Introduction To Mass Spectrometry

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 carbon12 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 C12. 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 pretreatment 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.

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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).

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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)

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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.)

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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.

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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 )

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The samples are usually deposited onto the plate in microliter or sub-microliter volumes; this process is called “spotting”. Spotting may be done by hand, or for high throughput applications automated plate spotters are available. MALDI plates are generally re-usable many times over, although they need to be cleaned thoroughly to avoid cross-contamination. Disposable plates are also available. Most lasers used for MALDI produce light in the near UV region; either nitrogen lasers (337 nm) or ND:YAG lasers (355nm). For some applications, infra-red lasers are used. The most common matrix material used for biochemical applications is alphacyano hydroxycinnamic acid, often called “CHC” or “alpha-cyano”. There are dozens of other matrix compounds which can be used. Any sample area on the plate which is struck by the laser, is rapidly depleted. Therefore the plate (or the laser beam) must be continually be moved to allow fresh area of sample to be exposed to the laser. A camera/video monitor combination is used to visualize the interaction of the sample plate with the laser; normally “burn spots” in the dried sample make it easy to see which areas have been desorbed. The screen shot below shows an example of typical MALDI source control software which incorporates a video image of an individual sample spot, along with a "roadmap" of the entire sample plate (Figure 9 below)…

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MALDI plates containing dried (crystallized) samples can usually be kept for long periods (days or weeks) without deterioration…for future re-analysis. Care must be taken to protect stored plates from dust and contamination. The type of matrix used, as well as the laser energy, strongly influences the amount of fragmentation which takes place during the ionization process. MALDI can also be done at atmospheric pressure (as opposed to in a vacuum). This so-called “AP-MALDI” has various advantages (e.g. fast plate loading) and disadvantages (e.g. more complex interface required, larger vacuum pumps etc.). Most MALDI sources currently used in biochemical analysis are of the vacuum type. Vacuum MALDI is very efficient and sensitive because it has no interface losses (i.e. losses due to transfer of the sample from atmospheric pressure into vacuum).

MALDI vs Electrospray (Nanospray)

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MALDI produces mostly singly charged ions; this yields simpler mass spectra, especially for high mass compounds (large peptides and small proteins). ESI produces a lot of multiply charged ions, so the spectra of high mass compounds can be very complex. BUT…a high mass range is not required to see them. It is this multiple-charging aspect of ESI that allows large biomolecules to be seen with quadrupole instruments of limited mass range; see Figure 10 below

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ESI does not give much source fragmentation, although the amount of fragmentation can be varied to a certain degree by adjusting the interface parameters (voltages). With MALDI, the laser energy density and type of matrix used can be used to control the degree of fragmentation. If the amount of sample is extremely limited, MALDI is a good choice, not only because of its high sensitivity, but because sample consumption is easily controlled and 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 UV light source (e.g. mercury lamp) rather than a pulsed laser. Photoionization is useful for some classes of compounds which do not ionize efficiently by electrospray, e.g. steroids, and polycyclic aromatic hydrocarbons.

ICP: this is an acronym for Inductively Coupled Plasma, a type of source used for inorganic analysis (e.g. metallomics). The sample is typically dissolved in water and introduced as a fine spray (mixed with argon gas), which is then dissociated and ionized by application of a very intense RF electric field at atmospheric pressure. The resulting argon plasma has a brilliant flame-like appearance. Compounds in the plasma are fully dissociated to form atomic ions. ICP sources are typically used for trace metals analysis, and for measuring levels of inorganic impurities and additives in silicon semiconductors. Figure 11 below shows the basic components of a typical ICP source.

