mass

Slide 1: 

An OverviewofLC/MS/MS

Why LC/MS? : 

Why LC/MS? LC – Offers chromatographic separation of compounds MS - compound identification MS/MS offers fragmentation for characterisation MS/MS - selectivity & specificity for quantitation

Information Expected From LC/MS/MS : 

Information Expected From LC/MS/MS Quantitative analysis. Accurate mass measurement. Metabolite identification. Impurity Detection at low level. Structure elucidation and Confirmation of the structure.

The General Mass Spectrometry : 

The General Mass Spectrometry

Slide 5: 

Mass Spectrometry Ionization Techniques Hard, Fragments Soft, Intact

Electron Ionization (EI) : 

Electron Ionization (EI)

Chemical Ionisation (CI) : 

Chemical Ionisation (CI) Low-mass, volatile, thermally stable organic compounds.

Fast Atom Bombardment (FAB) : 

Fast Atom Bombardment (FAB) Used to analyze nonvolatile, thermally unstable, and high-polarity compounds. The material to be analyzed is mixed with a non-volatile chemical protection environment called a matrix. Bombarded under vacuum with a high energy (4000 to 10,000 electron volts) beam of atoms. The atoms are typically from an inert gas such as argon or xenon. Common matrices include glycerol, thioglycerol, 3-nitrobenzyl alcohol (3-NBA), 18-Crown-6 ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine.

Slide 9: 

Atmospheric Pressure Ionization (API)

Various ionization methods : 

Various ionization methods

ESI : 

ESI

Slide 12: 

Mass Spectrometry

Slide 13: 

Electrospray Ionization

Slide 14: 

IonSpray - ESI

Turbo IonSpray : 

Turbo IonSpray

ESI : 

ESI

Slide 17: 

Mass Spectrometry

Chipcube interface : 

Chipcube interface

Multimode : 

Multimode

Slide 20: 

Mass Spectrometry

Slide 21: 

Mass Spectrometry

Slide 22: 

APCI Ionization Source

APCI : 

APCI

Heated Nebulizer (APCI) Inlet : 

Heated Nebulizer (APCI) Inlet

Heated Nebulizer (APCI) Inlet : 

Heated Nebulizer (APCI) Inlet Suitable for Nonpolar, thermally stable compounds Usually, Molecular Weight < 1000 amu Probe is heated to facilitate vaporization Requires nebulizer and auxiliary gas Requires corona discharge needle to produce ionization (APCI)

Slide 26: 

Mass Spectrometry Heated Nebulizer: Atmospheric Pressure Chemical Ionisation (APCI) corona discharge needle polar to unpolar and thermally stable compounds

Slide 27: 

Mass Spectrometry

Slide 28: 

Mass Spectrometry

Slide 29: 

Mass Spectrometry

Photo Ionization Source : 

Photo Ionization Source

APPI : 

APPI

APPI : 

APPI

Slide 33: 

Operating Principle Samples are sprayed with the aid of a nebulizing gas into a heated probe, as in APCI. A dopant compound is vaporized in the auxiliary gas within the heated nebulizer probe. In the ionization region, dopant molecules are predominantly ionized by UV radiation and form photoions. The photoions initiate a cascade of ion-molecule reactions leading to the formation of the ionized analytes in the form [MH]+ (by proton transfer) or [M]+• (by charge transfer).

Slide 34: 

The role of the Dopant The dopant acts as an intermediate in the ionization process Increases analyte ionization efficiency Two dopants were extensively tested Toluene Acetone

Slide 36: 

Mass Spectrometry Sensitivity Polar compounds: ESI > APCI > PhotoSpray Non Polar compounds: PhotoSpray > APCI > ESI Chemical Background ESI > APCI > PhotoSpray Suceptibility to the MP composition PhotoSpray > ESI > APCI

PhotoSpray Potential Application Areas : 

PhotoSpray Potential Application Areas Pharmaceutical Companies/CRO’s Steroids/Hormones/Nonpolar Vitamins Relative Nonpolar drugs Alternative Markets Petroleum/Oil Industry Polymer Analysis Food Analysis Sugars Carotenoids Essential Oils Environmental Analysis PAH’s

Slide 38: 

MALDI-TOF Mass Spectrometry

MATRIX : 

MATRIX

Slide 40: 

Mass Spectrometry

Slide 41: 

INTERFACE

Ion Transfer from Atmosphere to a High Vacuum Environment : 

Ion Transfer from Atmosphere to a High Vacuum Environment Problem of all conventional interface designs: Matrix, e.g., salt particles, may clog the orifice and/or form deposition inside the interface or further downstream How to overcome this problem: Off-axis spraying Z spray Orhtogonal spraying Still not good enough

How the curtain gas works : 

How the curtain gas works 1. Curtain Gas assists in droplet evaporation 2. Curtain Gas declusters ions

How the curtain gas works : 

How the curtain gas works

How the curtain gas works : 

How the curtain gas works

Slide 46: 

How the curtain gas works

How the curtain gas works : 

How the curtain gas works

RF Ion Guide (Q0) : 

RF Ion Guide (Q0)

Slide 49: 

Mass Spectrometry

Mass spectrometry : 

Mass spectrometry

Mass Accuracy & resolution : 

Mass Accuracy & resolution

Analyzers : 

