiss Federal Institute of Technology in Zurich. The solution, he adds, is to "lower the hurdle by providing access to technology more easily". To those ends, Seeberger is trying to develop user-friendly automated solutions for complicated procedures such as the synthesis of complex carbohydrates.Seeberger is not alone. Pauline Rudd, a professor of glycobiology at University College Dublin in Ireland and a principal investigator at the National Institute for Bioprocessing Research and Training, spent the better part of ten years refining chromatography approaches for glycosylation analysis. Now she and her colleagues are taking their approach to the next level, using high-performance liquid chromatography (HPLC) as the basis for a high-throughput pipeline for analysing glycans.Glycoproteins featuring N-linked glycans are first immobilized either in gels or on membranes, and the glycans are then released using an enzyme that cleaves the sugars from the proteins. The system examines the patterns of the glycans on the proteins by attaching fluorescent labels to the sugars, which Rudd says offers highly sensitive results during chromatography.Sweet analysisThe labelled sugars are run on a normal phase HPLC column and the resulting peaks are correlated to a pre-run dextran ladder, thereby assigning a 'glucose unit value' to each of the peaks. "We have a database that is automatically interrogated to give us a list of sugars that could have these particular glucose units," says Rudd. Using this information, a series of exoglycosidase digestions is performed and those data are fed back into the computer program to assign final structures.The researchers recently installed an automated liquid-handling platform from Hamilton Robotics of Reno, Nevada, so that they could do their glycan analyses in 96-well plates. "One analysis will take about eight hours, so the aim is to get it done by the end of the shift," says Rudd.Speed is important, Rudd notes, because the drug industry increasingly wants to monitor the glycosylation patterns of proteins. "When people want to achieve quality by design, they need to determine the optimal culture conditions and time for harvesting monoclonal antibodies," she says. "Therefore, they need to understand how the glycosylation changes over the course of production." Rudd says that her pipeline can test samples every hour, over a number of days or at different pH conditions to find those optimal points.Elizabeth Higgins, chief executive and founder of GlycoSolutions in Worcester, Massachusetts, feels that a different factor is driving the drug industry's interest in glycan analysis. GlycoSolutions offers glycomics services and analyses, and last year worked on 20 different glycosylation analysis projects for various pharmaceutical companies. Higgins says that the analyses were largely done to meet regulatory requirements. "Most companies we work with are driven by getting data for the Food and Drug Administration," she says.Another company working on high-throughput tools for analysing glycosylation patterns to aid drug development is Procognia, in Ashdod, Israel. Because many different glycoforms can exist, an extensive knowledge of glycosylation patterns and how they change during drug manufacturing is important for he development of biosimilar drugs, says Ilana Belzer, Procognia's vice-president of research and development. To tackle this issue, the firm has developed GlycoScope, a high-throughput workflow for glycosylation analysis platform that uses lectin (carbohydrate-binding protein) arrays and informatics tools to provide glycosylation fingerprints and glycan structures for glycoproteins.By providing values of molecular weight that can be used to deduce initial structures, mass spectrometry (MS) is yet another approach to glycan analysis. "MS is very good at defining the sugar profiles of cell surfaces," says James Paulson, a glycobiologist at the Scripps Research Institute who studies glycan binding proteins that mediate cellular communication in the immune system. With additional isolation and fragmentation using either matrix-assisted laser desorption/ionization or electrospray ionization followed by tandem MS (MS/MS), researchers can deconvolute the configuration of sugars, a process very similar to protein sequencing.The past few years have seen the arrival of many commercial programs and algorithms that assign glycan structures based on MS spectra. PREMIER Biosoft based in Palo Alto, California, sells SimGlycan, which uses MS/MS data to query a database of more than 8,000 theoretical glycan fragmentation patterns to generate a list of probable structures. Developers at the Palo Alto Research Center have designed new software packages that identify and annotate glycopeptides from a combination of single and tandem MS data.MS analysis works well early on, says Higgins, but can be dangerous when it comes to working out the exact sugar structure because researchers often make assumptions based on mass alone. But the main challenge in using mass spectrometry for glycan analysis is figuring out the linkages between sugars. This is complex because the system needs consistently to fragment the sugars at the correct point to show one sugar is linked to a certain position on another sugar, says Paulson. He adds that such consistency remains an issue.Rudd says that the HPLC approach along with enzyme digests can identify specific sugar linkages. HPLC columns can resolve the sugars on the basis of their conformations, and each monosaccharide contributes a specific incremental value to the retention time of an oligosaccharide. The pools of released sugars are treated with enzyme arrays in which each enzyme is highly specific for a particular monosaccharide in a particular linkage. The sequence and the linkage between sugars can be determined simultaneously for all the sugars in the pool. To help researchers interested in using this approach, Rudd's group recently made available the database GlycoBase and the analytical tool AutoGU to aid in the assignment of provisional structures based on HPLC profiles.Glycan arrays and bird fluTHERMO FISHER SCIENTIFICAdvances in mass spectrometry are improving analyses of sugar composition.The hunt for specific binding partners to various branched sugars or sugar-binding proteins called lectins requires a higher-throughput system. This can be achieved using glycan arrays. First described in 2002, these