Liquid chromatography-mass spectrometry

Harini Chandra

a This is a common analytical tool that combines physical separation by liquid chomatography with mass analysis and resolution by mass spectrometry. It is capable of separating and identifying complex mixtures for proteomics studies.

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1Master Layout

Protein Peptide fragments

MS/MS

In-gel digestion

Liquid chromatography

Tandem MS

Action Audio NarrationDescription of the actionOverview slide.

This slide must be shown as an overview along with narration of topic heading given in slide #1. The three boxes shown in blue must be tabs which will take the user to the correct module of the animation. (Part 1 – In-gel digestion; Part 2- Liquid chromatography; Part 3- Tandem MS)

LC-MS a common analytical tool that combines physical separation by liquid chomatography with mass analysis and resolution by mass spectrometry. It is capable of separating and identifying complex mixtures for proteomics studies.

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1Master Layout (Part 1)This animation consists of 3 parts:Part 1: In-gel digestionPart 2: Liquid chromatography Part 3: Tandem mass spectrometry

Cut gel into small pieces

Protein of interest

Trypsin

Peptide fragments

SDS-PAGE gel

Cutting

Tubes with buffer and proteolytic enzyme

Protein bands

2-DE gel

Definitions of the components:Part 1- In-gel digestion

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11. In-gel digestion: The process by which proteins separated by various electrophoretic techniques are dissolved in a suitable buffer and then fragmented by means of proteolytic enzymes such as trypsin.

2. Complete SDS-PAGE gel: The gel in which the protein mixture has been run after mixing with the anionic detergent, SDS, such that proteins are separated on the basis of their molecular weights to form unique bands. This is stained with a suitable dye that binds to the proteins thereby allowing the bands to be visualized.

3. Protein bands: The pattern of proteins that have been run on the gel and have been separated from each other based on their size.

4. Tubes with buffer & proteolytic enzyme: The fragmented gel pieces are placed in tubes containing a suitable buffer solution that will allow the protein to go into solution. The tubes also contain proteolytic enzymes, which will fragment the protein at specific sequences depending upon its specificity.

5. Trypsin: A proteolytic enzyme that cleaves proteins at the C terminal of arginine or lysine residues unless there is a proline present after either of these residues, thereby generating small peptide fragments.

Definitions of the components: Part 1- In-gel digestion

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17. Protein of interest: The protein whose sequence is to be determined and properties are to be studies by various MS techniques.

8. Peptide fragments: The smaller “pieces” of protein that are generated after cleavage at specific points by the proteolytic enzymes.

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4Action Audio NarrationDescription of the actionAs shown in the animation.

First show the blue rectangle with the band patterns along with the tubes containing blue solution below. This must be fragmented into small pieces along the dotted lines indicated with a knife or scissors (not shown here) and each small piece must then enter one test tube. A red box must then appear around the 4th tube and it must be enlarged to indicate that it is the protein of interest.

Electrophoretic separation of a protein mixture results in distinct protein bands. These proteins can be can be used for analytical purposes by carrying out in-gel digestion. The entire gel is fragmented into small pieces with each piece being dissolved in a suitable buffer.

Part 1, Step 1:

Complete SDS-PAGE gel

Protein bands

Protein of interest

Tubes with buffer

Complete 2-DE gel

Protein of interest

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4Action Audio NarrationDescription of the actionAs shown in the animation.

Show the tube on the left with its label. This must be zoomed into and the reactions must be shown in a stepwise manner as depicted in the animation.

The protein solution is treated with a reducing agent like dithiothreitol, which cleaves the disulphide bonds in the protein. This is followed by treatment with iodoacetamide, which alkylates the sulfhydryl groups thereby preventing reformation of the disulphide bonds.

Part 1, Step 2:

Addition of DTT & IAA

Reduction & alkylation of proteins

NH3 COO-

S S

NH3 COO-S

H

SH

NH3COO-

S

CH2CNH2

O

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CH2CNH2

O

DTT, 65oC

Iodoacetamide, 30oC

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4Action Audio NarrationDescription of the actionAs shown in the animation.

Show the tube on the left with the label followed. This must be zoomed into to show the brown rectangle with its labels. The orange pie object must then appear and move across the brown rectangle breaking it at the green point indicated by the black arrow as it moves along. It must not cleave the third green box. Once it has broken the brown box, the resulting fragments must be displayed.

Following cleavage of the disulphide bonds, the protein is treated with a proteolytic enzyme, the most commonly used one being trypsin. This cleaves the protein at specific residues thereby generating smaller peptide fragments. This tryptic digest is used for further purification and analysis.

