'Mass spectrometry' (previously called 'mass spectroscopy' ()
[1] or informally, '"mass-spec"' and 'MS') is an analytical technique used to measure the
mass-to-charge ratio of
ions. It is most generally used to find the composition of a physical sample by generating a
mass spectrum representing the masses of sample components. The mass spectrum is measured by a 'mass spectrometer'.
All mass spectrometers consist of three basic parts: an '
ion source', a 'mass analyzer', and a 'detector system'. The stages within the mass spectrometer are:
# Producing ions from the sample
# Separating ions of differing masses
# Detecting the number of ions of each mass produced
# Collecting the data and generating the mass spectrum
The technique has several applications, including:
★ identifying unknown
compounds by the mass of the compound molecules or their fragments
★ determining the
isotopic composition of elements in a compound
★ determining the
structure of a compound by observing its fragmentation
★ quantifying the amount of a compound in a sample using carefully designed methods (mass spectrometry is not inherently quantitative)
★ studying the fundamentals of
gas phase ion chemistry (the chemistry of ions and neutrals in vacuum)
★ determining other physical, chemical, or even biological properties of compounds with a variety of other approaches
History
In
1886,
Eugen Goldstein observed "rays" that travelled through the channels of a perforated
cathode in a low pressure
gas discharge and moved toward the anode, in the opposite direction to the negatively charged
cathode rays. Goldstein called these positively charged
anode rays "Kanalstrahlen" or 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 (''e/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
Wilhelm Wien by reducing the pressure to create a mass spectrograph. The processes that more directly gave rise to the modern version of the mass spectrometer were devised by
Arthur Jeffrey Dempster and
F.W. Aston in
1918 and
1919 respectively.
In
2002, the
Nobel Prize in Chemistry was received by John Bennett Fenn for the development of
electrospray ionization (ESI) and
Koichi Tanaka for the development of
soft laser desorption (SLD) in
1987. An improved SLD method,
matrix-assisted laser desorption/ionization (MALDI), was developed in 1987 by
Franz Hillenkamp and
Michael Karas.
[2]
Simplified working example
Schematics of a typical mass spectrometer
Different chemicals have different
masses, and this fact is used in a
mass spectrometer to determine what chemicals are present in a sample. For example, table salt (
Na Cl), may be
vaporized (turned into
gas) and ionized (broken down) into electrically charged particles (Na
+ and Cl
-), called
ions, in the first phase of the mass spectrometry. The
sodium ions are monoisotopic, with mass 23u.
Chloride ions have two
isotopes of mass 35u (~75%) and mass 37u (~25%). They also have a charge, which means that their speed and direction may be changed with an
electric or
magnetic field. The ions are accelerated to a high speed by an electric field after which they are directed into a magnetic field. The magnetic field applies a force to each ion perpendicular to the plane defined by the particles' direction of travel and the magnetic field lines. This force deflects the ions (makes them curve instead of traveling in a straight line) to differing degrees depending on their mass-to-charge ratio. The lighter ions are deflected more than the heavier ions because according to
Newton's second law of motion the acceleration of a particle is inversely proportional to its mass. Thus the magnetic field deflects the lighter ions more than the heavier ions. The detector measures the deflection of each resulting ion beam. From this measurement, the mass-to-charge ratios of all the ions produced in the source can be determined. From this information it is possible to determine the chemical composition of the original sample (i.e. that both sodium and chlorine are present in the sample) and the isotopic compositions of its constituents (i.e. whether the ratio of
35Cl to
37Cl has been changed by some process).
This example was of a
sector instrument, however there are many types of mass spectrometers. All of these have in common that they possess an 'Ion Source', that produces ions, an 'Analyzer' that sorts them in some way by their masses, and a 'Detector' that measures the relative intensities of different masses. The underlying principle of all mass spectrometers is that the paths of gas phase ions in electric and magnetic fields are dependent on their mass-to-charge ratios which is used by the analyzer to distinguish the ions from one another.
Instrumentation
Ion source
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 (due to
John Fenn) and
matrix-assisted laser desorption/ionization (MALDI, due to K. Tanaka and separately, M. Karas and F. Hillenkamp).