arrays feature different oligosaccharides or polysaccharides printed on slides or held in wells on a plate. "I think that glycan arrays have been a spectacular success over the past few years," says Paulson.Initially, the arrays contained relatively small numbers of glycans and were designed mainly to study the specificity of antibodies and carbohydrate-binding proteins. But Wilson is one of a number of researchers who realized that some of these arrays would prove useful for diverse applications relevant to their own research. He uses glycan arrays for studying how the influenza virus binds to cells.ETH-ZURICHAutomated carbohydrate synthesis could speed glycobiology research efforts.Some viruses, such as flu and HIV, attach themselves to host cells during the early stages of infection by binding to sugars on the cells' surface. Paulson, in fact, discovered in the 1980s that avian flu viruses recognize different sugar receptors from their human virus counterparts. For Wilson, glycan arrays offered a way to look in detail at the specificity of different flu strains for various sugars, especially the H5N1 strain of bird flu that emerged in 1997 as a worldwide health concern, as well as the strain that caused the human pandemic in 1918. "We have analysed 50 to 60 or maybe even more influenza haemagglutinin mutants on the array to look for how the 1918 and H5N1 influenza strains can convert from human-to-avian or avian-to-human receptor specificity," says Wilson. Work has gone far in explaining how mutations can change the sugars to which influenza strains bind, thereby interconverting the receptor-binding characteristics of avian strains and human strains.Wilson thinks that glycobiology is brought to the attention of a much wider audience when researchers use tools such as glycan arrays to work on well-known microorganisms such as the flu virus (see ). "There are a lot of other uses for these arrays, but everyone understands flu and the risks of bird flu," he says.The Consortium for Functional Glycomics (CFG), an effort funded by the US National Institute of General Medical Sciences, aims to provide unique resources for glycobiology research. Headed by Paulson, the consortium has expanded the number of glycans available for arrays. "There are now 480 glycans in the consortium library," says Paulson, "and they all have amino-terminal linkers that allow them to be printed on slides using standard robotics." The ease of generating and analysing these arrays is opening the field to ever more researchers who can now submit samples to the CFG for rapid analysis.Commercial developers also make high-content arrays with both glycans and carbohydrate-binding lectins attached to the surface. Robotic Labware Designs in Encinitas, California, offers printing services for glycan arrays as well as a series of preprinted glycan arrays. QIAGEN, headquartered in Hilden, Germany, provides the Qproteome GlycoArray kit for glycosylation analysis. This array and analysis software, developed by Procognia, contains a series of specific lectins that bind different monosaccharides, which allows researchers to determine the pattern and relative abundance of specific glycosylation epitopes in a glycoprotein.Although 480 glycans on one array might not seem impressive compared with DNA microarrays, which can contain over a million features, Paulson is unperturbed. For carbohydrate-binding proteins, which usually recognize and interact with the tips of glycans, 480 represents a reasonable approximation of the options. "If you only consider the tips, or the last six or seven sugars, then it is a very finite number of structures, in the order of 500," he says.P. RUDDRobotics are proving crucial in several high-throughput glycosylation analysis approaches.The problem for glycobiologists is how quickly carbohydrate diversity can grow when those 500 structures are attached to different branches on a single N-linked glycan. "If you allow any one of those structures to occur on any one of the four branches, you have this huge number of structures that could theoretically exist," says Paulson. And this is where glycan arrays run into a wall — researchers want this level of diversity on their arrays to help them understand how proteins and pathogens bind sugars, but generating such a diversity of glycans can be difficult.Although many groups still try to isolate sugars from natural sources to use in their research, most agree that improving synthesis methods and technology is essential to obtaining large quantities of diverse carbohydrates."I think that up to now, carbohydrate synthesis has been restricted to a relatively small group of experts who bring considerable technical knowledge to the table," says Seeberger. Even for experts, such synthesis can take a long time — weeks or even years when it comes to making complex carbohydrates or glycoconjugates. Seeberger and his group, along with a handful of other labs around the world, have been working to improve carbohydrate synthesis methods. Ultimately they hope to develop automated instruments that can synthesize carbohydrates much like DNA synthesizers currently produce nucleic acids.There are currently two main approaches to carbohydrate synthesis: solid-phase or one-pot synthesis. In 2001, Seeberger and his colleagues described an automated system that uses solid-phase synthesis for carbohydrates. A programmable one-pot synthesis approach, meanwhile, has been advanced by Chi-Huey Wong, from the Scripps Research Institute and Academia Sinica in Taipei, Taiwan, and his colleagues.Cooking up sugarsIn solid-phase synthesis, sugar building blocks are attached to a surface or a bead, which can be moved during the synthesis process to allow other monosaccharides to be added. The one-pot approach uses a computer program to determine which monosaccharides the next reagent is added and the mixture stirred. This process is repeated until an oligosaccharide is obtained. "What you save is the different work-up steps that often take much more time to achieve than the actual synthesis," says Seeberger of the one-pot approach. He adds that in this sense, both approaches cut down on the purification and separation steps in carbohydrate synthesis.Seeberger sees the building blocks as a big issue for both approaches. Unlike DNA, which has four nucleotide bases, or the 20 amino acids that comprise peptides, there are 10 common monosaccharides in humans and many more