Thermal Desorption: a general term for sources which use heat to convert solid samples to gas phase samples (and ions). There are many variations on this theme; sources may operate at atmospheric pressure, or in vacuum. Usually the source uses a secondary process (such as corona discharge APCI…see below) to generate ions. Thermal desorption sources are most often used for environmental and security screening applications, e.g. analysis of soils, dusts, fingerprints, and for polymer analysis. API: this is an acronym for Atmospheric Pressure Ionization, which encompasses several sub-types of ion sources. The electrospray source is a type of API (since it operates at atmospheric pressure), but electrospray is so popular that it is usually considered to be in a separate class by itself. The sources in use today, which are commonly referred to as “API sources” e.g. APCI tend to use an electrical discharge as the primary means by which ions are formed, while APPI (atmospheric photo ionization) uses photons. (In electrospray, there is NO electrical discharge, ions are formed by evaporation of charged droplets.) API sources require the sample to be in the gas phase before it can be ionized.

Some common types of CI sources are: • APCI: Atmospheric Pressure Chemical Ionization. A continuous electrical “corona” discharge is generated in the ion source, which causes the air molecules (nitrogen and oxygen) in the source to ionize. These ionized air molecules in turn transfer their energy to the sample molecules. This is a very “soft” ionization process, i.e., it causes minimal fragmentation of most sample molecules. APCI sources can analyze liquid samples (provided the liquid can be evaporated), and are the preferred source for direct ambient air analysis (for environmental and security applications). • CI: Chemical ionization. This is very similar to APCI, except that the ionization involves energy transfer to the sample from molecules other than air. That is, the electrical discharge ionizes an additive compound, or CI reagent, which in turn ionizes the sample. Depending on the additive used, the characteristics of the ionization may be varied, e.g. to selectively ionize only a certain class of compounds while leaving others as neutrals. CI reagents such as benzene, toluene, dichloromethane etc. have been used for specific applications. The level of CI reagent added is generally very low, in the parts per thousand to parts per million range. • Some types of CI sources run at reduced pressures; this allows a stronger electrical discharge to be produced, which in turn allows more inert compounds (such as pcb’s) to be ionized. CI sources are popular for environmental analysis (e.g. measurement of dioxins in soil and water). EI: Electron Ionization or Electron Impact. The earliest type of mass spec ion source; this is the source you will see in old mass spec textbooks. It is a rugged "workhorse" device with few adjustments and little to go wrong. The EI source is designed to ionize gas phase samples in a moderate to high vacuum. It works by bombarding the sample molecules with a beam of electrons. The electron beam tends to "knock off" electrons from sample molecules, forming positive ions. The energy of the electron beam is adjustable, but the "standard" setting is 70 eV…enough energy to ionize and fragment any organic molecule. In reality, the sample is usually extensively fragmented and the parent ion is often unseen. The EI source has a very low efficiency for producing negative ions. Due to the extensive fragmentation this type of source produces, it is rarely if ever used today for the analysis of biomolecules, although it is useful for the analysis of things like pcb's and dioxins. And the list goes on: even more mass spec ion sources, in brief… • FAB: Fast Atom Bombardment…a beam of high-energy atoms (usually Argon or Xenon) is directed onto a liquid-phase sample. The sample is usually mixed with a liquid "matrix" such as glycerol. The impact of the fast atoms causes desorption (ejection) and ionization of sample molecules from the matrix. FAB is similar to MALDI in that it generally produces a prominent parent ion peak with little fragmentation; it is useful for determining the molecular weights of large biomolecules. • MAB: Metastable Atom Bombardment…similar to FAB.

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FD: Field Desorption… a solid sample is ionized and desorbed from a speciallyprepared surface by application of a very high electric field.

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Laser Ionization or Laser Ablation… this is useful for analysis of metal surfaces. A pulsed laser beam is focused tightly onto a solid surface; this causes both vaporization and ionization of a thin layer of the sample surface.

