MICROBIAL PURE CULTURE

Isolation of Pure Culture

Microorganisms are generally found in nature (air, soil and water) as mixed populations. Even the diseased parts of plants and animals contain a great number of microorganisms, which differ markedly from the microorganisms of other environments. To study the specific role played by a specific microorganism in its environment, one must isolate the same in pure culture. Pure culture involves not only isolation of individual microorganisms from a mixed population, but also the maintenance of such individuals and their progenies in artificial media, where no other microorganisms find way to grow.

However, it is not easy to isolate the individual microorganisms from natural habitats and grow them under imposed laboratory conditions. For this, great deal of laboratory manipulation is required. If inoculums from any natural habitat is taken and allowed to grow in a culture medium, a large number of diverse colonies may develop that, due to crowdedness, may run together and, thereby, may lose individuality. Therefore, it is necessary to make the colonies well-isolated from each other so that each appears distinct, large and shows characteristic growth forms. Such colonies may be picked up easily and grown separately for detailed study. Several methods for obtaining pure cultures are in use. Some common methods are in everyday-use by a majority of microbiologists, while the others are methods used for special purposes.

Common Methods of isolation of pure culture

Pure culture of microorganisms that form discrete colonies on solid media, e.g., yeasts, most bacteria, many other microfungi, and unicellular microalgae, may be most commonly obtained by plating methods such as streak plate method, pour plate method and spread plate method.

But, the microbes that have not yet been successfully cultivated on solid media and are cultivable only in liquid media are generally isolated by serial dilution method.

Streak Plate Method

This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar medium. The successive streaks "thin out" the inoculums sufficiently and the microorganisms are separated from each other. It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. These plates are incubated to allow the growth of colonies. The key principle of this method is that, by streaking, a dilution gradient is established across the face of the Petri plate as bacterial cells are deposited on the agar surface. Because of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are deposited

Various methods of streaking

Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/ needle and restreaked onto fresh media to ensure purity.

Pour Plate Method

This method involves plating of diluted samples mixed with melted agar medium. The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C).
The bacteria and the melted medium are mixed well. The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity.

Pour plate method has certain disadvantages as follows: (i) the picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and (ii the microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms.

However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture.

Pour Plate Method

A. Media/dilution	B. Pouring of the plate; and	C. Colony development after incubation. Control consists of the sterilized plating medium alone

The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In contrast to pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium.

Spread Plate Method

In this method the mixed culture of microorganisms is not diluted in the melted agar medium (unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid, usually, water or physiological saline. A drop of so diluted liquid from each tube is placed on the centre of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-­rod.

The medium is now incubated. When the colonies develop on the agar medium plates, it is found that there are some plates in which well-isolated colonies grow. This happens as a result of separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate.

Serial Dilution Method

As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions.

Spread plate method

The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution. The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. For convenience, suppose we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms i.e., 100 microorganisms/ml of the liquid medium.

Serial dilution method

If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10 microorganisms/ ml. If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism. If this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism.

Special Methods of Isolation on of Pure Culture

1. Single Cell Isolation methods

An individual cell of the required kind is picked out by this method from the mixed culture
and is permitted to grow. The following two methods are in use.

(i) Capillary pipette method

Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile pipette drawn to a capillary. One then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillary pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture.

(ii) Micromanipulator method

Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial cell) from a hanging drop preparation. The micro-manipulator has micrometer adjustments by means of which its micropipette can be moved right and left, forward, and backward, and up and down. A series of hanging drops of a diluted culture are placed on a special sterile coverslip by a micropipette.

Now a hanging drop is searched, which contains only a single microorganism cell. This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip. When the number of cells increases in that drop as a result of multiplication, the drop is transferred to a culture tube having suitable medium. This yields a pure culture of the required microorganism.

The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. The disadvantages are that the equipment is expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason why this method is reserved for use in highly specialized studies.

2. Enrichment Culture Method

Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture. The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of known composition and specific condition of incubation favors the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms.

Proof of Purity of Cultures

Assuming that one has isolated a pure culture, how does one establish that it is pure? A pure culture is one in which the cells are all of one kind, i.e., demonstrate "likeness". Hence, the proof of purity of cultures consists of demonstrating the "likeness" of microorganisms in the culture. It is based on certain criteria as follows:
1.The microorganisms look alike microscopically and stain in the same fashion.

