Concepts in Mass Spectrometry

Concepts in Mass Spectrometry
Mass spectrometry (MS) is the technique for protein identification and analysis by production of charged molecular species in vacuum, and their separation by magnetic and electric fields based on mass to charge (m/z) ratio. MS has increasingly become the method of choice for analysis of complex protein samples in proteomics studies due to its ability to identify thousands of proteins. a Title of the concept Sandipan Ray, Suruchi Rao & Harini Chandra

Basic components of mass spectrometer
Master Layout (Part 1) Part 1: Fundamentals of Mass Spectrometry Part 2: Ionization techniques Part 3: Mass Analyzers Part 4: Tandem mass spectrometry 1 2 Basic components of mass spectrometer Creates ions Resolves ions Determines the mass of ions 3 Mass analyzer Ion source 4 Sample Mass detector 5

Definitions of the components: Part 1- Fundamentals of mass spectrometry
1. Mass spectrometry: A technique for production and detection of charged molecular species in vacuum, after their separation by magnetic and electric fields based on mass to charge (m/z) ratio. 2. Mass spectrometer: An instrument that produces charged molecular species in vacuum, separates them by means of electric and magnetic fields and measures the mass-to-charge ratios and relative abundances of the ions thus produced. 3. Protein sample: The protein whose sequence is to be analyzed must be broken down into peptide fragments, analyzed by mass spectrometry and the sequences then re-assembled so that the intact protein sequence is obtained. 4. Sample inlet: The first point of contact where the sample is introduced within the mass spectrometer either as liquid nano-droplets or as a mixture with matrix. 5. Ionization source: The ionization source is responsible for converting analyte molecules into gas phase ions in vacuum. The technology that enables this is termed soft ionization for its ability to ionize non-volatile biomolecules while ensuring minimal fragmentation and thus, easier interpretation. The most common ionization sources employed are Matrix Assisted Laser Desorption-Ionization (MALDI) and Electrospray Ionization (ESI). 2 3 4 5

Definitions of the components: Part 1- Fundamentals of mass spectrometry
6. Mass analyzer: The mass analyzer resolves the ions produced by the ionization source on the basis of their mass-to-charge ratios. Various characteristics such as resolving power, accuracy, mass range and speed determine the efficiency of these analyzers. Commonly used mass analyzer include Time of Flight (TOF), Quadrupole (Q) and ion trap. 7. Charged peptide fragments: The peptide fragments generated by the ionization source carry positive, negative as well as neutral charges. Sensitivity of detection for positive ions is higher than negative ions while neutral ions are not detected. 8. Detector: The ion detector determines the mass of ions that are resolved by the mass analyzer and generates data which is then analyzed. The electron multiplier is the most commonly used detection technique. 2 3 4 5

3 Part 1, Step 1: 1 2 4 5 Vacuum Envelope Detection Ionization
Mass Sorting (filtering) Ion Detector Ion Source Mass Analyzer 2 Forms ions (charged molecules) Sort Ions by Mass (m/z) Detects ions 3 Data Processing Data System Sample Inlet m/z 1000 2000 Mass Spectrum Relative Abundance 4 Action Description of the action Audio Narration As shown in animation. Show sequential appearance of each component as shown in the animation. The detailed definition of each component given in the previous 2 slides must be shown as a popup if the user moves his cursor to it. Mass spectrometer is an instrument that produces charged molecular species in vacuum, separates them by means of electric and magnetic fields and measures the mass-to-charge ratios and relative abundances of the ions thus produced. It is being increasingly used for detection and analysis of proteins from complex samples. 5

1 Master Layout (Part 2) 2 3 4 5 Types of ion sources
Part 1: Fundamentals of Mass Spectrometry Part 2: Ionization techniques Part 3: Mass Analyzers Part 4: Tandem mass spectrometry 2 Types of ion sources Matrix Assisted Laser Desorption Ionization (MALDI) 3 Electrospray Ionization (ESI) Fast Atom Bombardment (FAB) 4 Laser desorption (LD) Plasma desorption (PD) 5

