Fundamentals of Mass Spectrometry. Fundamentals of Mass Spectrometry. Learning Objective. Proteomics

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1 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. Learning Objective In this learning object, the learner will be able to, Define Describe Ionization techniques Recall Mass Analyzers, and, Recall Tandem mass spectrometry

2 Fundamentals of mass spectrometry 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.

3 Ionization techniques 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).

4 Ionization techniques In MALDI, the analyte of interest is mixed with an aromatic matrix compound like α-cyano-4hydroxycinnamic 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.

5 Ionization techniques 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.

6 Ionization techniques 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.

7 Mass analyzers 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.

8 Mass analyzers 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.

9 Mass analyzers 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.

10 Mass analyzers 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.

11 Mass analyzers 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.

12 Mass analyzers 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.

13 Tandem mass spectrometry 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.

14 Tandem mass spectrometry 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.

15 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-tocharge 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).

16 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.

17 Ionization techniques 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 matrixprotein 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 nonvolatile 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.

18 Ionization techniques 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

19 Mass analyzers 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 highfrequency 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 for 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

20 Mass analyzers 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. 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 detector thereby 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. The ion trap captures the ions, fragments ions of a particular m/z, and scans the fragments to generate tandem mass spectrum.

21 Tandem mass spectrometry 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 two mass analyzers carries out further fragmentation of the selected ions by collision against an inert gas like argon. 5. Mass analyzer #2: This mass analyzer resolves all the fragmented ions based on their mass-tocharge ratio.

22 Tandem mass spectrometry 6. Detector: The resolved ions are finally detected by a detector. Most commonly used detector is the electron multiplier tube.

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(Refer Slide Time 00:09) (Refer Slide Time 00:13)

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