2. When plated, all the colonies formed look alike.

3. Streaks, stabs, etc. are uniform.

4. Several isolated colonies perform identically, i.e., ferment the same sugars, and so on.

Capillary method for obtaining a single microbial cell

Maintenance and Preservation of Pure Cultures

Once a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure cultures free from contamination. Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium (subculturing) to allow continuous growth and viability of microorganisms. The transfer is always subject to aseptic conditions to avoid contamination.
Since repeated sub culturing is time consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time. In addition, there is a risk of genetic changes as well as contamination. Therefore, it is now being replaced by some modern methods that do not need frequent subculturing. These methods include refrigeration, paraffin method, cryopreservation, and lyophilization (freeze drying).

Refrigeration
Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime.
Paraffin Method

This is a simple and most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin in poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for several years.

Cryopreservation
Cryopreservation (i.e., freezing in liquid nitrogen at -196°C) helps survival of pure cultures for long storage times. In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol that prevent the formation of ice crystals and promote cell survival.
Lyophilization (Freeze-Drying)

In this method, the culture is rapidly frozen at a very low temperature (-70°C) and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the microbes go into dormant state and retain viability for years. Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in refrigerators. Freeze-drying method is the most frequently used technique by culture collection centers.

MASS SPECTROSCOPY

Mass spectrometry (MS) is an analytical technique for the determination of the elemental composition of a sample or molecule. It is also used for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios. In a typical MS procedure:

1. a sample is loaded onto the MS instrument, and undergoes vaporization.
2. the components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of positively charged particles (ions)
3. the positive ions are then accelerated by an electric field
4. computation of the mass-to-charge ratio (m/z) of the particles based on the details of motion of the ions as they transit through electromagnetic fields, and
5. detection of the ions, which in step 4 were sorted according to m/z.

MS instruments consist of three modules: an ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionization, move ions that exist in solution into the gas phase); a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields; and a detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present. The technique has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of gas phase ion chemistry (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.

Etymology

The word spectrograph has been used since 1884 as an "International Scientific Vocabulary". The linguistic roots are a combination and removal of bound morphemes and free morphemes which relate to the terms spectr-um and phot-ograph-ic plate. Early spectrometry devices that measured the mass-to-charge ratio of ions were called mass spectrographs which consisted of instruments that recorded a spectrum of mass values on a photographic plate. A mass spectroscope is similar to a mass spectrograph except that the beam of ions is directed onto a phosphor 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 oscilloscope. The use of the term mass spectroscopy is now discouraged due to the possibility of confusion with light spectroscopy. Mass spectrometry is often abbreviated as mass-spec or simply as MS. Thomson has also noted that a mass spectroscope is similar to a mass spectrograph except that the beam of ions is directed onto a phosphor screen. The suffix -scope here denotes the direct viewing of the spectra (range) of masses.

History

For more details on this topic, see History of mass spectrometry.

Replica of an early mass spectrometer

Francis William Aston won the 1922 Nobel Prize in Chemistry for his work in mass spectrometry

In 1886, Eugen Goldstein observed rays in gas discharges under low pressure that traveled away from the anode and through channels in a perforated cathode, opposite to the direction of negatively charged cathode rays (which travel from cathode to anode). Goldstein called these positively charged anode rays "Kanalstrahlen"; the standard translation of this term into English is "canal rays". Wilhelm Wien 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 J.J. Thomson later improved on the work of Wien by reducing the pressure to create a mass spectrograph.

The first application of mass spectrometry to the analysis of amino acids and peptides was reported in 1958. Carl-Ove Anderson highlighted the main fragment ions observed in the ionization of methyl esters.

Some of the modern techniques of mass spectrometry were devised by Arthur Jeffrey Dempster and F.W. Aston in 1918 and 1919 respectively. In 1989, half of the Nobel Prize in Physics was awarded to Hans Dehmelt and Wolfgang Paul for the development of the ion trap technique in the 1950s and 1960s. In 2002, the Nobel Prize in Chemistry was awarded to John Bennett Fenn for the development of electrospray ionization (ESI) and Koichi Tanaka for the development of soft laser desorption (SLD) and their application to the ionization of biological macromolecules, especially proteins. The earlier development of matrix-assisted laser desorption/ionization (MALDI) by Franz Hillenkamp and Michael Karas has not been so recognized despite the comparable (arguably greater) practical impact of this technique, particularly in the field of protein analysis. This is due to the fact that although MALDI was first reported in 1985, it was not applied to the ionization of proteins until 1988, after Tanaka's report.