Definitions of the components: Part 2 – Ionization techniques
1 1. Matrix Assisted Laser Desorption Ionization (MALDI): MALDI is an efficient process for generating gas-phase ion of peptides and proteins for mass spectrometric detection.Target plate with dried matrix-protein sample is exposed to short, intense pulses from a UV laser. 2. Electrospray Ionization (ESI): Ions are formed by spraying a dilute solution of analyte (sample) at atmospheric pressure from the tip of a fine metal capillary, creating a fine mist of droplets. The droplets are formed in a very high electric field and become highly charged. As the solvent evaporates, the peptide and protein molecules in the droplet pick up one or more protons from the solvent to form charged ions. 3. Fast Atom Bombardment (FAB): FAB is an ionization technique used in mass spectrometry. The material to be analyzed is mixed with a non-volatile chemical protection environment called a matrix and is bombarded under vacuum with a high energy beam of atoms. 4. Laser desorption (LD): Describes the process of directing laser light at a solid sample in order to generate sample ions in the gas phase. Also called laser ablation when used to gasify solid samples. 2 3 4 5

Definitions of the components: Part 2 – Ionization techniques
1 5. Plasma desorption (PD): plasma desorption mass spectrometry (PDMS) method allows the effective elimination of salt contaminants in the biomolecular film, which Is Important to Improve molecular Ion yields. 6. Matrix: Solution containing high concentration of a UV absorbing molecule deposited on sample plate along with samples. It is essential to select a matrix appropriate to the type of sample to be analysed. 7. Laser: Light amplification by stimulated emission of radiation is a mechanism for emitting electromagnetic radiation. 8. Target plate: Same as the sample plate. Samples are spotted on it. 9. Ion current: Flow of ions generated from peptides/proteins and moves towards sample orifice. 10. MS sample orifice: Small orifice of specific diameter for selective passage of specific ions from the ion current 2 3 4 5

3 Part 2, Step 1: 1 2 4 5 Types of ion sources
Matrix Assisted Laser Desorption Ionization (MALDI) 2 Electrospray Ionization (ESI) Fast Atom Bombardment (FAB) 3 Laser desorption (LD) Plasma desorption (PD) 4 Action Description of the action Audio Narration The main heading must appear after which each of the sub-headings below must be shown to appear sequentially. User must be allowed to choose any of the headings to read the definition of it as described in the previous slide. The first two must finally be highlighted and the user should be allowed to click on either of them which must re-direct the user to either of the next 2 slides depending on his choice. The ionization source is responsible for converting analyte molecules into gas phase ions in vacuum. This has been made possible by the development of soft ionization techniques, which ensures that the non-volatile protein sample is ionized without completely fragmenting it.The most commonly used ionization sources are Matrix Assisted Laser Desorption-Ionization (MALDI) and Electrospray Ionization (ESI). <As given in definitions in previous slides.> As shown in the animation 5

Matrix-assisted laser desorption ionization (MALDI)
Part 2, Step 2: 1 Matrix-assisted laser desorption ionization (MALDI) Flight tube 2 Laser 3 Generated ions Target plate Matrix &analyte 4 Action Description of the action Audio Narration Show the brown surface at the bottom with the grey projection, the violet rectangular box on the left and the grey tube on top. Show a red beam coming out of the violet rectangle and falling on the grey surface. Once it touches the surface, the small colored circles must appear from it, first the green ones followed by the red and then the blue. These must then move upwards towards the grey tube as shown. In MALDI, the analyte of interest is mixed with an aromatic matrix compound like a-cyano-4-hydroxycinnamic acid, sinapinic acid etc. This is then dissolved in an organic solvent and placed on a metallic sample plate. The evaporation of solvent leaves the analyte embedded in the matrix. The target plate is placed in a vacuum chamber with high voltage and short laser pulses are applied. The laser energy gets absorbed by the matrix and is transferred to the analyte molecules which undergo rapid sublimation resulting in gas phase ions. These ions then accelerate towards the mass analyzer based on their mass-to-charge ratio. As shown in the animation 5