in bacterial systems. Even more vexing when it comes to synthesis is the potential for branching of sugars. For example, glucose can link to another sugar at two points in its structure — a 1–6 linkage or a 1–4 linkage. This means that two different building blocks must be available for synthesis, which adds another level of complexity. The synthesis of monosaccharide building blocks was advanced recently when Shang-Cheng Hung in Taiwan and his colleagues reported a selective one-pot synthesis approach for the synthesis of highly functionalized, differentially protected monosaccharides.Some commercial companies are producing monosaccharide building blocks for chemical syntheses. Dextra Laboratories in Reading, UK, offers monosaccharides as well as various glycoconjugates and more complex N-linked oligosaccharides. And other companies such as Omicron Biochemicals of South Bend, Indiana, and GLYCOTEAM in Hamburg, Germany, offer carbohydrate chemical synthesis services.Chemical synthesis is not the only route to obtaining synthetic carbohydrates — researchers can also take advantage of nature's methods. "Enzymatic synthesis is one approach the CFG uses and that has enormously accelerated the rate at which you can synthesize complex natural sugars," says Paulson. But the approach is limited by the number of glycosyltransferase enzymes needed to synthesize all the carbohydrates researchers may be interested in. The number of glycosyltransferases needed for synthesis can be almost as daunting as the number of monosaccharide building blocks in chemical approaches. For example, GlycoGene, a company based in Ibaraki, Japan, offers enzymatic synthesis services to researchers through the use of more than 180 different glycosyltransferases. For this reason, the CFG has merged enzymology and chemistry in the production of many of the sugars on its glycan array.ETH-ZURICHPeter Seeberger is working on fresh approaches to carbohydrate synthesis.Although he is keen to see automated chemical synthesis up and running, Paulson sees gaps when it comes to the carbohydrates that can be synthesized with existing methods. "You cannot make everything you want now, although you can make some carbohydrates quickly and easily," he says. "The gaps are the key things and these might be what people are really interested in looking at."Despite this, Seeberger still sees access to tools as the greatest challenge in glycobiology at the moment. "When you think about genomics and proteomics, you can sequence and you can synthesize," he says. "But those two things are still not generally possible in glycobiology."The field of glycobiology is still finding its way 20 years after the word was first printed. Although advances in the analysis and synthesis of carbohydrates are leading to fresh insights, much remains to be discovered. But Dwek can sit back and take comfort in the knowledge that his word has blossomed into a field that continues to grow. "I think the future of glycobiology is very exciting," he says.ReferencesCampbell, M. P., Royle, L., Radcliffe, C. M., Dwek, R. A. & Rudd, P. M. Bioinformatics 24, ;1216 (2008).Plante, O. J., Palmacci, E. R. & Seeberger, P. H. Science 291, ;1527 (2001).&|&&|&&|&&|&&|Wang, C.-C. et al. Nature 446, 896&# (2007).&|&&|&&|&&|Hwang, G. M., Pang, L., Mullen, E. H. & Fainman, Y. IEEE Sens. J. 8, ;2079 (2008).
Nathan Blow is the technology editor for Nature and Nature Methods. MORE ARTICLES LIKE THIS These links to content published by NPG are automatically generated.NEWS AND VIEWSNature Biotechnology News and Views (01 Jun 2003)Nature Chemical Biology News and Views (01 Apr 2009)
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ADVERTISEMENTFrom Wikipedia, the free encyclopedia
mass spectrometer, model IMS 3f.
Orbitrap mass spectrometer.
Mass spectrometry (MS) is an analytical technique that ionizes
and sorts the
based on their . In simpler terms, a
measures the masses within a sample. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.
is a plot of the ion signal as a function of the . These spectra are used to determine the elemental or
of a sample, the masses of particles and of , and to elucidate the chemical structures of molecules, such as
and other .
In a typical MS procedure, a sample, which may be solid, liquid, or gas, is ionized, for example by bombarding it with electrons. This may cause some of the sample's molecules to break into charged fragments. These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same amount of deflection. The ions are detected by a mechanism capable of detecting charged particles, such as an . Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.
For more details on this topic, see .
Replica of J.J. Thomson's third mass spectrometer.
observed rays in
under low pressure that traveled away from the anode and through channels in a perforated , opposite to the direction of negatively charged
(which travel from cathode to anode). Goldstein called these positively charged
"Kanalstrahlen"; the standard translation of this term into English is "".
found that strong electric or magnetic fields deflected the canal rays and, in 1899, constructed a device with parallel electric and magnetic fields that separated the positive rays according to their charge-to-mass ratio (Q/m). Wien found that the charge-to-mass ratio depended on the nature of the gas in the discharge tube. English scientist
later improved on the work of Wien by reducing the pressure to create the mass spectrograph.
Calutron mass spectrometers were used in the Manhattan Project for uranium enrichment.
The word spectrograph had become part of the
by 1884. Early spectrometry devices that measured the mass-to-charge ratio of ions were called
which consisted of instruments that recorded a
of mass values on a . A mass spectroscope is similar to a mass spectrograph except that the beam of ions is directed onto a
screen. A mass spectroscope configuration was used in early instruments when it was desired that the effects of adjustments be quickly observed. Once the instrument was properly adjusted, a photographic plate was inserted and exposed. The term mass spectroscope continued to be used even though the direct illumination of a phosphor screen was replaced by indirect measurements with an . The use of the term mass spectroscopy is now discouraged due to the possibility of confusion with light . Mass spectrometry is often abbreviated as mass-spec or simply as MS.