Part 1, Step 3:

Protein of interest

Addition of Trypsin

Tryptic digestion

N-terminalC-terminal

Trypsin

Digested peptide fragments

K/R K/R K/R P

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1Master Layout (Part 2)

This animation consists of 3 parts:Part 1: In-gel digestionPart 2: Liquid chromatography Part 3: Tandem mass spectrometry

Solvent tray

HPLC Pump

Sample injector

Chromatography Column

Detector

High pressure liquid chromatography

Mobile phase

Sample vials

Data analysis

Definitions of the components:Part 2- Liquid chromatography

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1 1. Liquid chromatography: This is a chromatographic separation technique that separates molecules based on their differential adsorption and desorption between the stationary matrix phase in the column and the mobile phase.

2. High pressure liquid chromatography (HPLC): Finer stationary phase matrix molecules, packed very close to each other requires the application of pressure for the mobile phase and analyte molecules to pass through it, giving rise to the technique of HPLC. Better separation and resolution of the components is achieved by this technique.

3. Mobile phase: The solvent system or buffer that is used to elute out the molecules bound to the stationary phase matrix present in the column. The mobile phase is decided based on the nature of the analyte molecules and their interactions with the stationary phase. A combination of 2 or more solvents may also be used with their compositions adjusted such that it totals 100%.

4. Solvent tray: A tray in which the solvent bottles are placed.

5. HPLC Pump: A device that is used to mix solvents in the desired proportion and pump them through the column. The ‘dual pumps’ can mix two solvent systems while the quaternary pumps can mix up to four solvents. Flow rate of liquid passing through the column and pressure maintained are adjusted by modifying the settings of the pump through the HPLC software.

6. Sample vials: These are small 2-5 mL bottles in which the sample is placed in a suitable solvent system.

Definitions of the components:Part 2- Liquid chromatography

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1 7. Sample Injector: Injection of the sample into the column may be done either manually or by means of an automated system. A syringe draws out the sample from the sample vial (~1-5 L) and injects it directly into the packed chromatography column.

8. Chromatography column: The column consists of a packed stationary phase matrix that selectively adsorbs certain analyte molecules due to specific interactions. The bound analyte molecules can be eluted from the column by modifying the conditions and properties of the mobile phase. Commonly used column packing includes:

a. Reverse phase chromatography (RPC): The stationary phase in RPC consists of long, aliphatic carbon chains possessing a highly hydrophobic nature. Molecules are bound on the column by means of hydrophobic interactions and are eluted out when the solvent polarity is modified.

b. Strong cation exchange (SCX): SCX consists of a stationary phase matrix made up of negatively charged sulphonic acid groups which bind the oppositely charged peptide molecules. These can be eluted out using a positively charged mobile phase which binds the analyte molecules more firmly.

9. Detector: The eluted analyte molecules pass from the column outlet into a flow cell present in the detector. UV is the most commonly used detector for protein molecules which absorb strongly at 280 nm.

10. Data analysis: A graph of peak intensity against retention time is obtained after detection which can be thoroughly analyzed using various tools available on the software systems.

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4Action Audio NarrationDescription of the actionAs shown in the animation.

First show the bottles placed in a tray as shown above followed by the box on the right indicating the ‘pump’. The circle on the box must be zoomed into and the cube with coloured circles on it must be shown. Tubing must be shown connected to each of these circles and liquids of different colours must be shown to enter these circles as depicted in animation.

A typical liquid chromatography setup consists of the solvent bottles, degassifier, dual or quaternary pump, sample injector, column and detector. Different solvents can be placed in the solvent bottles depending upon the purification requirement. These solvents are mixed in the desired ratio and pumped into the column during elution after removal of any trapped air inside it by means of the de-gassifier.

Part 2, Step 1:

Solvent trayHPLC Pump

Mobile phase

Pump inlet

Pump outlet – mixed solvents

Solvent bottles

Flow adjustment valves

Solvent flow to pump

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4Action Audio NarrationDescription of the actionAs shown in the animation.

Show the setup on the left with all its labels. The second and third boxes must be zoomed into to show the figures on the right. The ‘injector’ must enter the sample bottle with its plunger down. It must remain in this bottle for a couple of seconds and the plunger must be shown to move up. This must then move and be injected into the column. Liquid must be shown to flow through the tube connecting the ‘pump’ and ‘column’. Once the liquid flows, the colour in the column must change and the liquid must be shown to pass through the tubing at the outlet.

