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The following article is intended for biologists and biochemists who are interested in knowing the basics of how mass spectrometers work. It provides a very general and descriptive introduction to mass spectrometry, with an absolute minimum of math and physics knowledge required.
An Introduction to Mass Spectrometry The following article is intended for biologists and biochemists who are interested in knowing the basics of how mass spectrometers work. It provides a very general and descriptive introduction to mass spectrometry, with an absolute minimum of math and physics knowledge required. 1. Definition of Mass Spectrometry Mass spectrometry is a chemical analysis technique which is based on the measurement of the mass (atomic or molecular weight) of molecules or atoms. 2. Applications of Mass Spectrometry Mass spectrometry is widely used today in many diverse areas. Some examples of current applications are: • environmental: analysis of air, water and soil samples for trace contaminants • pharmaceutical: drug development and quality control • biological research: determination of protein and peptide structure • semiconductor electronics: determination of levels of additives and impurities in silicon wafers • metallurgy: determination of levels of trace elements in metals and metal ores • astrochemistry: measurement of composition of planetary atmospheres and surfaces (e.g. NASA Mars Rover) • food: analysis of pesticide residues on fruits and vegetables • security: explosives and contraband drug detection • military: mobile detection of biological and chemical agents (e.g. bacteria, nerve gas) • medical: screening of newborn babies for genetic disorders • sports: screening of athletes (and race horses) for performance-enhancing drugs 3. Basic Concepts and Definitions Ion: a molecule (or atom) which has either a positive or negative electrical charge. The amount of charge is "quantized", and is reported in integer units. For example, an ion may have a charge of +1 or +2 or -3 units, but not + ½ or -1½ etc. Proton: fundamental atomic particle which has, by definition, an electrical charge of +1 unit. A neutral molecule which (for example) gains one extra proton will have an overall electrical charge of +1; if it gains 2 protons it will have a charge of +2; and so on. Protons may be either gained or lost from molecules. Electron: fundamental atomic particle which by definition has an electrical charge of -1 unit. Same behavior as "proton" above, but oppositely charged, i.e. if a neutral molecule gains one electron (or alternatively loses one proton!) it attains a charge of -1. Neutral: in mass spectrometry, this refers to any particle (usually a molecule) which has no electrical charge. Mass: in mass spectrometry, a synonym for "molecular weight" (or atomic weight); usually symbolized by "m" in equations. Has units of "Da" (Daltons) or "amu" (atomic mass units). One amu is defined as 1/12 of the mass of a carbon 12 atom (see "isotopes" below). The prefix "k" denotes "1000" e.g. "40 kDa" indicates a molecular weight of 40,000 Da. Charge: in mass spectrometry, the quantized amount of charge on an ion, e.g. +1, -2; usually symbolized by the letter "z" in equations. Mass to Charge Ratio: usually written as m/z, this is simply the molecular weight of an ion divided by the number of charges it carries. (Note that even if the charge is negative, i.e. -2, the value of m/z is still normally written as a positive number.) All common mass spectrometric techniques are based on the use of electromagnetic fields to separate ions. Ions are actually separated on the basis of their mass to charge ratio, not on the basis of their mass. However if the charge on an ion is known, its mass can be readily determined. Mass Spectrum: the data output of a mass spectrometer is most frequently presented as a graph of ion population versus mass (Figure 1 below). The largest peak in a mass spectrum (e.g. at m/z 570.8 in the figure below) is referred to as the base peak. Isotopes: Most elements consist of atoms with several stable masses or isotopes. Different isotopes of any given element have the same number of protons, but different numbers of neutrons. By far the most common example of this in biochemical mass spectrometry is carbon. The most common stable isotope of carbon (~ 99% natural abundance) has an atomic mass of 12; it is commonly referred to as "carbon 12" or C 12 . There is also a stable isotope of carbon with a mass of 13 (~ 1% abundance), commonly referred to as "carbon 13". Therefore in a mass spectrum of a carbon-containing compound, peaks due to BOTH of these naturally occurring isotopes are observed; the relative intensity of the peaks due to carbon-12 and -13 depends on the number of carbon atoms in the molecule (ion) being analyzed (Figure 2, below). Mass Resolution: the resolution, R, of a mass spectrometer is defined by R = m / Δm where m is the ion mass and Δm is the width of the corresponding peak in the mass spectrum. An instrument with a resolution of 1000 (at mass 1000) can clearly separate an ion (peak) at mass 1000 from an adjacent peak at mass 1001 or 999. AC: alternating current; an electrical potential which varies with time in a regular periodic fashion. In the mass spec world, the term "RF" (radio frequency) is often used interchangeably with the term AC. DC: direct current; an electrical potential which does not vary in a periodic fashion; it may, however, be "ramped"…increased or decreased in a smooth, controlled manner. 