Modern techniques of mass spectrometry were devised by
in 1918 and 1919 respectively.
were used for separating the
developed by
during the . Calutron mass spectrometers were used for
established during World War II.
In 1989, half of the
was awarded to
for the development of the ion trap technique in the 1950s and 1960s.
In 2002, the
was awarded to
for the development of
for the development of
(SLD) and their application to the ionization of biological macromolecules, especially proteins.
Schematics of a simple mass spectrometer with sector type mass analyzer. This one is for the measurement of carbon dioxide
ratios () as in the
A mass spectrometer consists of three components: an ion source, a mass analyzer, and a detector. The
converts a portion of the sample into ions. There is a wide variety of ionization techniques, depending on the phase (solid, liquid, gas) of the sample and the efficiency of various ionization mechanisms for the unknown species. An extraction system removes ions from the sample, which are then targeted through the mass analyzer and onto the detector. The differences in masses of the fragments allows the mass analyzer to sort the ions by their mass-to-charge ratio. The detector measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present. Some detectors also give spatial information, e.g., a multichannel plate.
The following example describes the operation of a spectrometer mass analyzer, which is of the
type. (Other analyzer types are treated below.) Consider a sample of
(table salt). In the ion source, the sample is
(turned into ) and ionized (transformed into electrically charged particles) into
(Cl-) ions. Sodium atoms and ions are , with a mass of about 23 u. Chloride atoms and ions come in two isotopes with masses of approximately 35 u (at a natural abundance of about 75 percent) and approximately 37 u (at a natural abundance of about 25 percent). The analyzer part of the spectrometer contains
fields, which exert forces on ions traveling through these fields. The speed of a charged particle may be increased or decreased while passing through the electric field, and its direction may be altered by the magnetic field. The magnitude of the deflection of the moving ion's trajectory depends on its mass-to-charge ratio. Lighter ions get deflected by the magnetic force more than heavier ions (based on , F = ma). The streams of sorted ions pass from the analyzer to the detector, which records the relative abundance of each ion type. This information is used to determine the chemical element composition of the original sample (i.e. that both sodium and chlorine are present in the sample) and the isotopic composition of its constituents (the ratio of 35Cl to 37Cl).
source at the
linear accelerator
is the part of the mass spectrometer that ionizes the material under analysis (the analyte). The ions are then transported by
to the mass analyzer.
Techniques for ionization have been key to determining what types of samples can be analyzed by mass spectrometry.
are used for
and . In chemical ionization sources, the analyte is ionized by chemical ion-molecule reactions during collisions in the source. Two techniques often used with
biological samples include
(invented by ) and
(MALDI, initially developed as a similar technique "Soft Laser Desorption (SLD)" by K. Tanaka for which a Nobel Prize was awarded and as MALDI by M. Karas and F. Hillenkamp).
Quadrupole mass spectrometer and electrospray ion source used for Fenn's early work
In mass spectrometry, ionization refers to the production of gas phase ions suitable for resolution in the mass analyser or mass filter. Ionization occurs in the . There ar each has advantages and disadvantages for particular applications. For example,
(EI) gives a high degree of fragmentation, yielding highly detailed mass spectra which when skilfully analysed can provide important information for structural elucidation/characterisation and facilitate identification of unknown compounds by comparison to mass spectral libraries obtained under identical operating conditions. However, EI is not suitable for coupling to , i.e. , since at atmospheric pressure, the filaments used to generate electrons burn out rapidly. Thus EI is coupled predominantly with , i.e. , where the entire system is under high vacuum.
Hard ionization techniques are processes which impart high quantities of residual energy in the subject molecule invoking large degrees of fragmentation (i.e. the systematic rupturing of bonds acts to remove the excess energy, restoring stability to the resulting ion). Resultant ions tend to have m/z lower than the molecular mass (other than in the case of proton transfer and not including isotope peaks). The most common example of hard ionization is electron ionization (EI).
Soft ionization refers to the processes which impart little residual energy onto the subject molecule and as such result in little fragmentation. Examples include
(ESI), and
Inductively coupled plasma ion source
(ICP) sources are used primarily for cation analysis of a wide array of sample types. In this source, a plasma that is electrically neutral overall, but that has had a substantial fraction of its atoms ionized by high temperature, is used to atomize introduced sample molecules and to further strip the outer electrons from those atoms. The plasma is usually generated from argon gas, since the first ionization energy of argon atoms is higher than the first of any other elements except He, O, F and Ne, but lower than the second ionization energy of all except the most electropositive metals. The heating is achieved by a radio-frequency current passed through a coil surrounding the plasma.
Others include , ,
Mass analyzers separate the ions according to their . The following two laws govern the dynamics of charged particles in electric and magnetic fields in vacuum:
{\displaystyle \mathbf {F} =Q(\mathbf {E} +\mathbf {v} \times \mathbf {B} )}
{\displaystyle \mathbf {F} =m\mathbf {a} }
( of motion in non-relativistic case, i.e. valid only at ion velocity much lower than the speed of light).