The sample injector system may be automatic or manual. The automatic sampler uses a syringe to inject the sample placed in a vial directly into the column. Once the sample is injected, mobile phase flows into the column through the pump. The column consists of a stationary matrix that preferentially binds certain analytes. Outlet from the column enters the flow cell where it is detected.

Part 2, Step 2:

HPLC Pump

LC ColumnSample injector

Sample vials

Injector

Column

Column inlet from pump

Column outlet to detector

Sample

Mobile phase

Column elution

Pump

Sample injection & column elution

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4Action Audio NarrationDescription of the actionAs shown in the animation.

Show the column on top. The brown region must be zoomed into and first the figure on the left must be depicted followed by the blue dialogue box. This must then disappear and the figure on the right must appear followed by the red dialogue box.

Various stationary phase matrices are available that separate the components of the mixture based on different principles. One of the commonly used matrices, the strong cation exchanger, separates charged peptides based on their electrostatic interactions with negatively charged sulphonic acid groups on the resin surface. Reverse phase chromatography is another commonly used tool, which uses a hydrophobic matrix consisting of long aliphatic carbon chains. These retain analytes on the basis of their hydrophobic interactions and can be eluted by changing the polarity of the solvent. Nano-liquid chromatography, which makes use of C-18 capillary columns, has gained popularity for proteomic studies due to their ability to achieve fine separation.

Part 2, Step 3: Column types

Strong cation exchanger (SCX)Reverse phase column (RPC)

Sulphonic acid groups Long hydrophobic

aliphatic chains

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

Based on electrostatic attraction between negative sulphonic acid and positive peptide. Elution caused by addition of positively charged mobile phase.

Based on hydrophobic interactions between analyte & stationary phase. Elution brought about by modifying mobile phase polarity.

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4Action Audio NarrationDescription of the actionAs shown in the animation.

The setup on the left must be shown along with the computer and its graph display. The grey boxes must be zoomed into to show the ‘column on the right, the cube (detector) with the circle on top and the grey ‘flow cell’ cuboid. A tube must connect the ‘column’ and ‘flow cell’ through which a liquid must pass. Once it passes, the flow cell colour must change. The circle on top must then emit a glow. Once this happens, the graph on top right must also appear.

The separated components pass from the column outlet into the flow cell present in the detector. The most commonly used detector for protein analysis is the UV detector which analyzes the protein absorbance at 280 nm and plots a graph of retention time against intensity. Each peak corresponds to a particular analyte in the sample mixture.

Part 2, Step 4: Sample detection

Data analysisUV detector

Flow cell

Column

Chromatography Column

Detector

Retention time

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1Master Layout (Part 3)

This animation consists of 3 parts:Part 1: In-gel digestionPart 2: Liquid chromatography Part 3: Tandem mass spectrometry

Ion

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Precursor Ion Selector

Collision

C

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Mass

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Ma

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Generates and

Accelerates Ions

Separates Ions

Induces Fragmentati

on of Precursor

Re-Accelerates Fragments

Separates Fragments

Definitions of the components:Part 3 – Tandem mass spectrometry

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11. 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.

Definitions of the components:Part 3 – Tandem mass spectrometry

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15. 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.

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4Action Audio NarrationDescription of the actionAs 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.

Part 3, Step 1:

Generated ions

Matrix &analyteMatrix &analyteTarget plate

LaserLaser

Flight tube

Ion Source

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Ion current

Sample orifice

Protein/peptide sample

~2.2 kV

Capillary tipSpray needle

MALDI ESI

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Types of mass analyzer

Time-of-Flight (TOF)

Ion traps Quadrupole (Q)

Ion cyclotron Resonance

(ICR)Orbitrap Magnetic

sector

Action Audio NarrationDescription of the actionAs 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.

Part 3, Step 2:

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Flight Tube

Detector

Ion

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Time of Flight (TOF)

Part 3, Step 3:

Action Audio NarrationDescription of the actionSeparation 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.

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Ion-trap mass analyzer

Fragment ions

Ring electrode

End cap electrode

Part 3, Step 4:

Action Audio NarrationDescription of the actionSeparation 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.

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Quadrupole mass analyzer

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m3

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Detector

Ion source

RF (radio frequency) mode:

Allows ions of any m/z ratio to pass through

Part 3, Step 5:

Action Audio NarrationDescription of the actionMovement of colored circles. As shown in the animation.

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.

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Quadrupole mass analyzer

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m3

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m4m2

Detector

Ion source

Scanning mode:

Ions of selected m/z ratio are allowed through to the detector

Part 3, Step 6:

Action Audio NarrationDescription of the actionMovement of the colored circle. As shown in the animation.

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.

Action Audio Narration

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4Description of the action

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.