6. 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 is: a mixture of ions of varying mass and charge, contained within a small area within a high vacuum, is subjected to a strong electric field for a very short period of time (i.e., a "pulse"). This pulsed field is applied such that all the ions begin to move in the same direction, due to electrostatic force (attraction and/or repulsion). Uncharged molecules are not affected. Consider Newton's third law: f = ma, or rearrange to get a = f/m. This just means that the acceleration (a) of an object is equal to the applied force (f) divided by the mass (m). In the case of ions, the applied force (f) due to the pulsed electric field is the same for all ions which have the same charge. Ions which are, say, doubly charged, experience twice the force as singly charged ions. And of course, we may have a wide range of masses (m) for the ions in the mixture. The end result is that, following application of a brief electric field pulse, a mixture of ions of various masses and charge states is set into motion, in accordance with a = f/m; therefore, ions with the lowest m are accelerated to the greatest speed during the duration of the pulse. For equal m, ions with multiple charges are accelerated proportionally more than singly charged ions (Figure 12 below).

If the ions are now allowed to "drift" through space under high vacuum conditions, they will begin to separate (in space) according to the speed to which they were initially accelerated by the pulse. The lighter (and/or more highly charged) ions are traveling faster, and "pull ahead" of the heavier ions which are moving more slowly. (This is analogous in some ways to the separation of compounds as they flow down the length of a chromatographic column, although the mechanism of separation is of course different). If we place an ion detector at a fixed distance from the ion source (pulsed field), and monitor the arrival times of ions following the initial pulse, we find of course that the lightest (and/or most highly charged) ions arrive first, followed in sequence by heavier or less charged ions. This record of number of ions detected versus arrival time is the basis of a time of flight mass spectrum.

Analogous to chromatography, a longer flight time generally results in greater separation (resolution) between similar compounds (masses). In practice, the total ion flight distance (path) is usually between 100 and 300 cm for commercial TOF mass spectrometers. This length is a practical compromise based on the fact that lab instruments need to be of a reasonable size, and also that there are many other factors influencing resolution besides length of the flight path. Making the flight path longer offers minimal improvement in resolution, beyond a certain point. The common components of a time-of-flight mass spec are: (Figure 13 below)

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Ion Source: usually a MALDI-type source, but others may be used. See section on ion sources for more details… Ion Accelerator: the unit which applies the pulsed electric field to the mixture of ions from the source. Usually consists of an array of stacked metal plates and metal meshes (grids) (Figure 14 below).

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Ion Reflector: sometimes called by other names, such as "ion mirror" or "reflectron". 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 of the microchannel plate (mcp) variety. An mcp is a very thin, flat glass plate with many microscopic channels; the channels are coated internally with a material which emits electrons when struck by ions (or electrons). Ions strike one side of the plate, causing electrons to be released. The electrons "bounce" along through the channels in the plate, eventually emerging from the other side, where they are collected and counted. Two stacked mc plates are usually used, to give higher signal gain. See photos and schematic diagram following. Note that the mcp detector responds best to ions which strike it at high velocity; if an ion is traveling too slowly when it strikes the detector, it may not eject any electrons, and therefore will not be detected. Timing and Data systems: in modern TOF instruments the timing of the accelerating pulses, and detection of the ions, is all computer controlled. High resolution TOF instruments require fast (and expensive) timing and data systems.

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High voltage power supplies: for large, heavy biomolecules in particular, very strong electric fields are required to accelerate the ions to a reasonable velocity. (Recall that "slow" ions are poorly detected.) In practice, this means that very high voltages are usually applied in the accelerator and reflector

regions…generally in the range of 4 to 30 kV. The higher the accelerating voltage, the better the performance for large molecules…but with a corresponding increase in size and cost of the mass spec. Types of TOF: •