Here F is the force applied to the ion, m is the mass of the ion, a is the acceleration, Q is the ion charge, E is the electric field, and v × B is the
of the ion velocity and the magnetic field
Equating the above expressions for the force applied to the ion yields:
{\displaystyle (m/Q)\mathbf {a} =\mathbf {E} +\mathbf {v} \times \mathbf {B} .}
is the classic equation of motion for . Together with the particle's initial conditions, it completely determines the particle's motion in space and time in terms of m/Q. Thus mass spectrometers could be thought of as "mass-to-charge spectrometers". When presenting data, it is common to use the (officially)
m/z, where z is the number of
(e) on the ion (z=Q/e). This quantity, although it is informally called the mass-to-charge ratio, more accurately speaking represents the ratio of the mass number and the charge number, z.
There are many types of mass analyzers, using either static or dynamic fields, and magnetic or electric fields, but all operate according to the above differential equation. Each analyzer type has its strengths and weaknesses. Many mass spectrometers use two or more mass analyzers for . In addition to the more common mass analyzers listed below, there are others designed for special situations.
There are several important analyser characteristics. The
is the measure of the ability to distinguish two peaks of slightly different m/z. The mass accuracy is the ratio of the m/z measurement error to the true m/z. Mass accuracy is usually measured in
or . The mass range is the range of m/z amenable to analysis by a given analyzer. The linear dynamic range is the range over which ion signal is linear with analyte concentration. Speed refers to the time frame of the experiment and ultimately is used to determine the number of spectra per unit time that can be generated.
ThermoQuest AvantGarde sector mass spectrometer
For more details on this topic, see .
A sector field mass analyzer uses an electric and/or magnetic field to affect the path and/or
particles in some way. As shown above,
bend the trajectories of the ions as they pass through the mass analyzer, according to their mass-to-charge ratios, deflecting the more charged and faster-moving, lighter ions more. The analyzer can be used to select a narrow range of m/z or to scan through a range of m/z to catalog the ions present.
For more details on this topic, see .
(TOF) analyzer uses an
to accelerate the ions through the same , and then measures the time they take to reach the detector. If the particles all have the same , the
will be identical, and their
will depend only on their . Ions with a lower mass will reach the detector first.
For more details on this topic, see .
use oscillating electrical fields to selectively stabilize or destabilize the paths of ions passing through a
field created between 4 parallel rods. Only the ions in a certain range of mass/charge ratio are passed through the system at any time, but changes to the potentials on the rods allow a wide range of m/z values to be swept rapidly, either continuously or in a succession of discrete hops. A quadrupole mass analyzer acts as a mass-selective filter and is closely related to the , particularly the linear quadrupole ion trap except that it is designed to pass the untrapped ions rather than collect the trapped ones, and is for that reason referred to as a transmission quadrupole. A magnetically enhanced quadrupole mass analyzer includes the addition of a magnetic field, either applied axially or transversely. This novel type of instrument leads to an additional performance enhancement in terms of resolution and/or sensitivity depending upon the magnitude and orientation of the applied magnetic field. A common variation of the transmission quadrupole is the triple quadrupole mass spectrometer. The “triple quad” has three consecutive quadrupole stages, the first acting as a mass filter to transmit a particular incoming ion to the second quadrupole, a collision chamber, wherein that ion can be broken into fragments. The third quadrupole also acts as a mass filter, to transmit a particular fragment ion to the detector. If a quadrupole is made to rapidly and repetitively cycle through a range of mass filter settings, full spectra can be reported. Likewise, a triple quad can be made to perform various scan types characteristic of .
For more details on this topic, see .
works on the same physical principles as the quadrupole mass analyzer, but the ions are trapped and sequentially ejected. Ions are trapped in a mainly quadrupole RF field, in a space defined by a ring electrode (usually connected to the main RF potential) between two endcap electrodes (typically connected to DC or auxiliary AC potentials). The sample is ionized either internally (e.g. with an electron or laser beam), or externally, in which case the ions are often introduced through an aperture in an endcap electrode.
There are many mass/charge separation and isolation methods but the most commonly used is the mass instability mode in which the RF potential is ramped so that the orbit of ions with a mass a & b are stable while ions with mass b become unstable and are ejected on the z-axis onto a detector. There are also non-destructive analysis methods.
Ions may also be ejected by the resonance excitation method, whereby a supplemental oscillatory excitation voltage is applied to the endcap electrodes, and the trapping voltage amplitude and/or excitation voltage frequency is varied to bring ions into a resonance condition in order of their mass/charge ratio.
(CIT) is a derivative of the quadrupole ion trap where the electrodes are formed from flat rings rather than hyperbolic shaped electrodes. The architecture lends itself well to miniaturization because as the size of a trap is reduced, the shape of the electric field near the center of the trap, the region where the ions are trapped, forms a shape similar to that of a hyperbolic trap.
is similar to a quadrupole ion trap, but it traps ions in a two dimensional quadrupole field, instead of a three-dimensional quadrupole field as in a 3D quadrupole ion trap. Thermo Fisher's LTQ ("linear trap quadrupole") is an example of the linear ion trap.
A toroidal ion trap can be visualized as a linear quadrupole curved around and connected at the ends or as a cross section of a 3D ion trap rotated on edge to form the toroid, donut shaped trap. The trap can store large volumes of ions by distributing them throughout the ring-like trap structure. This toroidal shaped trap is a configuration that allows the increased miniaturization of an ion trap mass analyzer. Additionally all ions are stored in the same trapping field and ejected together simplifying detection that can be complicated with array configurations due to variations in detector alignment and machining of the arrays.