ElectrosprayIonization (ESI)
Part 2, Step 3: 1 ElectrosprayIonization (ESI) 2 Ion current Protein/peptide sample + c v 3 Capillary tip Spray needle Sample orifice ~2.2 kV 4 Action Description of the action Audio Narration As shown in the animation. First show the tube on the left along with the blue box which is connected to a supply as shown. As well as the red ‘M’ shaped structure on the other end. Next show a liquid entering the leftmost end of the tube which must move along the entire length of the tube and once it reaches tip, show a spray of small dots/circles coming out of the tip which must move towards the gap in the middle of the ‘M’ shape with smallest ones passing through the small gap first. In ESI, the sample is present in the liquid form and ions are created by spraying a dilute solution of the analyte at atmospheric pressure from the tip of a fine metal capillary, creating a mist of droplets. The droplets are formed in a very high electric field and become highly charged. As the solvent evaporates, the peptide and protein molecules in the droplet pick up one or more protons from the solvent to form charged ions. These ions are then accelerated towards the mass analyzer depending upon their mass and charge. 5

3 Part 2, Step 4: 1 2 4 5 Comparison between MALDI and ESI Action
1. Sample analysis Simple peptide mixture Analysis of complex sample 2. Bias Polar/charged peptides Nonpolar peptides 3. Effect of salts Salt tolerant Salt sensitive 4. Liquid chromatography Offline Online, analysis can be coupled to LC 5. Sequence coverage Less More 6. Nobel prize Chemistry, 2002 2 3 4 Action Description of the action Audio Narration As shown in the animation. Show appearance of each row of the column one at a time. MALDI and ESI both have their pros and cons and can be used for the analysis of different types of protein samples. Development of both there techniques were awarded the Nobel Prize in 2002. 5

1 Master Layout (Part 3) 2 3 4 5 Types of mass analyzer
Part 1: Fundamentals of Mass Spectrometry Part 2: Ionization techniques Part 3: Mass Analyzers Part 4: Tandem mass spectrometry Types of mass analyzer 2 3 Time-of- Flight (TOF) Ion traps Quadru pole (Q) Ion cyclotron Resonance (ICR) Orbitrap Magnetic sector 4 5

Definitions of the components: Part 3 – Mass analyzers
1 1. Time-of-Flight (TOF): This is a mass analyzer in which the flight time of the ion from the source to the detector is correlated to the m/z of the ion. 2. Ion traps: An ion trap makes use of a combination of electric and magnetic fields that captures ions in a region of a vacuum system or tube. It traps ions using electrical fields and measures the mass by selectively ejecting them to a detector. This analyzer typically has lower resolution. 3.Quadrupole: Quadrupole mass analyzers use oscillating electrical fields to selectively stabilize or destabilize the paths of ions passing through a radio frequency (RF) quadrupole field. 4. Ion cyclotron resonance (ICR): A high-frequency mass spectrometer in which the specific ions to be detected are selected by setting a value of the quotient mass/charge,, after which they absorb maximum energy through the effect of a high-frequency electric field and a constant magnetic field perpendicular to the electric field. 5. Orbitrap: An orbitrap is a type of MS analyzer that consists of an outer barrel-like electrode and a coaxial inner spindle-like electrode that form an electrostatic field with quadro-logarithmic potential distribution. Ions that get injected tangentially into the electric field cycle around the central electrode rings and oscillate along the central spindle. The ions are detected by means of the frequency of their harmonic oscillations, which in turn is dependent on the m/z ratio. 2 3 4 5