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linear: the first and simplest type of TOF mass spec; the ions travel in a straight line from the ion source to the ion detector. Unless the flight path is very long, resolution will be limited. However, this type of instrument is the simplest (translation: lowest cost), is easy to tune and use, can have a very large mass range (300k Daltons or more). reflectron: mentioned above. In addition to effectively lengthening the ion flight path, the reflector also helps compensate for variations in ion motion in the source, improving resolution even further. Multiple reflections of the ion beam can be used (e.g. 3 reflectors can be used to create a "W" shaped flight path) to get a very long flight path in a compact instrument. The drawback is cost, complexity, sensitivity and difficulty in tuning the instrument (one or more reflectors). The longer the flight path the better the resolution but sensitivity is decreased as some ions are lost along the way. TOF-TOF: this is basically two separate TOF mass specs connected in series, with an ion fragmentation stage in between. Following the initial TOF stage, rather than striking an ion detector, ions enter a timed “selection stage” or “gate”, which either allows ions to pass, or rejects them, based on their flight times (and thus on their m/z ratio). Selected ions, which are allowed to pass this gate, enter a fragmentation cell, where they collide with inert gas molecules. Following fragmentation, the mixture of ions enters a second acceleration stage, after which the flight path is similar to conventional TOF instruments (Figure 15 below):

axial vs orthogonal ion injection: the traditional TOF instrument uses a pulsed (MALDI) ion source which is located "in line" (on-axis or axial) with the initial

flight path of the ions; the ions travel in a straight line from the moment they are created until they are accelerated into the flight tube; as in Figures 12 and 16. A newer type of TOF instrument injects the ions into the accelerator stage at a 90 degree angle ("orthogonal") to the flight path, as in Figure 13 for example. This allows the use of continuous ion sources (such as electrospray) in addition to MALDI, and also offers some advantages in terms of improved resolution. TOF in Biochemistry: Time of Flight mass specs are very popular in the biochemical field 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 filter assembly is often called a "rod set" or a "quad".) The rods are most commonly cylindrical, although sometimes they have a hyperbolic cross section (Figure 17 below).

A combination of alternating-current (AC) and direct-current (DC) 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 length of the rod set. Figure 18 below is an "artist's concept" of the flight path of the ions…

In the simplest mode of operation, the AC and DC voltages applied to the rods are kept at a constant level; in this case, only one relatively narrow range of mass/charge ratios is "stable" within the quad. These ions (if they exist) can pass freely through the quad and exit the other end; all other ions are lost: either they are ejected out the sides of the quad (i.e. between the gaps between the electrodes) or they strike the electrodes and are neutralized. Two other common modes of quadrupole operation are scanning (where the AC and DC voltages are varied but the ratio between them is maintained at a constant value), and peak hopping (where the voltages "jump" between a series of values which are stable for particular ions of interest. The scanning mode of operation is the most common, particularly for unknown samples, and produces what is immediately recognized as a 'mass spectrum". The peak hopping (or multiple ion monitoring) mode is used for quantitation of samples in which the ions of interest are known, e.g. detection of environmental pollutants. The most popular configuration of the quadrupole mass spec is the so-called triple quad, (see Figure 19 below). As the name implies, three quadrupoles are arranged in series, so that sample ions pass through all of them sequentially on their path from the ion source to the detector. The three quads are often referred to as Q1, Q2 and Q3. (Note that on some instruments, the middle unit (Q2) is not actually a quadrupole (4-rod array): it can be a "hexapole" (6 rods), "octopole" (8 rods), or some other structure, such as an array of ringshaped electrodes.). The vacuum interfaces used with triple quad instruments are usually optimized for use with electrospray sources. (If quadrupoles are used in the interface region, they are sometimes referred to as Q0 or Q00.) Triple quad ion detectors come in various shapes and forms, but fundamentally are of either the discrete-dynode variety, or the continuous-dynode (Channeltron) type (Figure 20).

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The operation of a quadrupole MS/MS instrument is basically as follows. Ions of a particular mass (m/z) of interest are selected in Q1; all other ions are rejected. The selected ions (sometimes called parent ions or precursor ions) are injected into Q2,