As with the toroidal trap, linear traps and 3D quadrupole ion traps are the most commonly miniaturized mass analyzers due to their high sensitivity, tolerance for mTorr pressure, and capabilities for single analyzer tandem mass spectrometry (e.g. product ion scans).
Orbitrap mass analyzer
For more details on this topic, see .
instruments are similar to
mass spectrometers (see text below). Ions are
trapped in an orbit around a central, spindle shaped electrode. The electrode confines the ions so that they both orbit around the central electrode and oscillate back and forth along the central electrode's long axis. This oscillation generates an
in the detector plates which is recorded by the instrument. The frequencies of these image currents depend on the mass to charge ratios of the ions. Mass spectra are obtained by
of the recorded image currents.
Orbitraps have a high mass accuracy, high sensitivity and a good dynamic range.
Fourier transform ion cyclotron resonance mass spectrometer
For more details on this topic, see .
(FTMS), or more precisely
MS, measures mass by detecting the
produced by ions
in the presence of a magnetic field. Instead of measuring the deflection of ions with a detector such as an , the ions are injected into a
(a static electric/magnetic ) where they effectively form part of a circuit. Detectors at fixed positions in space measure the electrical signal of ions which pass near them over time, producing a periodic signal. Since the frequency of an ion's cycling is determined by its mass to charge ratio, this can be
by performing a
on the signal.
has the advantage of high sensitivity (since each ion is "counted" more than once) and much higher
and thus precision.
(ICR) is an older mass analysis technique similar to FTMS except that ions are detected with a traditional detector. Ions trapped in a
are excited by an RF electric field until they impact the wall of the trap, where the detector is located. Ions of different mass are resolved according to impact time.
A continuous dynode particle multiplier detector.
The final element of the mass spectrometer is the detector. The detector records either the charge induced or the current produced when an ion passes by or hits a surface. In a scanning instrument, the signal produced in the detector during the course of the scan versus where the instrument is in the scan (at what m/Q) will produce a , a record of ions as a function of m/Q.
Typically, some type of
is used, though other detectors including
are also used. Because the number of ions leaving the mass analyzer at a particular instant is typically quite small, considerable amplification is often necessary to get a signal.
are commonly used in modern commercial instruments. In
and , the detector consists of a pair of metal surfaces within the mass analyzer/ion trap region which the ions only pass near as they oscillate. No direct current is produced, only a weak AC image current is produced in a circuit between the electrodes. Other inductive detectors have also been used.
Tandem mass spectrometry for biological molecules using ESI or MALDI
is one capable of multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation. For example, one mass analyzer can isolate one
from many entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by
(CID). A third mass analyzer then sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time, as in a . There are various methods for
molecules for tandem MS, including
(SID). An important application using tandem mass spectrometry is in .
Tandem mass spectrometry enables a variety of experimental sequences. Many commercial mass spectrometers are designed to expedite the execution of such routine sequences as
and precursor ion scanning. In SRM, the first analyzer allows only a single mass through and the second analyzer monitors for multiple user-defined fragment ions. SRM is most often used with scanning instruments where the second mass analysis event is
limited. These experiments are used to increase specificity of detection of known molecules, notably in pharmacokinetic studies. Precursor ion scanning refers to monitoring for a specific loss from the precursor ion. The first and second mass analyzers scan across the spectrum as partitioned by a user-defined m/z value. This experiment is used to detect specific motifs within unknown molecules.
Another type of tandem mass spectrometry used for
(AMS), which uses very high voltages, usually in the mega-volt range, to accelerate negative ions into a type of tandem mass spectrometer.
When a specific configuration of source, analyzer, and detector becomes conventional in practice, often a compound
arises to designate it, and the compound acronym may be better known among nonspectrometrists than the component acronyms. The epitome of this is , which simply refers to combining a
source with a
mass analyzer. The MALDI-TOF moniker is more widely recognized by the non-mass spectrometrists than MALDI or TOF individually. Other examples include , ,
and . Sometimes the use of the generic "MS" actually connotes a very specific mass analyzer and detection system, as is the case with AMS, which is always sector based.
Certain applications of mass spectrometry have developed monikers that although strictly speaking would seem to refer to a broad application, in practice have come instead to connote a specific or a limited number of instrument configurations. An example of this is , which refers in practice to the use of a limited number of sector
this name is used to refer to both the application and the instrument used for the application.
An important enhancement to the mass resolving and mass determining capabilities of mass spectrometry is using it in tandem with
and other separation techniques.
A gas chromatograph (right) directly coupled to a mass spectrometer (left)
Main article:
A common combination is
chromatography-mass spectrometry (GC/MS or GC-MS). In this technique, a
is used to separate different compounds. This stream of separated compounds is fed online into the
is applied. This filament emits electrons which ionize the compounds. The ions can then further fragment, yielding predictable patterns. Intact ions and fragments pass into the mass spectrometer's analyzer and are eventually detected.
conservation scientist performing .
Main article:
Similar to gas chromatography MS (GC/MS), liquid chromatography-mass spectrometry (LC/MS or LC-MS) separates compounds chromatographically before they are introduced to the ion source and mass spectrometer. It differs from GC/MS in that the mobile phase is liquid, usually a mixture of
and organic , instead of gas. Most commonly, an
source is used in LC/MS. Other popular and commercially available LC/MS ion sources are
and . There are also some newly developed ionization techniques like .