4. Essential Components of a Mass Spectrometer - (Figure 3, below) Vacuum Mass spectrometry is normally performed under high vacuum conditions. This is done because the ion filtering techniques used in mass specs are only effective under conditions where molecules do not undergo collisions with other molecules. Generally, a pressure lower than about 5 x 10 -5 torr (torr = 1/760 of an atmosphere = 133.322 Pa) is required for optimum performance of quadrupole and ion trap instruments; time of flight instruments normally require even lower pressures for operation (10 -6 torr range) due to their longer ion flight paths and higher mass ranges. If the pressure in the mass spec is too high, both sensitivity and mass resolution will be compromised. In modern mass specs, a combination of turbomolecular and roughing pumps are used to generate the required high vacuum conditions. It is worth noting that an ion separation technique known as Ion Mobility Spectrometry (IMS), which is similar in some ways to mass spec, does not require a vacuum. Rather, it is typically performed at atmospheric pressure. IMS offers sensitivity comparable to mass spec, but it is rarely used in biochemical applications due to its limited resolution and mass range. The beauty of IMS is that bulky and expensive vacuum pumps are not required, making it ideal for mobile applications. Ion Source and Vacuum Interface Since mass specs filter ions only, the sample molecules of interest must be ionized before they can be selected and detected. This is accomplished in an ion source, of which there are many types…discussed in more detail later in Section 5. Many ion sources operate at pressure higher than the pressure required by the mass spec analyzer. In this case, a "vacuum interface" stage is required to transfer the ions from the relatively high pressure of the source, to the very low pressure of the mass analyzer. A vacuum interface is basically a device which separates ions (i.e., the sample) from unwanted neutral gas molecules. There are many types of interfaces; most of the newer ones incorporate "proprietary" technology. An exception is the traditional MALDI ion source (discussed below), which forms ions under high vacuum conditions and therefore does not require a vacuum interface. No interface is 100% efficient; some ions are always lost. Ion Separator (Mass Analyzer) Ions are separated according to their mass-to-charge ratio. Common separation techniques are discussed in detail below in Section 6. Ion Detector The most common types of ion detectors in use today are based on the collision of ions with "active" surfaces. An active surface is most commonly a material which, when struck by an ion with sufficient velocity, releases one or more electrons. These electrons are then amplified and detected; the number of electrons produced and detected is proportional to the number of ions striking the detector. Some detectors are based on surfaces which emit light (photons) when they are struck by ions; the light is then converted to electrons in a secondary process. Data System The data system (computer + software) is responsible for controlling the operating parameters of the mass spec, and presenting the data to the operator. In the most basic sense, the data system scans the ion separator (keeping track of the mass at any given point in time), and correlates the quantity of ions detected with the selected mass. Additional Mass Spec Stages, Components and Peripherals Many mass spec components may be employed beyond the basics described above. For example, fragmentation / reaction stages are often employed to "break up" large ions into smaller fragments; this yields additional structural information beyond the simple molecular weight of the compound. In conjunction with this fragmentation, it is common to employ multiple sequential stages of mass separation. Put simply, an ion of interest is selected, fragmented, and the resulting ionized fragments are then analyzed in a second mass analysis step. This process is commonly referred to as MS/MS or (an older term) Tandem MS; (Figure 4 below). Multiple Ion Sources: many mass specs can be fitted with interchangeable ion sources, to optimize their performance for particular tasks or types of samples. Sample Prep and Separation devices: mass specs are commonly used with sample pre- treatment and pre-separation equipment such as: liquid chromatographs; gas chromatographs; gel electrophoresis; and a host of automated peripherals such as gel cutters, extractors, autosamplers, plate spotters, flow splitters, UV detectors…the list goes on. Although the same mass spectrometer may be used to analyze widely varying types of samples (e.g. air, blood samples, soil), the sample prep equipment and introduction procedure normally must be optimized for each sample type. Also, a general goal of sample prep is to present the mass spec with the cleanest sample possible (after all, who wants mud in their ion source…?) 