which is called a collision cell (or something similar). An inert gas such as nitrogen or argon is added to Q2, with the result that incoming ions collide with the inert gas molecules and undergo fragmentation. The degree of fragmentation can be controlled by varying (1) the amount of gas in Q2, (2) the type of gas in Q2, and (3) the speed with which the ions enter Q2 (the "ion energy"). The ion fragments produced are sometimes called daughter ions or product ions (but usually just fragment ions). Q2 is a nonresolving quad, which means that it can only contain and transmit ions, but it cannot mass-select them. The fragment ions produced in Q2 are then passed into Q3, which is a resolving quad like Q1. Here, masses of interest can again be selected…either by means of full mass scans, or monitoring of selected ions. The power of triple quad MS/MS lies in its selectivity, which results in a very low background noise level. Triple quad MS/MS is particularly suited to quantitative pharmaceutical and environmental analysis at ultra-trace levels. MS/MS is what made quadrupole mass specs famous; the first commercial MS/MS instruments (developed in the early 1980s) were all triple quads. Today, TOF, Ion Trap, and Hybrid mass specs also offer MS/MS capabilities, often combined with other advantages such as fast scanning and wide mass ranges. Quads in Biochemistry: Quadrupole mass specs are very popular for a number of reasons: they are generally compact, and are available with a very wide range of price and performance characteristics. The quadrupole MS is in general very rugged and reliable, 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 structure, where they are confined or trapped by DC and AC electric fields. A fundamental difference between quadrupole and ion trap systems is that the quadrupole mass filter is a "flow through" system, in which the ions being filtered are (normally) in constant forward motion through the electrode array. In the ion trap, by definition, ions are "trapped" for a period of time (which can be up to several seconds), with no forward motion, before they are released for detection. The selection or mass filtering of the ions occurs during the time in which they are trapped. By varying (scanning) the intensity of the AC electric field(s), ions of specific mass-to-charge ratios become resonant, and are selectively ejected (scanned out) from the trap region. A unique advantage of most ion traps is that a stream of incoming ions can be accumulated for a period of time, then scanned out all at once, in order to improve sensitivity for ultra-trace analysis. Types of ion traps:

•

3D ion traps: in these systems, the ions move in a three-dimensional pattern during the trapping/filtering stage. The most common (and original) type of 3D trap is the quadrupole ion trap, which physically does not look much like a quadrupole rod set, EXCEPT in cross-section where the two are very similar! The ion confinement cell consists of a ring-shaped electrode with curved "cap" electrodes above and below the ring…Figure 21 below:

The sketch above shows only the mass analyzer section. The complete instrument looks more like this…(Figure 22, below)

•

A recently commercialized type of 3D ion trap system, the Orbitrap, is based in part on the Kingdon trap, originally developed in 1923, although it also has some characteristics of both 3D quadrupole traps and FT-ICR traps. The Orbitrap ion

confinement cell consists of specially-shaped central and outer electrodes which are fabricated with very high precision…see Figure 23 below…

•

In the Orbitrap, ion oscillations in the z-direction (as indicated on diagram) are detected as electrical 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 traps: in these instruments, the ions move in an essentially linear pattern during the trapping/filtering stage. The ion confinement cell usually looks much like a quadrupole rod array, with either hyperbolic or round rods. The ions are confined in a narrow beam along the central axis of the trap. When it is time to detect the ions, they may be ejected from the trap either through the ends of the rod array (i.e. along the central axis, "axial ejection"), or through the sides of the rod array (through slots cut in the rods, "radial ejection"). The following diagrams (Figures 24-26) tell the whole story…

• . •

•

It should be noted that a conventional quadrupole rod set can be also used as a linear ion trap; this allows some mass specs to use the same rod array in two different modes of mass filtering operation (quadrupole filtering OR ion trapping). fragmentation (MS-MS and MSn) in ion traps: most ion trap instruments have the capability of performing MS/MS type analyses. However, the mechanism by which this is done differs from that of triple quad instruments: in a triple quad, the fragmentation occurs continuously, i.e. it is sequential in space. In an ion trap, the fragmentation is sequential in time. The basic process for MS/MS in an ion trap is as follows. A mixture of sample ions is admitted to the trap and retained. Next, ions of unwanted m/z are selectively ejected from the trap, while the desired sample ions are retained. Then follows a fragmentation step, in which the AC fields are varied to increase the energy (motion) of the trapped ions. Normally there is a low pressure of inert gas (helium or nitrogen) present in the trap at this point. The net effect is similar to the Q2 of a triple quad instrument: the rapidly-moving sample ions collide with the inert gas molecules in the trap and become fragmented. Finally, the AC fields are varied so as to "scan out" the fragments from the trap. Note that, if desired, selected fragments may be retained in the trap and subjected to a second fragmentation step (or even a third). This ability to retain and repeatedly fragment sample ions is unique to ions traps, and is usually known as MSn where "n" is the number of m/z selection steps. MSn can be useful for structural analysis of complex biomolecules.