Main article:
Capillary electrophoresis–mass spectrometry (CE-MS) is a technique that combines the liquid separation process of
with mass spectrometry. CE-MS is typically coupled to electrospray ionization.
Main article:
Ion mobility spectrometry-mass spectrometry (IMS/MS or IMMS) is a technique where ions are first separated by drift time through some neutral gas under an applied electrical potential gradient before being introduced into a mass spectrometer. Drift time is a measure of the radius relative to the charge of the ion. The
of IMS (the time over which the experiment takes place) is longer than most mass spectrometric techniques, such that the mass spectrometer can sample along the course of the IMS separation. This produces data about the IMS separation and the mass-to-charge ratio of the ions in a manner similar to .
The duty cycle of IMS is short relative to liquid chromatography or gas chromatography separations and can thus be coupled to such techniques, producing triple modalities such as LC/IMS/MS.
Mass spectrum of a peptide showing the isotopic distribution
Mass spectrometry produces various types of data. The most common data representation is the .
Certain types of mass spectrometry data are best represented as a . Types of chromatograms include
(TIC), and
(SRM), among many others.
Other types of mass spectrometry data are well represented as a three-dimensional . In this form, the mass-to-charge, m/z is on the x-axis, intensity the y-axis, and an additional experimental parameter, such as time, is recorded on the z-axis.
Mass spectrometry data analysis is specific to the type of experiment producing the data. General subdivisions of data are fundamental to understanding any data.
Many mass spectrometers work in either negative ion mode or positive ion mode. It is very important to know whether the observed ions are negatively or positively charged. This is often important in determining the neutral mass but it also indicates something about the nature of the molecules.
Different types of ion source result in different arrays of fragments produced from the original molecules. An electron ionization source produces many fragments and mostly single-charged (1-) radicals (odd number of electrons), whereas an electrospray source usually produces non-radical quasimolecular ions that are frequently multiply charged. Tandem mass spectrometry purposely produces fragment ions post-source and can drastically change the sort of data achieved by an experiment.
Knowledge of the origin of a sample can provide insight into the component molecules of the sample and their fragmentations. A sample from a synthesis/manufacturing process will probably contain impurities chemically related to the target component. A crudely prepared biological sample will probably contain a certain amount of salt, which may form
with the analyte molecules in certain analyses.
Results can also depend heavily on sample preparation and how it was run/introduced. An important example is the issue of which matrix is used for MALDI spotting, since much of the energetics of the desorption/ionization event is controlled by the matrix rather than the laser power. Sometimes samples are spiked with sodium or another ion-carrying species to produce adducts rather than a protonated species.
Mass spectrometry can measure molar mass, molecular structure, and sample purity. Each of these questions requires a different ex therefore, adequate definition of the experimental goal is a prerequisite for collecting the proper data and successfully interpreting it.
electron ionization mass spectrum
Main article:
Since the precise
of a molecule is deciphered through the set of fragment masses, the interpretation of
requires combined use of various techniques. Usually the first strategy for identifying an unknown compound is to compare its experimental mass spectrum against a library of mass spectra. If no matches result from the search, then manual interpretation or
must be performed. Computer simulation of
and fragmentation processes occurring in mass spectrometer is the primary tool for assigning structure or peptide sequence to a molecule. An
structural information is fragmented
and the resulting pattern is compared with observed spectrum. Such simulation is often supported by a fragmentation library that contains published patterns of known decomposition reactions.
taking advantage of this idea has been developed for both small molecules and .
Analysis of mass spectra can also be spectra with . A mass-to-charge ratio value (m/z) with only integer precision can represent an immense number of theoretically pos however, more precise mass figures significantly reduce the number of candidate . A computer algorithm called formula generator calculates all molecular formulas that theoretically fit a given
with specified tolerance.
A recent technique for structure elucidation in mass spectrometry, called , identifies individual pieces of structural information by conducting a search of the
of the molecule under investigation against a library of the
of structurally characterized precursor ions.
Particle Analysis by Laser Mass Spectrometry
high-altitude research aircraft
Mass spectrometry has both
uses. These include identifying unknown compounds, determining the
composition of elements in a molecule, and determining the
of a compound by observing its fragmentation. Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of
(the chemistry of ions and neutrals in a vacuum). MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds.
As an analytical technique it possesses distinct advantages such as: Increased sensitivity over most other analytical techniques because the analyzer, as a mass-charge filter, reduces background interference, Excellent specificity from characteristic fragmentation patterns to identify unknowns or confirm the presence of suspected compounds, Information about molecular weight, Information about the isotopic abundance of elements, Temporally resolved chemical data.
A few of the disadvantages of the method is that often fails to distinguish between optical and geometrical isomers and the positions of substituent in o-, m- and p- positions in an aromatic ring. Also, its scope is limited in identifying hydrocarbons that produce similar fragmented ions.