Definitions of the components: Part 3 – Mass analyzers
1 6. Magnetic sector: Double-focusing magnetic sector mass spectrometers make use of static electric or magnetic fields for detection of ions. They provide high sensitivity, high resolution, and a reproducibility that is unmatched in any other kind of mass analyzer. 7. Flight tube: A connecting tube between the ion source and detector within which the ions of different size and charge migrate to reach the detector. 8. Collision cell: A device that selects a specific ion and further fragments into smaller ions. 9. Reflector: A component of the mass spectrometer that reflects ions towards the detectorthereby compensating for minor differences in kinetic energy. 10. Fragment ions: An electrically charged dissociation product of an ionic fragmentation. 11. End cap and ring electrode: Components of ion-trap mass analyzer. The analyzer consists of a chamber surrounded by a ring electrode and two end-cap electrodes. They Ion trap captures the ions, fragments ions of a particular m/z, and scans the fragments to generate tandem mass spectrum. 2 3 4 5

3 Part 3, Step 1: 1 2 4 5 Types of mass analyzer Action
cyclotron Resonance (ICR) Orbitrap Magnetic sector Time-of- Flight (TOF) Quadru pole (Q) 3 Ion traps 4 Action Description of the action Audio Narration As shown in the animation. Show appearance of the main heading followed by the headings below. The definitions given in the previous slide must appear as a popup when the user clicks on any of the headings. Finally the first two headings must get highlighted and clicking on either of these must re-direct the user to the next two slides. The mass analyzer resolves the ions produced by the ionization source on the basis of their mass-to-charge ratios. Various characteristics such as resolving power, accuracy, mass range and speed determine the efficiency of these analyzers. Commonly used mass analyzer include Time of Flight (TOF), Quadrupole (Q) and ion trap. 5

3 Part 3, Step 2: 1 2 4 5 Time of Flight (TOF) Action
Flight Tube 3 Ion Source Detector 4 Action Description of the action Audio Narration Separation of colored circles. As shown in the animation. First show the grey tube along with the ‘detector’ and ‘ion source’ at each end. Next show the circles of different sizes and colors appearing which must then move towards the ‘detector’ at different speeds with the smallest green circles moving fastest followed by red and finally blue. The time of flight analyzer accelerates charged ions generated by the ionization source along a long tube known as the flight tube. Ions are accelerated at different velocities depending on their mass to charge ratios. Ions of lower masses are accelerated to higher velocities and reach the detector first. The TOF analyzer is most commonly used with MALDI ionization source since MALDI tends to produce singly charge peptide ions. The time of flight under such circumstances is inversely proportional to square root of molecular mass of the ion. 5

Ion-trap mass analyzer
Part 3, Step 3: 1 Ion-trap mass analyzer Ring electrode 2 Fragment ions 3 End cap electrode 4 Action Description of the action Audio Narration Separation of colored circles. As shown in the animation. First show the big orange circle entering middle of the grey region as shown. This must then be fragmented and smaller colored circles must be shown to appear just outside the grey area. An ion trap makes use of a combination of electric and magnetic fields and captures ions in a region of a vacuum system or tube. It traps ions using electrical fields and measures the mass by selectively ejecting them to a detector. 5

Quadrupole mass analyzer
Part 3, Step 4: 1 Quadrupole mass analyzer RF (radio frequency) mode: Allows ions of any m/z ratio to pass through 2 Detector - - 3 Ion source - m2 m1 m4 m3 - 4 Action Description of the action Audio Narration First show the four parallel rods, the ‘ion source’ and ‘detector’. Show the colored circles coming out of the ion source and all of them moving towards the detector with slightly different speeds. Quadrupole mass analyzers use oscillating electrical fields to selectively stabilize or destabilize the paths of ions passing through a radio frequency (RF) quadrupole field. The quadrupole mass analyzer can be operated in either the radio frequency or scanning mode. In the RF mode, ions of all m/z are allowed to pass through which are then detected by the detector. Movement of colored circles. As shown in the animation. 5