Inductively coupled plasma sources are used primarily for metal analysis on a wide array of sample types. 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 ionisation.
[3]
Mass analyzer
Mass analyzers separate the ions according to their
mass-to-charge ratio. All mass spectrometers are based on dynamics of charged particles in electric and magnetic fields in vacuum where the following two laws apply:
:
(
Lorentz force law)
:
(
Newton's second law of motion)
where 'F' is the force applied to the ion, ''m'' is the mass of the ion, 'a' is the acceleration, ''q'' is the ionic 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 of charged particles. Together with the particle's initial conditions it completely determines the particle's motion in space and time and therefore is the basis of every mass spectrometer. It immediately reveals that two particles with the same physical quantity ''
m/q'' behave exactly the same. Thus all mass spectrometers actually measure ''m/q'' and strictly speaking should be called mass-to-charge spectrometers. When presenting data, it is common to use the (officially) dimensionless ''m/z'' (called
mass-to-charge ratio, although (more accurately) it represents the ratio of the mass number and the charge number), where z is the number of
elementary charges (''e'') on the ion (z=q/e).
There are many types of mass analyzers, using either static or dynamic fields, and magnetic or electric fields, but all operate according to this same law. 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 other less common ones designed for special situations.
Sector
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 change the direction of ions that are accelerated through the mass analyzer. The ions enter a magnetic or electric field which bends the ion paths depending on their mass-to-charge ratios, deflecting the more charged and faster-moving, lighter ions more. The ions eventually reach the detector and their relative abundances are measured. The analyzer can be used to select a narrow range of m/q or to scan through a range of m/q to catalog the ions present.
[4]
Time-of-flight
Perhaps the easiest to understand is the
Time-of-flight (TOF) analyzer. It 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, then their
kinetic energies will be identical, and their
velocities will depend only on their
masses. Lighter ions will reach the detector first.
[5]
Quadrupole
Quadrupole mass analyzers use oscillating electrical fields to selectively stabilize or destabilize ions passing through a
radio frequency (RF)
quadrupole field. 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 operates without trapping the ions. A common variation of the quadrupole is the triple quadrupole.
Quadrupole ion trap
The
quadrupole ion trap works on the same physical principles as the QMS, 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
are stable while ions with mass
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.
[6][7]
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 (LTQ) is similar to a QIT, but traps ions in a 2D quadrupole field, instead of a 3D quadrupole field as in a QIT. Ions can be stored along the entire length of the LTQ which results in a higher ion capacity.
Fourier transform ion cyclotron resonance
Fourier transform mass spectrometry, 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 cyclical 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 high resolution and thus precision.
[8][9]
Ion cyclotron resonance 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 with ions of different mass being resolved in time.
Orbitrap
The
Orbitrap is the most recently introduced mass analyser. In the Orbitrap, 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 in the Orbitrap. 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.
[10]
Detector
The final element of the mass spectrometer is the detector. The detector records the charge induced or 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, significant amplification is often necessary to get a signal.
Microchannel Plate Detectors are commonly used in modern commercial instruments.
[11] 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.
[12]
Tandem MS (MS/MS)
Tandem mass spectrometry involves multiple steps of mass selection or analysis, usually separated by some form of fragmentation. A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry. 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 catalogs 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.
[13]
Tandem mass spectrometry enables a variety of experiments. Although it allows for many uniquely designed experiments some types of experiments are commonly used and built into many commercial mass spectrometers. Examples of these include single reaction monitoring (SRM), multiple reaction monitoring (MRM) and precursor ion scan. In single reaction monitoring the first analyzer allows only a single mass through and the second analyzer monitors for a specifically defined fragment ion. MRM is nearly identical except the second analyzer monitors multiple user defined fragment ions. These monikers 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 such as 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 separated by a user defined m/z value. This experiment is used to detect specific motifs within unknown molecules.