Mass spectrometer to determine the 16O/18O and 12C/13C isotope ratio on biogenous carbonate
Main article:
Mass spectrometry is also used to determine the
composition of elements within a sample. Differences in mass among isotopes of an element are very small, and the less abundant isotopes of an element are typically very rare, so a very sensitive instrument is required. These instruments, sometimes referred to as isotope ratio mass spectrometers (IR-MS), usually use a single magnet to bend a beam of ionized particles towards a series of
which convert particle impacts to . A fast on-line analysis of
content of water can be done using , FA-MS. Probably the most sensitive and accurate mass spectrometer for this purpose is the
(AMS). This is because it provides ultimate sensitivity, capable of measuring individual atoms and measuring nuclides with a dynamic range of ~1015 relative to the major stable isotope. Isotope ratios are important markers of a variety of processes. Some isotope ratios are used to determine the age of materials for example as in . Labeling with stable isotopes is also used for protein quantification. (see
Several techniques use ions created in a dedicated ion source injected into a flow tube or a drift tube:
(SIFT-MS), and
(PTR-MS), are variants of
dedicated for trace gas analysis of air, breath or liquid headspace using well defined reaction time allowing calculations of analyte concentrations from the known reaction kinetics without the need for internal standard or calibration.
is an instrument that combines
mass spectrometry and field-evaporation microscopy to map the location of individual atoms.
Main article:
Pharmacokinetics is often studied using mass spectrometry because of the complex nature of the matrix (often blood or urine) and the need for high sensitivity to observe low dose and long time point data. The most common instrumentation used in this application is
with a . Tandem mass spectrometry is usually employed for added specificity. Standard curves and internal standards are used for quantitation of usually a single pharmaceutical in the samples. The samples represent different time points as a pharmaceutical is administered and then metabolized or cleared from the body. Blank or t=0 samples taken before administration are important in determining background and ensuring data integrity with such complex sample matrices. Much attention is paid to the linearity o however it is not uncommon to use
with more complex functions such as quadratics since the response of most mass spectrometers is less than linear across large concentration ranges.
There is currently considerable interest in the use of very high sensitivity mass spectrometry for
studies, which are seen as a promising alternative to .
Main article:
Mass spectrometry is an important method for the characterization and
of proteins. The two primary methods for ionization of whole proteins are
(MALDI). In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as "" strategy of protein analysis. The top-down approach however is largely limited to low-throughput single-protein studies. In the second, proteins are enzymatically digested into smaller
or , either in
separation. Other proteolytic agents are also used. The collection of peptide products are then introduced to the mass analyzer. When the characteristic pattern of peptides is used for the identification of the protein the method is called
(PMF), if the identification is performed using the sequence data determined in
analysis it is called . These procedures of protein analysis are also referred to as the "" approach. A third approach however is beginning to be used, this intermediate "middle-down" approach involves analyzing proteolytic peptide larger than the typical tryptic peptide.
Mass spectrometry (MS), with its low sample requirement and high sensitivity, has been predominantly used in
for characterization and elucidation of . Mass spectrometry provides a complementary method to
for the analysis of glycans. Intact glycans may be detected directly as singly charged ions by
(MALDI-MS) or, following permethylation or peracetylation, by
(ESI-MS) also gives good signals for the smaller glycans. Various
are now available which interpret MS data and aid in Glycan structure characterization.
NASA's Phoenix Mars Lander analyzing a soil sample from the "Rosy Red" trench with the TEGA mass spectrometer
As a standard method for analysis, mass spectrometers have reached other planets and moons. Two were taken to
by the . In early 2005 the
mission delivered a specialized
instrument aboard the
through the atmosphere of , the largest moon of the planet . This instrument analyzed atmospheric samples along its descent trajectory and was able to vaporize and analyze samples of Titan's frozen, hydrocarbon covered surface once the probe had landed. These measurements compare the abundance of isotope(s) of each particle comparatively to earth's natural abundance. Also on board the
spacecraft is an ion and neutral mass spectrometer which has been taking measurements of Titan's atmospheric composition as well as the composition of ' plumes. A
mass spectrometer was carried by the
launched in 2007.
Mass spectrometers are also widely used in space missions to measure the composition of plasmas. For example, the Cassini spacecraft carries the Cassini Plasma Spectrometer (CAPS), which measures the mass of ions in Saturn's .
Mass spectrometers were used in hospitals for respiratory gas analysis beginning around 1975 through the end of the century. Some are probably still in use but none are currently being manufactured.
Found mostly in the , they were a part of a complex system, in which respired gas samples from patients undergoing
were drawn into the instrument through a valve mechanism designed to sequentially connect up to 32 rooms to the mass spectrometer. A computer directed all operations of the system. The data collected from the mass spectrometer was delivered to the individual rooms for the anesthesiologist to use.
The uniqueness of this magnetic sector mass spectrometer may have been the fact that a plane of detectors, each purposely positioned to collect all of the ion species expected to be in the samples, allowed the instrument to simultaneously report all of the gases respired by the patient. Although the mass range was limited to slightly over 120 , fragmentation of some of the heavier molecules negated the need for a higher detection limit.
The primary function of mass spectrometry is as a tool for chemical analyses based on detection and quantification of ions according to their mass-to-charge ratio. However, mass spectrometry also shows promise for material synthesis. Ion soft landing is characterized by deposition of intact species on surfaces at low kinetic energies which precludes the fragmentation of the incident species. The soft landing technique was first reported in 1977 for the reaction of low energy sulfur containing ions on a lead surface.
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