Ion Traps in Biochemistry: Ion Traps are one of the most popular types of mass spec for biochemical applications. They generally offer compact size, good sensitivity, fast scanning, MS/MS and MSn capabilities, adequate mass range (for peptides anyways), and resolution ranging from good to amazing depending on the type of instrument and the scanning mode. The most common type of ion source used is some sort of electrospray. Cost of these instruments scales with their capability, but in general they are among the most affordable types of mass spectrometer. Fourier Transform (FTMS, FT-ICR) The Fourier Transform mass spectrometer is a type of ion trap. Ions to be analyzed are injected into a cube-shaped cell whose walls are composed of three types of opposing metal plates: trapping, excitation, and detection. In addition, a very strong DC (constant) magnetic field is applied to the cell. When applied in the proper proportions, the combination of electric and magnetic fields causes the ions to be trapped in the center of the cell. If an RF signal is applied to the "excitation" plates, the trapped ions move in a circular (orbital) path within the cell. The frequency of the orbital motion of any particular ion is directly related to its mass/charge ratio. A given ion may orbit for one second or more…a very long time in the mass spec world….before colliding with a background gas molecule. An FT-ICR cell is shown schematically in Figure 27 below…

As in the Orbitrap, a very high vacuum in the ICR cell is required to minimize the frequency of these ion-molecule collisions, thereby enhancing instrument performance. The FTMS differs from other types of mass spectrometers (except its cousin, the Orbitrap) in that it does not have a conventional ion detector which converts ions to electrons upon impact. Rather, the FT technique relies on a "non-destructive" detection technique called Ion Cyclotron Resonance (ICR). Since the ions are moving, charged particles, as they "orbit" within the cell they induce an electrical potential in the "detection" plates. This electrical signal is amplified, then analyzed using the mathematical principle of Fourier Transform, which basically means that all the different frequency components of the detected electrical signal are analyzed and deconvoluted to yield a mass spectrum. (The frequency with which an ion "orbits" in the ICR trap is inversely proportional to its m/z ratio, e.g. heavy ions orbit more slowly than lighter ones. The more ions there are at a particular m/z the stronger the detected signal at that particular frequency. hence the ICR cell yields information on both the mass/charge and relative abundance of ions.) The capabilities of FTMS are impressive: the resolution of modern instruments ranges from 100000 to beyond 1 million, with excellent mass accuracy (low ppm or better) and very high sensitivity (attomoles in some cases). Depending on the instrument, biofriendly ion sources such as nano-electrospray and MALDI may be used. FTMS is primarily used for structure elucidation work, as opposed to quantitation. A very strong magnetic field is required to produce a detectable ICR signal with currently available electronics. This translates into a requirement for a very large magnet. The magnet structure on an FT-ICR instrument typically weighs between 50 and 400 kilograms. Some instruments (see Figure 28 below) come with ladders as standard equipment, for inspection and servicing of the upper parts of the magnet structure.. The magnets used are superconducting, which means that they need to be cooled with liquid nitrogen and helium. A very high vacuum (about 10-10 torr) is also required in the ICR cell, which necessitates a complex multi-stage pumping system.