Common Mass Spectrometer Configurations & Techniques
When all of the elements (source, analyzer and detector) of a mass spectrometer are combined to form a complete instrument and the specific configuration becomes common a new name, often an abbreviation of one or more of the internal components, becomes attached to the specific configuration and can become, within certain circles, more well known than the specific internal components. The most ubiquitous example 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, however, often more widely recognized by the non-mass spectrometrist scientist than MALDI or TOF individually as if inseparable. 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 implies a very specific mass analyzer and detection system as with AMS, which is always sector based. In other cases there are common configurations that may be implied but not necessarily.
Certain applications of mass spectrometry have developed monikers that although technically referring to a broad application also tend to indicate a specific or a limited number of instrument configurations. An example of this is
isotope ratio mass spectrometry (IRMS). Despite only specifically indicating an application, the use of a limited number of sector based mass analyzers is implied and the name is used to refer to both the application and the instrument used for the application.
Other Separation Techniques Combined with Mass spectrometry
An important enhancement to the mass resolving and determining capacity of mass spectrometry is the combination of mass spectrometry with analysis techniques that the resolve mixtures of compounds in a sample based on other characteristics before introduction into the mass spectrometer.
Gas chromatography/MS
: ''See also the main article on
Gas chromatography-mass spectrometry''
A common form of mass spectrometry 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 on-line 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 analyser and are eventually detected.
[14]
Liquid chromatography/MS
: ''See also the main article on
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 combination of
water and organic
solvents, instead of gas. Most commonly, an
electrospray ionization source is used in LC/MS.
IMS/MS
Ion mobility spectrometry/mass spectrometry is a technique where ions are first separated by drift time through some pressure of neutral gas given an electrical potential gradient before being introduced into a mass spectrometer.
The drift time is a measure of the radius relative to the charge of the ion. The duty cycle of IMS (time over which the experiment takes place) is longer than most mass spectrometers 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.
[15]
The duty cycle of IMS is short relative to liquid chromatography or gas chromatography separations and can thus be coupled to such techniques producing triply hyphenated techniques such as LC/IMS/MS.
Data and analysis
Data representations
Mass spectrometry produces various types of data. The most ubiquitous 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
contour map of mass-to-charge on one axis, intensity on another and an additional experimental parameter (often time) on the third axis, thus producing a three dimensional surface.
Data analysis
'Basics'
Mass spectrometry data analysis is a complicated subject matter that is very specific to the type of experiment producing the data. There are several general subdivisions of data that are fundamental to beginning to understand 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.
There are many different types of ion sources that behave very differently from each other. A source such as an electron ionization source produces many fragments and mostly odd electron species with one charge, whereas a source such as an electrospray source usually produces quasimolecular even electron species that 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. For example, if the sample is coming from a synthesis/manufacturing process impurities are likely to be present that are related to the major component. If the sample is a relatively crude preparation of a biological sample, the sample likely contains a certain amount of salt that may form
adducts with the analyte molecules in certain analyses.
Results can also depend heavily on how was the sample prepared and how was it run/introduced. An important example is which matrix was 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 most commonly overlooked basic question by non-mass spectrometrists trying to use mass spectrometry or interact with a mass spectrometrist is what is the over-arching goal of the project. To interpret data one must know the desired outcome (and have collected the right data in the first place). There are many bits of information that can be gleaned from mass spectrometry data, such as the masses of the molecules, the purity of the sample, and the structure of the molecules. Each of these questions requires a different approach. Simply asking for a "mass-spec" will most likely not answer the real question at hand.
Applications
Isotope ratio MS: isotope dating and tracking
Mass spectrometer to determine the 16O/18O and 12C/13C isotope ratio on biogenous carbonate
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. Labelling with stable isotopes is also used for protein quantification. (see
Protein quantitation 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 articles: 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 articles: 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.
[16][17][18]
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.
Mass spectrometry of proteins
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 analyser. This approach is referred to as "top-down" strategy of protein analysis. In the second, proteins are enzymatically digested into smaller
peptides using an agent such as
trypsin or
pepsin. Other proteolytic agents are also used. The collection of peptide products are then introduced to the mass analyser. This
peptide mass fingerprinting (PMF) approach of protein analysis is also referred to as the "bottom-up" approach.