Simplified example

Schematics of a simple mass spectrometer with sector type mass analyzer. This one is for the measurement of Carbon dioxide isotope ratios (IRMS) as in the carbon-13 urea breath test

The following example describes the operation of a spectrometer mass analyzer, which is of the sector type. (Other analyzer types are treated below.) Consider a sample of sodium chloride (table salt). In the ion source, the sample is vaporized (turned into gas) and ionized (transformed into electrically charged particles) into sodium (Na⁺) and chloride (Cl^-) ions. Sodium atoms and ions are monoisotopic, with a mass of about 23 amu. Chloride atoms and ions come in two isotopes with masses of approximately 35 amu (at a natural abundance of about 75 percent) and approximately 37 amu (at a natural abundance of about 25 percent). The analyzer part of the spectrometer contains electric and magnetic 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 Newton's second law of motion, 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).

Instrumentation

Ion source technologies

The ion source is the part of the mass spectrometer that ionizes the material under analysis (the analyte). The ions are then transported by magnetic or electric fields to the mass analyzer.

Techniques for ionization have been key to determining what types of samples can be analyzed by mass spectrometry. Electron ionization and chemical ionization are used for gases and vapors. In chemical ionization sources, the analyte is ionized by chemical ion-molecule reactions during collisions in the source. Two techniques often used with liquid and solid biological samples include electrospray ionization (invented by John Fenn) and matrix-assisted laser desorption/ionization (MALDI, developed by K. Tanaka and separately by M. Karas and F. Hillenkamp^{[citation needed]}).

Inductively coupled plasma (ICP) sources are used primarily for cation analysis of a wide array of sample types. In this type of Ion Source Technology, a 'flame' of 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 glow discharge, field desorption (FD), fast atom bombardment (FAB), thermospray, desorption/ionization on silicon (DIOS), Direct Analysis in Real Time (DART), atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), spark ionization and thermal ionization (TIMS).

Ion Attachment Ionization is a newer soft ionization technique that allows for fragmentation free analysis.

Mass analyzer technologies

Mass analyzers separate the ions according to their mass-to-charge ratio. The following two laws govern the dynamics of charged particles in electric and magnetic fields in vacuum:

(Lorentz force law);

(Newton's second law 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 x B is the vector cross product of the ion velocity and the magnetic field

Equating the above expressions for the force applied to the ion yields:

This differential equation is the classic equation of motion for charged particles. 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) dimensionless m/z, where z is the number of elementary charges (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 tandem mass spectrometry (MS/MS). In addition to the more common mass analyzers listed below, there are others designed for special situations.

Sector

For more details on this topic, see sector instrument.

A sector field mass analyzer uses an electric and/or magnetic field to affect the path and/or velocity of the charged particles in some way. As shown above, sector instruments 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.

Time-of-flight

For more details on this topic, see time-of-flight mass spectrometry.

The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions through the same potential, and then measures the time they take to reach the detector. If the particles all have the same charge, the kinetic energies will be identical, and their velocities will depend only on their masses. Lighter ions will reach the detector first.

Quadrupole

For more details on this topic, see Quadrupole mass analyzer.

Quadrupole mass analyzers use oscillating electrical fields to selectively stabilize or destabilize the paths of ions passing through a radio frequency (RF) quadrupole field. Only a single mass/charge ratio is passed through the system at any time, but changes to the potentials on magnetic lenses allows 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 Quadrupole ion trap, 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 common variation of the quadrupole is the triple quadrupole. Triple quadrupole mass spectrometers have three quadrupoles arranged parallel to incoming ions. The first quadrupole acts as a mass filter. The second quadrupole acts as a collision cell where selected ions are broken into fragments. The resulting fragments are scanned by the third quadrupole.

Quadrupole ion trap

For more details on this topic, see quadrupole ion trap.

The quadrupole ion trap works on the same physical principles as the quadrupole mass analyzer, but the ions are trapped and sequentially ejected. Ions are created and trapped in a mainly quadrupole RF potential and separated by m/Q, non-destructively or destructively.

There are many mass/charge separation and isolation methods but 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.

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.

The cylindrical ion trap mass spectrometer is a derivative of the quadrupole ion trap mass spectrometer.

Linear quadrupole ion trap

A linear quadrupole ion 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 quadrupole ion trap. Thermo Fisher's LTQ ("linear trap quadrupole") is an example of the linear ion trap.

Fourier transform ion cyclotron resonance

A FT-ICR mass spectrometer

For more details on this topic, see Fourier transform mass spectrometry.

Fourier transform mass spectrometry (FTMS), or more precisely Fourier transform ion cyclotron resonance MS, measures mass by detecting the image current produced by ions cyclotroning in the presence of a magnetic field. Instead of measuring the deflection of ions with a detector such as an electron multiplier, the ions are injected into a Penning trap (a static electric/magnetic ion trap) 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 deconvoluted by performing a Fourier transform on the signal. FTMS has the advantage of high sensitivity (since each ion is "counted" more than once) and much higher resolution and thus precision.