Quadrupole mass analyzer
Part 3, Step 5: 1 Quadrupole mass analyzer Scanning mode: Ions of selected m/z ratio are allowed through to the detector 2 Detector - - 3 Ion source - m2 m1 m4 m3 - 4 Action Description of the action Audio Narration First show the four parallel rods, the ‘ion source’ and ‘detector’. Show the colored circles coming out of the ion source and only the blue circle moving towards the detector . In the scanning mode, the quadrupole analyzer selects ions of a specific m/z value as set by the user. A range can also be entered in which case only those specific ions satisfying the criteria will move towards the detector and the rest are filtered out. Movement of the colored circle. As shown in the animation. 5

1 Part 3, Step 6: 2 3 4 5 MALDI TOF ESI Ion Trap ESI Q-TOF Action
Typical MS configurations MALDI TOF 2 ESI Ion Trap ESI Q-TOF 3 4 Action Description of the action Audio Narration Movement of the colored circle into the grooves. As shown in the animation. First show the first blue circle and red ‘TOF’ figure. The circle must then move and enter into the groove of the red shape. Next, the second pair must appear and the circle must enter into the groove. The same thing must be done for all four pairs. (Last line of audio narration must coincide with appearance of each pair. The ionization source and mass analyzer can be combined in different ways to give varying configurations for the mass spectrometer. Some of the most commonly used MS configurations are MALDI with TOF, ESI with Ion Trap, ESI with Q & TOF and MALDI with Ion Trap. 5

1 Master Layout (Part 4) 2 3 4 5 Analyzer #2 Detector Ion Source
Part 1: Fundamentals of Mass Spectrometry Part 2: Ionization techniques Part 3: Mass Analyzers Part 4: Tandem mass spectrometry Tandem MS/MS Spectrometer 2 Precursor Ion Selector Detector 3 Analyzer#1 Mass Analyzer #2 Mass Ion Source Collision Cell 4 Generates and Accelerates Ions Separates Ions Induces Fragmentation of Precursor Re-Accelerates Fragments Separates Fragments 5

Definitions of the components: Part 4 – Tandem mass spectrometry
1 1. Tandem MS/MS spectrometer: This is a MS device that makes use of a combination of ion source and two mass analyzers, separated by a collision cell, in order to provide improved resolution of the fragment ions. The mass analyzers may either be the same or different. The first mass analyzer usually operates in a scanning mode in order to select only a particular ion which is further fragmented and resolved in the second analyzer. This can be used for protein sequencing studies. 2. Ion source: The ion source generates ion fragments from the intact protein or peptide which then enter the mass analyzer. Most commonly used ion sources are MALDI and ESI. 3. Mass analyzer #1: The first mass analyzer is usually set to operate in a scanning mode such that it allows only ions of a specific m/z value or within a particular range to move ahead. Q and TOF are commonly used. 4. Collision cell: The collision cell placed in between the first mass analyzer and the second ion source carries out further fragmentation of the selected ions by collision against an inert gas like argon. 2 3 4 5

Definitions of the components: Part 4 – Tandem mass spectrometry
1 5. Mass analyzer #2: This mass analyzer resolves all the fragmented ions based on their mass-to-charge ratio. 6. Detector: The resolved ions are finally detected by a detector. Most commonly used detector is the electron multiplier tube. 2 3 4 5

3 Part 4, Step 1: 1 2 4 5 Triple quadrupole Action
Relative abundance m/z Detector 2 Ions of selected m/z + + 3 Q2 – Collision cell Q3 – RF mode Peptide ions Q1 – Scanning mode Fragmented ions 4 Action Description of the action Audio Narration As shown in animation. First show the grey rods, the cube and the blue ‘detector’. Next show the green & pink ions on the left. The ions must move towards the first set of rods & only the pink ions must be allowed through the opening. These must enter the orange cube. In this, they must get fragmented into smaller pieces and must come out of the other end as shown. These smaller pieces must fly through the second set of rods and enter the detector. As each of the fragments reaches the detector, the graph on the right must start appearing from left to right until all the fragments have been detected. The triple quadrupole consists of two sets of parallel metallic rods interspersed by a collision cell. The first quadrupole scans the ions coming from the ionization source and allows only ions of a particular m/z ratio to pass through. These ions enter the collision cell where they are fragmented by collision against an inert gas like argon. The smaller fragments then enter the third quadrupole which scans all the ions in the radio frequency mode to generate a spectrum based on the varying behavior of ions in an oscillating electrical field. 5