Whole protein mass analysis is primarily conducted using either
time-of-flight (TOF) MS, or
Fourier transform ion cyclotron resonance (FT-ICR). These two types of instrument are preferable here because of their wide mass range, and in the case of FT-ICR, its high mass accuracy. Mass analysis of proteolytic peptides is a much more popular method of protein characterization, as cheaper instrument designs can be used for characterization. Additionally, sample preparation is easier once whole proteins have been digested into smaller peptide fragments. The most widely used instrument for peptide mass analysis are the MALDI
time-of-flight instruments as they permit the acquisition of PMFs at high pace (1 PMF can be analysed in approx. 10 sec). Multiple stage quadrupole-time-of-flight and the
quadrupole ion trap also find use in this application.
Protein and peptide fractionation coupled with mass spectrometry
Proteins of interest to biological researchers are usually part of a very complex mixture of other proteins and molecules that co-exist in the biological medium. This presents two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture contains roughly equal amounts of constituents, while in biological samples, different proteins tend to be present in widely differing amounts. If such a mixture is ionized using electrospray or MALDI, the more abundant species have a tendency to "drown" or suppress signals from less abundant ones. The second problem is that the mass spectrum from a complex mixture is very difficult to interpret because of the overwhelming number of mixture components. This is exacerbated by the fact that enzymatic digestion of a protein gives rise to a large number of peptide products.
To contend with this problem, two methods are widely used to fractionate proteins, or their peptide products from an enzymatic digestion. The first method fractionates whole proteins and is called
two-dimensional gel electrophoresis. The second method,
high performance liquid chromatography is used to fractionate peptides after enzymatic digestion. In some situations, it may be necessary to combine both of these techniques.
Gel spots identified on a 2D Gel are usually attributable to one protein. If the identity of the protein is desired, the gel spot can be excised, and digested proteolytically. The peptide masses resulting from the digestion can be determined by mass spectrometry using
peptide mass fingerprinting. If this information does not allow unequivocal identification of the protein, its peptides can be subject to
tandem mass spectrometry.
Characterization of protein mixtures using HPLC/MS is also called ''shotgun proteomics'' and ''mudpit''. A peptide mixture that results from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography. The eluent from the chromatography stage can be either directly introduced to the mass spectrometer through electrospray ionization, or laid down on a series of small spots for later mass analysis using MALDI.
Protein identification
There are two main ways MS is used to identify proteins.
Peptide mass fingerprinting (mentioned in the previous section) uses the masses of proteolytic peptides as input to a search of a database of predicted masses that would arise from digestion of a list of known proteins. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample.
Full MS and MS2 spectra of a peptide.
Tandem MS is becoming a more popular experimental method for identifying proteins. Collision-induced dissociation is used in mainstream applications to generate a set of fragments from a specific peptide ion. The fragmentation process primarily gives rise to cleavage products that break along peptide bonds. Because of this simplicity in fragmentation, it is possible to use the observed fragment masses to match with a database of predicted masses for one of many given peptide sequences. Tandem MS of whole protein ions has been investigated recently using
electron capture dissociation and has demonstrated extensive sequence information in principle but is not in common practice. This is sometimes referred to as the "top-down" approach in that it involves starting with the whole mass and then pulling it apart rather than starting with pieces (proteolytic fragments) and piecing the protein back together using
De novo repeat detection (bottom-up).
A number of different algorithmic approaches have been described to identify peptides and proteins from tandem mass spectrometry (MS/MS), peptide de novo sequencing and sequence tag based searching.
Other existing mass spec analysis software include:
For PMF searches refer to:
Peptide mass fingerprinting
For protein identification through peptide fragment fingerprinting (MS/MS) see these web resources :
Mascot
OMSSA
PeptideSearch
X!Tandem
Peptide de novo sequencing:
LuteFisk
PepNovo
Sherenga
Peptide sequence tag based searching:
SPIDER
InsPecT
Mascot
GutenTAG
[19]
Protein quantitation
Several recent methods allow for the quantitation of proteins by mass spectrometry. Typically, stable (e.g. non-radioactive) heavier
isotopes of
carbon (
13C) or
nitrogen (
15N) are incorporated into one sample while the other one is labeled with corresponding light isotopes (e.g.