Ion cyclotron resonance (ICR) is an older mass analysis technique similar to FTMS except that ions are detected with a traditional detector. Ions trapped in a Penning trap 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.

Orbitrap

For more details on this topic, see Orbitrap.

Very similar nonmagnetic FTMS has been performed, where ions are electrostatically 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 image current 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 Fourier transformation of the recorded image currents.

Similar to Fourier transform ion cyclotron resonance mass spectrometers, Orbitraps have a high mass accuracy, high sensitivity and a good dynamic range.

Detector

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 mass spectrum, a record of ions as a function of m/Q.

Typically, some type of electron multiplier is used, though other detectors including Faraday cups and ion-to-photon detectors 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. Microchannel plate detectors are commonly used in modern commercial instruments. In FTMS and Orbitraps, 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 DC current is produced, only a weak AC image current is produced in a circuit between the electrodes. Other inductive detectors have also been used.

Analysers characteristics

Mass resolving power

The mass resolving power is the measure of the ability to distinguish two peaks of slightly different m/z.

Mass accuracy

The mass accuracy is the ratio of the m/z measurement error to the true m/z. Usually measured in ppm or milli mass units.

Mass range

The mass range is the range of m/z amenable to analysis by a given analyzer.

Linear dynamic range

The linear dynamic range is the range over which ion signal is linear with analyte concentration.

Speed

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.

Tandem mass spectrometry

Main article: Tandem mass spectrometry

A tandem mass spectrometer 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 peptide 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 collision-induced dissociation (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 quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD). An important application using tandem mass spectrometry is in protein identification.

Tandem mass spectrometry enables a variety of experimental sequences. Many commercial mass spectrometers are designed to expedite the execution of such routine sequences as single reaction monitoring (SRM), multiple reaction monitoring (MRM), and precursor ion scan. In SRM, the first analyzer allows only a single mass through and the second analyzer monitors for a single user defined fragment ion. MRM allows for multiple user defined fragment ions. SRM and MRM are most often used with scanning instruments where the second mass analysis event is duty cycle limited. These experiments are used to increase specificity of detection of known molecules, notably in pharmacokinetic studies. Precursor ion scan 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 radiocarbon dating is Accelerator Mass Spectrometry (AMS), which uses very high voltages, usually in the mega-volt range, to accelerate negative ions into a type of tandem mass spectrometer.

Common mass spectrometer configurations and techniques

When a specific configuration of source, analyzer, and detector becomes conventional in practice, often a compound acronym arises to designate it, and the compound acronym may be better known among nonspectrometrists than the component acronyms. The epitome of this is MALDI-TOF, which simply refers to combining a matrix-assisted laser desorption/ionization source with a time-of-flight mass analyzer. The MALDI-TOF moniker is more widely recognized by the non-mass spectrometrists than MALDI or TOF individually. Other examples include inductively coupled plasma-mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), Thermal ionization-mass spectrometry (TIMS) and spark source mass spectrometry (SSMS). 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 isotope ratio mass spectrometry (IRMS), which refers in practice to the use of a limited number of sector based mass analyzers; this name is used to refer to both the application and the instrument used for the application.

Chromatographic techniques combined with mass spectrometry

An important enhancement to the mass resolving and mass determining capabilities of mass spectrometry is using it in tandem with chromatographic separation techniques.

Gas chromatography

A gas chromatograph (right) directly coupled to a mass spectrometer (left)

See also: Gas chromatography-mass spectrometry

A common combination is gas chromatography-mass spectrometry (GC/MS or GC-MS). In this technique, a gas chromatograph is used to separate different compounds. This stream of separated compounds is fed online into the ion source, a metallic filament to which voltage 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.

Liquid chromatography

See also: Liquid chromatography-mass spectrometry

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 water and organic solvents, instead of gas. Most commonly, an electrospray ionization source is used in LC/MS. There are also some newly developed ionization techniques like laser spray.

Ion mobility

See also: Ion mobility spectrometry-mass spectrometry

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 duty cycle 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 LC/MS.

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.

Data and analysis

Mass spectrum of a peptide showing the isotopic distribution

Data representations

See also: Mass spectrometry data format

Mass spectrometry produces various types of data. The most common data representation is the mass spectrum.

Certain types of mass spectrometry data are best represented as a mass chromatogram. Types of chromatograms include selected ion monitoring (SIM), total ion current (TIC), and selected reaction monitoring chromatogram (SRM), among many others.

Other types of mass spectrometry data are well represented as a three-dimensional contour map. 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.