Tandem MS/MS - Triple quadrupole

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Q1 – Scanning mode

Q2 – Collision cell

Q3 – RF mode

Peptide ions

Ions of selected m/z

Fragmented ions

Detector

m/z

Part 3, Step 7:

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Reflector

TOF 2TOF 1

Collision cell

Tandem MS/MS - MALDI-TOF-TOF-MS

Part 3, Step 8:

Action Audio NarrationDescription of the actionAs 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.

Detector

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Tandem MS/MS – ESI-Q-TOF

Part 3, Step 9:

Action Audio NarrationDescription of the actionAs shown in animation.

First show all the components of the instrument – the syringe, four rods, cube, blue rectangle, gray square with the dotted lines & the detector. Next show appearance of the coloured circles. Only the red one must move through the rods and after entering the rectangular box, they must be fragmented to give smaller circles. These must migrate through the blue tube and get reflected to reach the ‘detector’. The smallest circles must move the fastest while the largest must move slowest.

ESI-Q-TOF is a commonly used tandem MS configuration that first selects ions in the radio frequency mode. The selected peptide is then fragmented in the collision cell and the resulting ions are accelerated and resolved on the basis of their time of flight.

Quadrupole (scanning mode)

Collision cell TOF tube Reflector

Detector

ESIm3

m1

m4

m2

Interactivity option 1:Step No: 1

Boundary/limitsInteracativity Type Options Results

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Drag and drop.User must drag & drop the ‘proteins’ shown on either side to the correct part of the figure shown in the centre as depicted in the animation.

User must drag and drop the ‘proteins’ shown on either side of the column to the correct region of the column. Protein A must be dragged to the brown region of the column while protein B must be dragged to the yellow region. If the user does this correctly, a message saying ‘right answer’ must appear. Otherwise a message saying ‘wrong answer’ must appear.

Multidimensional Protein Identification Technology (MudPIT) is a widely adopted strategy that carries out two consecutive protein separations based on different principles as shown in the figure below. Shown on either side is a protein with different properties. Drag & drop the protein that will interact with the SCX & RP regions of the column respectively.

CH3

CH3

CH3

CH3

Protein AProtein B

Questionnaire1. Which of the following enzymes cleaves proteins at the C-terminal of arginine and lysine

residues?

Answers: a) Chymotrypsin b) Proteinase c) Trypsin d) Pepsin

2. The function of DTT during in-gel digestion of proteins is:

Answers: a) Oxidation of disulphide bonds b) Cleavage at N-terminal of amino acids c) Cleavage at C-terminal of amino acids d) Reduction of disulphide bonds

3. Reverse phase chromatography is based on the principle of:

Answers: a) Hydrophobic interactions b) Ionic interactions c) Covalent bonding

d) Hydrogen bonding

4. Detection of proteins can be carried out at:

Answers: a) 420 nm b) 280 nm c) 260 nm d) 320 nm

5. Which of the following is not a mass analyzer?

Answers: a) Time-of-Flight (TOF) b) Quadrupole (Q) c) Ion traps d) MALDI

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Links for further readingResearch papers:

• Link, A. J., Jennings, J. L. & Washburn, M. P. Analysis of protein composition using multidimensional chromatography and mass spectrometry. In Current protocols in protein science (ed. J.E.Coligan et al.), chapter 23, pp. 1-25. John Wiley and Sons, New York.•Link, A. J. et al. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 1999, 17:676-682.•Aebersold, R., Mann, M., “Mass spectrometry-based proteomics.” Nature. 2003,422(6928),198-207.•Karas, M., Hillenkamp, F., (1988). "Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons". Anal. Chem. 1988, 60, 2299–2301.•Washburn, M. P., Wolters, D. and Yates, J. R. III Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 2001, 19:242-247.•Dongre, A. R., Jones, J. L., Somogyi, A., Wysocki, V. H. Influence of peptide composition, gas-phase basicity, and chemical modification on fragmentation efficiency: Evidence for the mobile proton model. J. Am. Chem. Soc. 1996, 118: 8365-8374.

Books:•Link, A. J. & LaBaer, J. Proteomics – A Cold Spring Harbor Laboratory Course Manual. Experiment 4, 5 & 6.

Links for further reading

Website:

1. www.msterms.comAn IUPAC sponsored site to “To update and extend the definitions of terms related to the field of mass spectrometry”..2. www.cshprotocols.org3. OSCAR Animations – a) Concepts in Mass Spectrometry

b) Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) c) Protein separation techniques: basic electrophoresis d) Protein separation techniques: two dimensional gel electrophoresis