5. Types of Ion Sources There are many types of mass spec ion sources. The two ion sources used most often in biochemical applications are electrospray and MALDI. Electrospray-type sources: these sources are designed for the direct analysis of liquids, such as a continuous flow of effluent from an LC column or discrete liquid samples produced by various separatory techniques (gel electrophoresis etc.). In general, electrospray-type sources produce ions by spraying or atomizing a liquid sample under the influence of a high DC voltage. For production of positive ions, the sample is flowed through an electrically conductive tube of small inner diameter (typically 100 um) under pressure from a liquid pump (LC pump, syringe pump etc.). A high positive voltage (typically +5000 V) is applied to the tube, and the outlet of the tube is positioned close to a metal “plate” which forms the first inlet stage of the mass spec; the plate is kept at a much lower potential (typically +500 V). The liquid becomes electrically charged by being in contact with the walls of the sprayer tube; once the liquid reaches the exit of the tube, it is virtually “sucked out” of the tube by the strong electrostatic attraction of the nearby plate. (In Figure 5 below, this electrostatic spraying process is assisted by an additional inert "sprayer gas"…more details follow…) The liquid droplets evaporate, and as they do, sample ions are ejected from the droplets; this process is called ion evaporation…(Figure 6, below) Electrospray details and Jargon: • No single electrospray source design can operate with maximum efficiency over the extremely wide range of liquid flow rates (and sample volumes) which need to be analyzed in biochemical labs. Therefore, many variations of the basic electrospray source have been developed over the years, each one optimized for particular applications. • The basic electrospray source was originally developed for use with liquid flow rates in the low microliter-per-minute range (0.5 to 20 µL/min). • In some source designs, pressurized gas is used to assist with the spraying of the sample. This tends to give a more consistent and stable spray pattern, especially at higher liquid flow rates (above 20 µL/minute), which in turn improves signal stability, sensitivity and signal/noise ratio. Double-click the window below to see gas-pressure-assisted electrospray in action…(Video 1) • At higher liquid flow rates, the volume of liquid being sprayed is too great to evaporate at normal lab temperatures. This results in low sensitivity and/or unstable ion signals. The most common cure for this problem is the addition of HEAT, to speed up the evaporation of the sample droplets. The higher the liquid flow rate, and the greater the proportion of water in the sample, the more heat is required. (Figure 7 below: example of a heated electrospray source.) • Heat can be applied to the sample by various means; generally the goal is to heat the gas surrounding the sample, to speed evaporation and desolvation. Most of the commercially-available heated sources have proprietary designs, and come with cool names such as TurboSpray, IonMax and so forth. By varying the amount of heat applied, and applying a pressurized gas to assist with the spray process, the flow rate range over which the electrospray source is efficient can be extended up to 1 ml/minute and beyond; this allows the entire output of high-flow LC columns to be analyzed without flow splitting. • Going in the other direction: if the inner diameter of the electrospray tube is reduced, along with the dead volume of the liquid handling system, the operational flow rate can be reduced to the sub-microliter-per-minute range. With a very fine spray tip, flow rates of a few nanoliters per minute can be achieved. Electrospray sources of this type are often referred to as “nanospray” sources. These sources are very efficient, since the low flow of liquid evaporates readily, and the sprayer tip can be positioned very close to (or even inside) the sampling orifice of the mass spec. Laser Desorption sources: The often-used term "MALDI" is an acronym for Matrix Assisted Laser Desorption Ionization. The basic principle of MALDI is that the sample (analyte) is mixed with a compound called a matrix, the purpose of which is to strongly absorb laser light. In almost all cases, the sample and the matrix are prepared in the form of separate solutions; the two solutions are mixed together, and the mixture is then deposited on a solid surface and allowed to dry (form crystals). For analysis, the dried sample/matrix mixture is inserted into the source region of the mass spec, which is (usually) maintained at a moderate to high vacuum. A pulsed laser beam is focused onto a tiny area of the sample; the matrix compound is chosen so as to strongly absorb the laser light. The laser pulse causes a small region of the matrix compound to instantaneously vaporize, taking the sample with it. The matrix compound also transfers energy into the sample molecules, sufficient to ionize them. The result of each laser “shot” is a “plume” of ionized sample and matrix molecules; the ions are directed into the mass spectrometer by electrostatic fields (lenses, grids etc. as required) for mass filtering….see Figure 8 below. Since MALDI is in general a pulsed ionization technique, it is well suited to time of flight mass spectrometers, which by their nature require pulsed ion sources. MALDI details and jargon: • The surface upon which the sample/matrix mixture is deposited is usually called a “plate”; the most common MALDI plate material is stainless steel, although many other materials can also be used (glass, gold, silicon etc.)