3 Part 4, Step 2: 1 2 4 5 MALDI-TOF-TOF-MS Action
Detector 2 3 TOF 1 TOF 2 Reflector Collision cell 4 Action Description of the action Audio Narration As shown in animation. First show the black boxes with the components inside and the red rectangle. Next show a beam emerging from the rectangle which must fall on the dotted lines on the left. When this happens, the small circles must appear which must move towards the purple box in the centre, with the smallest moving the fastest. Next, the yellow circle alone must move forward and be shown to fragment into smaller particles coming out of the purple box. These particles must then fly towards the ‘reflector’ and then reach the detector, with the smallest moving fastest. This is another common tandem MS configuration in which the ions are first resolved on the basis of their time of flight in the first TOF analyzer. The selected ions enter the collision cell where they are further fragmented. The fragmented ions are accelerated and further resolved on the basis of their m/z values in the second TOF tube, after which they are detected. 5

Interactivity option 1:Step No: 1
Shown below are the components for an ESI-Q-TOF MS. Assemble the components in their correct positions & then click on the ESI tube to view the ion generation & detection. 2 Detector 3 m2 m1 m3 ESI m4 Quadrupole (scanning mode) Collision cell TOF tube Reflector 4 Interacativity Type Options Boundary/limits Results Once the user has assembled components & clicked on the grey tube, the colored circles must be generated. Only the red circle must move through the 4 rods & into the cube. Here it must be fragmented & disappear & the small pink circles must appear simultaneously. These circles must then move through the next tube towards the ‘detector’ as shown with the smallest circle moving fastest & largest one moving slowly. User must drag & drop the components shown above into their correct positions indicated by the dotted lines. Once this is done, user must click on the ‘ESI’ tube. 5 Drag and drop.

Questionnaire 1. Which are the two types of ionization sources used for the Mass Spectrometric analysis of biological samples? Answers: a) Fast Atom Bombardment and Chemical Ionization b) Electron Transfer Dissociation and Collision Induced Dissociation c) Matrix Associated Laser Desorption Ionization and Electrospray Ionization d) Electron Transfer Dissociation and Matrix Associated Laser Desorption Ionization 2. In case of a complex protein sample, which pre-fractionation step is commonly used in association with ESI? Answers: a) Western Blotting b) Thin Layer Chromatography (TLC) c) 2-D Electrophoresis (2DE) d) ‏Liquid Chromatography (LC) 1 2 3 4 5

Questionnaire 1 3. Which of the following Mass Analyzers uses the Tandem-in-time technique to collect fragmentation data? a) TOF-TOF b) Quadrupole-TOF c) Linear Ion Trap d) Triple Quadrupole 4. Which of the following MS configurations can be used for the identification of a specific protein from within a mixture? a) MALDI-TOF b) ESI-TOF c) None of the above d) Both of the above 5. Which of the following is not a mass analyzer? a) Time-of-Flight (TOF) b) Quadrupole (Q) c) Ion traps d) MALDI 6. Which of the following is not a ionization source? a) Fast Atom Bombardment (FAB) b) Matrix Assisted Laser Desorption Ionization (MALDI) c) Ion cyclotron resonance (ICR) d) Electrospray Ionization (ESI) 2 3 4 5

Links for further reading
Research papers: Aebersold, R., Mann, M., “Mass spectrometry-based proteomics.” Nature. 2003,422(6928), Karas, M., Hillenkamp, F., (1988). "Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons". Anal. Chem. 1988, 60, 2299–2301. Website: An IUPAC sponsored site to “To update and extend the definitions of terms related to the field of mass spectrometry”..

Concepts in Mass Spectrometry

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