12C and
14N). The two samples are mixed before the analysis. Peptides derived from the different samples can be distinguished due to their mass difference. The ratio of their peak intensities corresponds to the relative abundance ratio of the peptides (and proteins). The most popular methods for isotope labeling are
SILAC (stable isotope labellings with amino acids in cell culture), trypsin-catalyzed
18O labeling, ICAT (isotope coded affinity tagging), ITRAQ (isotope tags for relative and absolute quantitation).
[20]
“Semi-quantitative” mass spectrometry can be performed without labeling of samples. Typically, this is done with MALDI analysis (in linear mode). The peak intensity, or the peak area, from individual molecules (typically proteins) is here correlated to the amount of protein in the sample. However, the individual signal depends on the primary structure of the protein, on the complexity of the sample, and on the settings of the instrument. Other types of "label-free" quantitative mass spectrometry, uses the spectral counts (or peptide counts) of digested proteins as a means for determining relative protein amounts.
Protein structure
Characteristics indicative of the
3 dimensional structure of proteins can be probed with mass spectrometry in various ways.
[21] By using chemical crosslinking to couple parts of the protein that are close in space, but far apart in sequence, information about the overall structure can be inferred. By following the
exchange of amide protons with
deuterium from the solvent, it is possible to probe the solvent accessibility of various parts of the protein.
[22]
Space exploration
As a standard method for analysis several 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.
[23]
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),
[24] 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 likely still in use but none is currently being manufactured.
[25]
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.
This magnetic sector mass spectrometer's uniqueness 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 the all of the patient respired gases. 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.
[26]
See also
★
Mass spectrometry software
★
Electron spectrometer
★
Atom probe
★
Calutron
★
Helium mass spectrometer
★
MALDI imaging
★
Membrane introduction mass spectrometry
★
Secondary ionisation
★
Taylor cone
Manufacturers of Mass Spectrometry Equipment
★
Agilent Technologies
★
Bruker Daltonics
★
CovalX
★
JEOL
★
LECO Corporation
★
MDS SCIEX / Applied Biosystems
★
PerkinElmer
★
Shimadzu Corporation
★
Thermo Fisher Scientific
★
Waters Corporation
★
Varian
References
1. Murray, K. K., et al., Standard Definition of Terms relating to Mass Spectrometry (Provisional) IUPAC Analytical Chemistry Division (2006).
2. Measuring Mass: From Positive Rays to Proteins by Michael A. Grayson (Editor) (ISBN 0-941901-31-9)
3. Mass spectrometry with ion sources operating at atmospheric pressure, A. P. Bruins, , , Mass Spectrometry Reviews, 1991
4. Extending the mass range of a sector mass spectrometer, John S Cottrell, Roger J Greathead, Mass Spectrometry Reviews Vol 5, 1986. pp 215-247
5. Time-of-flight mass analyzers
H. Wollnik, Mass Spectrometry Reviews, Vol 12, 1993, pp 89-114
6. Paul W., Steinwedel H. (1953). "Ein neues Massenspektrometer ohne Magnetfeld". RZeitschrift für Naturforschung A '8' (7): 448-450
7. Quadrupole ion trap mass spectrometry: a view at the turn of the century, R. E. March, , , International Journal of Mass Spectrometry, 2000
8. Fourier transform ion cyclotron resonance spectroscopy, M. B. Comisarow and A. G. Marshall, , , Chemical Physics Letters, 1974
9. Fourier transform ion cyclotron resonance mass spectrometry: a primer, Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S., , , Mass Spectrometry Reviews, 1998
10. The Orbitrap: a new mass spectrometer, Q. Hu, R. J. Noll, H. Li, A. Makarov, M. Hardman and R. G. Cooks, , , Journal of Mass Spectrometry, 2005
11. A comparison between ion-to-photon and microchannel plate detectors, F. Dubois, R. Knochenmuss, R. Zenobi, A. Brunelle, C. Deprun and Y. L. Beyec, , , Rapid Communications in Mass Spectrometry, 1999
