Giới thiệu về phổ khối 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.

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)

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

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 )

• 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 cyano hydroxycinnamic acid, often called “CHC” or “alpha-cyano” There are dozens of other matrix compounds which can be used

alpha-• 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)…

• 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)

• 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

• ESI does not give much source fragmentation, although the amount of

fragmentation can be varied to a certain degree by adjusting the interface

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

• FD: Field Desorption… a solid sample is ionized and desorbed from a

specially-prepared surface by application of a very high electric field

• 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)

• 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)

• 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