Besides the requirements for a lab with a very solid floor, and plenty of space, the final requirement for the potential FTMS owner is for very deep pockets. These instruments are among the most costly of current mass spectrometers, and require careful tuning and meticulous maintenance if they are to perform at their full potential. Most FTMS instruments today are hybrids which employ additional mass selection stages prior to the final FT-ICR cell; see the following section for details. FTMS in Biochemistry: the FTMS is in many ways the biochemist's Dream Machine: the ultimate in resolution, fast scanning, high sensitivity, wide mass range… but unfortunately, also large in size and high in cost. It can also be finicky and difficult to tune, operate and maintain. FTMS is still more of a "cutting edge research" instrument than a 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 works as follows. A beam of ions (from an ion source), traveling in a straight line, is injected into a variable magnetic field (see Figure 29 below)…

The influence of the magnetic field causes the flight path of the ions to be deflected into an arc; the radius of the arc for any particular ion depends on its mass/charge ratio. The ions exit the magnetic field and are allowed to continue on their new flight path to an ion detector. Since the detector is far "off axis" from the original flight path of the ion beam, and has a very small acceptance area, only those ions which have been deflected by just the right amount by the magnetic field are able to strike the detector, and be counted; the rest are lost. So by varying (scanning) the strength of the magnetic field, ions of different mass/charge ratios may be focused onto the detector. A variation on the magnetic sector mass spec is the double focusing instrument (see Figure 30 below); here, the ions to be filtered pass through both a DC magnetic field, and a DC electric field, one after another in series. The net effect is to achieve a much higher mass resolution than would be possible with either field separately.

The “classic” mass specs of the 1960’s were magnetic sector instruments, usually equipped with an EI (electron impact) ion source. Advantages and disadvantages: sector instruments, especially the double focusing types, are noted for their high resolution capabilities. Until the advent of techniques such as FTMS, sector instruments were the high resolution champions of the mass spec world. On the minus side however…high-performance sector instruments in general are large, heavy, complex, expensive, and sensitive to their environment. They do not interface efficiently to pulsed ion sources such as MALDI. For most applications, they have been superseded by newer techniques such as FTMS (for ultra high resolution work) and quads/ion traps (for general 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 stages to allow MS/MS type experiments. A Q-TOF type instrument is basically a triple quad instrument in which q3 has been replaced by a TOF analyzer. This allows for very

fast, wide-mass-range, high-resolution detection of fragment ions from biomolecules such as peptides. One drawback of this technology is that the mass range of the instrument in MS/MS mode is limited by the mass range of the initial quadrupole stage, which in today’s commercial instruments is usually around 6000 m/z. (Figure 31 below: Q-TOF type hybrid mass spec schematic.)

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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…

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Trap-TOF: An ion trap mass analyzer followed by a TOF analyzer. Similar overall to the Q-TOF, however the ion trap cell is often capable of higher resolution at a given mass than a quadrupole.

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Q-Trap: Quadrupole section followed by a linear ion trap. There is normally a fragmentation cell between the two sections. Can be thought of a triple quad in which Q3 acts as a linear ion trap. The ion trap provides higher resolution and faster scanning capability than the Q3 it replaces. Note that some "dual mode” instruments use a conventional Q3 which can also run in ion trap mode.

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Trap-Trap: this officially counts as a hybrid ONLY IF the two sequential ion traps use different technologies. The LTQ-Orbitrap from Thermo Electron is a recently introduced example of this type of instrument.

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Q-FTMS: basically, a triple quad instrument where Q3 is replaced by an FT-ICR cell. Similar in concept to the Q-TOF hybrid, however the FT-ICR stage allows for even higher fragment ion resolution than a TOF stage. The large magnet associated with the FT stage makes these instruments large, heavy, and costly. Still…look at the smiling faces of the Q-FTMS users in Figure 32 below, and you will agree: the performance makes it all worthwhile…

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Trap-FTMS: similar to above, with an ion trap providing the initial stage of ion selection (and fragmentation). The Thermo Electron LTQ-FT is a commercially available example of this type of hybrid instrument.

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Nomenclature note: sometimes the above abbreviations are written as QqTOF, QqTrap, etc; the lower-case "q" denotes the presence of a non-mass-selective ion transfer or fragmentation stage (collision cell).