Data analysis

Basics

Mass spectrometry data analysis is a complicated subject that is very specific to the type of experiment producing the data. There are general subdivisions of data that 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-) species, whereas an electrospray source usually produces quasimolecular species which may be multiply charged. Tandem mass spectrometry purposely produces fragment ions post-source and can drastically change the sort of data achieved by an experiment.

By understanding the origin of a sample, certain expectations can be assumed as to 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 relatively crudely prepared biological sample will probably contain a certain amount of salt, which may form adducts with the analyte molecules in certain analyses.

Results can also depend heavily on how the sample was prepared 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.

The greatest source of trouble when non-mass spectrometrists try to conduct mass spectrometry on their own or collaborate with a mass spectrometrist is inadequate definition of the research goal of the experiment. Adequate definition of the experimental goal is a prerequisite for collecting the proper data and successfully interpreting it. Among the determinations that can be achieved with mass spectrometry are molecular mass, molecular structure, and sample purity. Each of these questions requires a different experimental procedure. Simply asking for a "mass spec" will most likely not answer the real question at hand.

Interpretation of mass spectra

Main article: Mass spectrum analysis

Since the precise structure or peptide sequence of a molecule is deciphered through the set of fragment masses, the interpretation of mass spectra 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 the search comes up empty, then manual interpretation^[34] or software assisted interpretation of mass spectra are performed. Computer simulation of ionization and fragmentation processes occurring in mass spectrometer is the primary tool for assigning structure or peptide sequence to a molecule. An a priori structural information is fragmented in silico 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. Software taking advantage of this idea has been developed for both small molecules and proteins.

Another way of interpreting mass spectra involves spectra with accurate mass. A mass-to-charge ratio value (m/z) with only integer precision can represent an immense number of theoretically possible ion structures. More precise mass figures significantly reduce the number of candidate molecular formulas, albeit each can still represent large number of structurally diverse compounds. A computer algorithm called formula generator calculates all molecular formulas that theoretically fit a given mass with specified tolerance.

A recent technique for structure elucidation in mass spectrometry, called precursor ion fingerprinting identifies individual pieces of structural information by conducting a search of the tandem spectra of the molecule under investigation against a library of the product-ion spectra of structurally characterized precursor ions.

Applications

Isotope ratio MS: isotope dating and tracking

Mass spectrometer to determine the ¹⁶O/¹⁸O and ¹²C/¹³C isotope ratio on biogenous carbonate

Main article: Isotope ratio mass spectrometry

Mass spectrometry is also used to determine the isotopic 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 Faraday cups which convert particle impacts to electric current. A fast on-line analysis of deuterium content of water can be done using Flowing afterglow mass spectrometry, FA-MS. Probably the most sensitive and accurate mass spectrometer for this purpose is the accelerator mass spectrometer (AMS). 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 carbon dating. Labeling with stable isotopes is also used for protein quantification. (see protein characterization below)

Trace gas analysis

Several techniques use ions created in a dedicated ion source injected into a flow tube or a drift tube: selected ion flow tube (SIFT-MS), and proton transfer reaction (PTR-MS), are variants of chemical ionization 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.

Atom probe

Main article: Atom probe

An atom probe is an instrument that combines time-of-flight mass spectrometry and field ion microscopy (FIM) to map the location of individual atoms.

Pharmacokinetics

Main article: Pharmacokinetics

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 LC-MS with a triple quadrupole mass spectrometer. 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 insuring data integrity with such complex sample matrices. Much attention is paid to the linearity of the standard curve; however it is not uncommon to use curve fitting 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 microdosing studies, which are seen as a promising alternative to animal experimentation.

Protein characterization

Main article: Protein mass spectrometry

Mass spectrometry is an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (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 "top-down" strategy of protein analysis. In the second, proteins are enzymatically digested into smaller peptides using proteases such as trypsin or pepsin, either in solution or in gel after electrophoretic 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 peptide mass fingerprinting (PMF), if the identification is performed using the sequence data determined in tandem MS analysis it is called de novo sequencing. These procedures of protein analysis are also referred to as the "bottom-up" approach.

Space exploration

As a standard method for analysis, mass spectrometers have reached other planets and moons. Two were taken to Mars by the Viking program. In early 2005 the Cassini-Huygens mission delivered a specialized GC-MS instrument aboard the Huygens probe through the atmosphere of Titan, the largest moon of the planet Saturn. 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 onboard the Cassini-Huygens spacecraft is an ion and neutral mass spectrometer which has been taking measurements of Titan's atmospheric composition as well as the composition of Enceladus' plumes.

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 magnetosphere.

Respired gas monitor

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 operating room, they were a part of a complex system, in which respired gas samples from patients undergoing anesthesia 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 u, fragmentation of some of the heavier molecules negated the need for a higher detection limit.