12. An inductive detector for time-of-flight mass spectrometry, M. A. Park, J. H. Callahan and A. Vertes, , , Rapid Communications in Mass Spectrometry, 1994
13. Linked-scan techniques for MS/MS using tandem-in-space instruments, Robert K. Boyd, , , Mass Spectrometry Reviews, 1994
14. Eiceman, G.A. (2000). Gas Chromatography. In R.A. Meyers (Ed.), ''Encyclopedia of Analytical Chemistry: Applications, Theory, and Instrumentation'', pp. 10627. Chichester: Wiley. ISBN 0-471-97670-9
15. Two-dimensional separations with electrospray ionization ambient pressure high-resolution ion mobility spectrometry/quadrupole mass spectrometry, L. M. Matz, G. R. Asbury and H. H. Hill, , , Rapid Communications in Mass Spectrometry, 2002
16. Increasing Speed and Throughput When Using HPLC-MS/MS Systems for Drug Metabolism and Pharmacokinetic Screening, Y. Hsieh and W.A. Korfmacher, Current Drug Metabolism Volume 7, Number 5, 2006, Pp. 479-489
17. Covey TR, Lee ED, Henion JD. 1986. High-speed liquid chromatography/tandem mass spectrometry for the determination of drugs in biological samples. Anal Chem 58:2453-2460.
18. Thermospray liquid chromatography/mass spectrometry determination of drugs and their metabolites in biological fluids. Covey TR et al. Anal Chem. 1985 Feb;57(2):474-81
19. Automated protein identification by tandem mass spectrometry: Issues and strategies, P. Hernandez, M. Müller and R. D. Appel, , , Mass Spectrometry Reviews, 2006
20. Proteolytic 18O-labeling strategies for quantitative proteomics, M. Miyagi and K. C. S. Rao, , , Mass Spectrometry Reviews, 2007
21. Probing noncovalent structural features of proteins by mass spectrometry, Z. Zhang, D. L. Smith, , , Mass Spectrometry Reviews, 1994
22. Hydrogen exchange mass spectrometry for the analysis of protein dynamics, T. E. Wales and J. R. Engen, , , Mass Spectrometry Reviews, 2006
23. Ions in space, S. Petrie and D. K. Bohme, , , Mass Spectrometry Reviews, 2007
24. http://caps.space.swri.edu/
25. Expired gas monitoring by mass spectrometry in a respiratory intensive care unit. Riker JB, Haberman B. Crit Care Med. 1976 Sep-Oct;4(5):223-9
26. Applications of respiratory mass spectrometry to intensive care, J. W. W. Gothard, C. M. Busst, M. A. Branthwaite, N. J. H. Davies and D. M. Denison, , , Anaesthesia, 1980
Bibliography
★ McLafferty, F. W. and Turecek, F., ''Interpretation of Mass Spectra'', University Science Books; 4th edition (May, 1993) ISBN 0-935702-25-3
★ Tuniz, C., et al., "Accelerator Mass Spectrometry: Ultrasensitive Analysis for Global Science", CRC Press, (1998) ISBN 0-8493-4538-3
★ Muzikar, P., et al., "Accelerator Mass Spectrometry in Geologic Research", Geological Society of America Bulletin v. 115 (2003) p. 643 - 654.
★
Mass Spectrometry: Principles and Applications, Edmond de Hoffman, , , John Wiley and Sons, 2001, ISBN 0-471-48566-7
External links
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Science Aid: Mass Spectrometry Easy to understand resource for high school level
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Overview of resources on Mass Spectrometry
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A History of Mass Spectrometry (Scripps)
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Mass spectrometer simulation An interactive application simulating the console of a mass spectrometer
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Nature Protocols An on-line resource containing protein - mass spectrometry methods
;Professional organizations
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American Society for Mass Spectrometry
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Australian and New Zealand Society for Mass Spectrometry
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British Mass Spectrometry Society
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Canadian Society for Mass Spectrometry
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International Mass Spectrometry Society
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