…(Figure 8 below ) [...]... Note that not all TOF mass specs use an ion reflector; when a reflector is not used, the genre is known as "linear TOF" The main purpose of the ion reflector is to lengthen the ion flight path (to improve mass resolution), without making the instrument physically larger Physically, the ion reflector looks much like the accelerator, only much larger Ion Detector: the ion detectors used in TOF are usually... in today’s commercial instruments is usually around 6000 m/z (Figure 31 below: Q-TOF type hybrid mass spec schematic.) • Note from the Legal Department: the term Q-TOF is a registered trademark of Waters Corporation (Micromass) It tends to be widely (if illegally) used to refer to ALL quadrupole-TOF hybrid instruments… • Trap-TOF: An ion trap mass analyzer followed by a TOF analyzer Similar overall to. .. lab workhorse at this point in time Electromagnetic Sector The electromagnetic sector mass spectrometer, often called a sector or magnetic sector instrument, although infrequently used today in biochemistry, deserves to be mentioned because it is the "original" type of mass spec used in chemical laboratories The original, and simplest, form of sector MS is the magnetic single focusing instrument; it... purpose mass spec) Hybrid Mass Spectrometers For purposes of this article, we will consider a “hybrid” mass spec to be one which combines two or more mass filtering technologies Many combinations are possible; some types of hybrid mass specs in use today include… • Q-TOF: A Quadrupole mass analyzer followed by a Time-Of-Flight analyzer An ion fragmentation stage is inserted between the two mass filter... Types of Mass Analyzers Now that the introductory material is out of the way, you are ready to learn some details about the different types of mass specs in use today In no time at all, you will be familiar with all sorts of cool acronyms and what they mean Prepare to impress your colleagues with your new-found knowledge!! Time of Flight (TOF) The basic principle of Time of Flight (TOF) mass spectrometry. .. due to their large mass range, very fast "scanning" and generally good resolution and sensitivity A typical high-end TOF instrument achieves a mass resolution of 10,000, with very high mass accuracy, and femtomole detection limits for peptides Quadrupole The basis of the quadrupole mass spec is a mass filter consisting of four parallel, electrically-conductive electrodes or "rods" (In MS-speak, this mass. .. unused sample deposited on the plate is easily stored for reanalysis Other Types of Ion Sources Used in Mass Spectrometry: Photoionization: photoionization involves the use of ultraviolet light to ionize the sample The distinction from MALDI is that in photoionization the sample absorbs the light directly whereas in MALDI the matrix absorbs the light Photoionization sources usually employ a continuous... voltages are applied to these electrodes The ions which are to be filtered (according to their mass -to- charge ratio) are injected into one end of this electrode array, and (begin to) travel down the central axis of the quad Once inside the quad, the ions are influenced by the combined electric field of the AC and DC voltages, and follow a complex pattern of motion as they continue to travel down the... "workhorse" of today's mass spec world However in the world of biochemistry, where samples with wide mass ranges are the order of the day, the quadrupole mass spec is currently less popular than other MS techniques, such as MALDI-TOF and some types of ion traps Ion Trap Structurally, an ion trap mass spec is most closely related to quadrupole instruments In general, sample ions are injected into an electrode... signals in the split outer electrode (very similar to FTMS…see next section) These signals are amplified and subjected to Fourier Transform processing to yield a conventional mass spectrum The Orbitrap cell requires a very high vacuum (10-9 torr) in order to operate efficiently It is capable of producing very high resolution mass spectra (over 100000), with a mass range of over 6000 Da • 2D (linear) ion . general and descriptive introduction to mass spectrometry, with an absolute minimum of math and physics knowledge required. 1. Definition of Mass Spectrometry Mass spectrometry is a chemical. based on the measurement of the mass (atomic or molecular weight) of molecules or atoms. 2. Applications of Mass Spectrometry Mass spectrometry is widely used today in many diverse areas all TOF mass specs use an ion reflector; when a reflector is not used, the genre is known as "linear TOF". The main purpose of the ion reflector is to lengthen the ion flight path (to