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In general, all hybrid instruments (plus TOF-TOF, which by definition is not a hybrid) are intended to offer improved MS/MS performance over triple quadrupole instruments, in certain areas: namely scan speed and resolution for wide mass range scans. Most hybrid instruments are aimed at proteomics applications such as protein and peptide sequencing. They are not generally

considered “quantitative” instruments…for accurate quantitation, the triple quad still rules. •

Because of the complexity of combining two mass filter technologies, and the requirement for high performance, hybrid instruments as a rule tend to be large, complicated and costly. But their flexibility and high performance has made them a common sight in most proteomics labs.

OK: So Which Type of Mass Spec is the Best ? The fact that there are so many different types of mass specs on the market today indicates that no one type is best for all situations and applications. Table 1 below summarizes in GENERAL terms some of the advantages of the different types of mass specs. Of course there are always exceptions, and technology is always advancing… Characteristics high efficiency for wide mass range scans high efficiency for targeted compound monitoring accurate quantitation

MS Type Ion Trap FTMS

Quad

TOF

Mag Sector

Hybrid







some



most





some

some



some

high sensitivity

 

rarely

rarely

rarely



some



some



some

 



most



  

high mass accuracy



high MW compounds



some



some

high resolution



most

some





most

very fast scanning modest size/weight ruggedness/mobility low cost





most

most

some

most

Some

some

most



some



Some

some

some

  

some some rarely

  

7. Some Additional Mass Spec Resources on the Internet (These are mainly introductory-level sites, but some have additional links to more advanced resources.) • • • • • • •

Mass Spectrometry Primer (from Waters) Mass Spectrometers - A Short Explanation for the Absolute Novice (from JEOL) ASMS "What is Mass Spectrometry" tutorial Introduction to Mass Spectrometry (from the University of Arizona) Characteristics of different mass analyzers (from The Fraunhofer Institute) Little Encyclopedia of Mass Spectrometry (at the University of Heidelberg) Mass Spectrometry Resource (at the University of Bristol)

8. Acknowledgements for Figures and Photos Figures 1, 8, 10, 14, 15, 20, 24, 29, Table 1, Video 1 - W. Fisher, prepared for this article Figures 2-7, 9, 12, 13, 17-19 - Applied Biosystems / MDS Sciex, http://www3.appliedbiosystems.com/applicationstechnologies/MassSpectrometry/index.h tm Figure 11 - Thomas, R. Spectroscopy 16, 26 (2001) http://spectroscopyonline.findanalytichem.com/spectroscopy/data/articlestandard/spectros copy/452001/1097/article.pdf Figure 16 - Huang, L., Baldwin, M.A., Maltby, D.A., Medzihradszky, K.F., Baker, P.R., Allen, N., Rexach, M., Edmondson, R.D., Cambell, J., Juhasz, P., Martin, S.A., Vestal, M.L., Burlingame, A.L. Molecular & Cellular Proteomics 1, 434-450 (2002) http://www.mcponline.org/cgi/content/abstract/1/6/434 Figure 21, 22, 25, 26, 30 - Thermo Electron Corporation, http://www.thermo.com/com/cda/landingpage/0,,391,00.html?ca=ms Figure 23 – Hu, Q., Noll, R.J., Li, H., Makarov, A., Hardman, M., Cooks, R.G. J. Mass Spectrom. 40, 430-443 (2005) http://www3.interscience.wiley.com/journal/110471441/abstract?CRETRY=1&SRETRY =0 Figure 27 - University of Bristol, http://www.chm.bris.ac.uk/ms/theory/theory.html Figure 28 - University of California at Berkeley - http://berkeley.edu/ Figure 31 - Vacuum Technology Incorporated, http://www.vacuumtechnology.com/PRODUCTS/MASS-SPECTROMETRY/

Figure 32 - Boston University School of Medicine http://www.bumc.bu.edu/ftms/research/esi-qqftms/