Analyzers Time of Flights mass spectrometer – SELDI, MALDI, QTOF Quadrupole mass spectrometer – Single Quad, Triple Quad Ion traps – 3D Ion trap, Linear ion trap FT-MS, FT-ICR (Fourier)

3D Ion trap Mass Spectrometry : 

3D Ion trap Mass Spectrometry

Slide 54: 

3D Ion trap Mass Spectrometry

Linear ion trap : 

Linear ion trap

Linear ion trap : 

Linear ion trap

Single Quadrupole MS : 

Single Quadrupole MS

Schematics of a Triple Quadrupole MS : 

RF Ion Guide: Focusses ion beam into Q1 Q1: Analyzer Quadrupole 1 can be scanned or set to pass one specific m/z Q2: Collision Cell can be pressurized with N2 for fragmentation of ions can be at low pressure for bandpassing all ions Q3: Analyzer Quadrupole 3 can be scanned or set to pass one specific m/z Schematics of a Triple Quadrupole MS

Triple Quadrupole MS : 

Triple Quadrupole MS

Product Ion Scan : 

Product Ion Scan Quadrupole 1 Collision Cell (Q2) Quadrupole 3

Slide 61: 

MS/MS - Product Ion Scan After identification, the precursor ion is sent into the collision cell for fragmentation Q1 is fixed, Q3 scans a defined mass range Provides structural information and identification of product ions

Slide 62: 

MS/MS - Precursor Ion Scan (cont.)

MS/MS - Precursor Ion Scan : 

MS/MS - Precursor Ion Scan Precursor ion scan Q1 scans a defined mass range, Q3 is fixed Used to determine the “origin” of particular product ion(s) created in the collision cell Frequently used for drug metabolite identification (common product ion observed in the metabolites)

Slide 64: 

MS/MS Constant Neutral Loss (cont.)

MS/MS Constant Neutral Loss : 

MS/MS Constant Neutral Loss Neutral loss scan: Q1 & Q3 both scan a given mass range but with a constant difference between the ranges scanned Spectrum indicates which ions lose a neutral species equal to Q1 - Q3 difference

Multiple Reaction Monitoring (MRM) : 

Multiple Reaction Monitoring (MRM)

MS/MS - Multiple reaction Monitoring (MRM) : 

MS/MS - Multiple reaction Monitoring (MRM) If Q1 and Q3 width=0, then MRM Many precursor to product ion pairs can be monitored MRM analysis is the best way to maximize signal/noise ratio of compounds MRM used primarily for quantitation studies

Schematics of a Q-TRAP : 

RF Ion Guide: Focusses ion beam into Q1 Q1: Analyzer Quadrupole 1 can be scanned or set to pass one specific m/z Q2: Collision Cell can be pressurized with N2 for fragmentation of ions can be at low pressure for bandpassing all ions Q3: Analyzer Quadrupole 3 can be scanned or set to pass one specific m/z Schematics of a Q-TRAP

QTRAP MSMS : 

QTRAP MSMS

Q-TOF MS/MS : 

Q-TOF MS/MS

Slide 71: 

MALDI TOF

Slide 72: 

MALDI-TOF Mass Spectrometry

MALDI-TOF/TOF : 

MALDI-TOF/TOF

Advantages : 

Advantages

SELDI-TOF : 

SELDI-TOF

Proteinchip surfaces : 

Proteinchip surfaces

SELDI-TOF Reader : 

SELDI-TOF Reader

Protein Sequencing by Collision Induced Dissociation : 

Protein Sequencing by Collision Induced Dissociation

Slide 79: 

Detector Key part of detector is dynode Dynode is electron-multiplying electrode Electron impact stimulates release of multiple electrons which cascade down voltage difference of multiple dynodes

Mass Spec Ion Detectors : 

Mass Spec Ion Detectors Faraday Cup Electron Multiplier and Channel Electron Multiplier Microchannel Plate Daly Detector (Scintillation Counter or Photomultiplier)

Faraday Cup : 

Faraday Cup Simplest Ion Detector Good for checking ion transmission Disadvantage: very poor sensitivity

Electron Multipliers : 

Electron Multipliers

Electron Multipliers : 

Electron Multipliers Advantages: very high current gain increases sensitivity; fast Disadvantage: short lifetime Electron multipliers are essential to almost all ion detectors Electrons amplified by a cascade effect Also called channeltron Used Quadrupole and ion trap

Microchannel Plate Ion Detector : 

Microchannel Plate Ion Detector

Microchannel Plate Ion Detector : 

Microchannel Plate Ion Detector Array of tiny channel electron multipliers (usually glass capillaries coated with electron-emissive material) - 12.5–25 μm in diameter and 1–2 mm long. Around 10 million tubes are stacked side by side to form a thin plate structure. Advantages: Fast - time response <1 ns, High sensitive Disadvantages: expensive, short lifetime Used in TOF MS

Photomultiplier (Daly detector) : 

Photomultiplier (Daly detector)

Photomultiplier (Daly detector) : 

Photomultiplier (Daly detector) Also called daly detector or scintillation counter Metal dynode emits secondary electrons Secondary electrons hit phosphorus screen and trigger photon emission; photon abundance measured by photomultiplier Advantage: keep detector in vacuum -- no contamination, low noise and long lifetime Disadvantage: cannot be exposed to light

collision-induced dissociation (CID) : 

collision-induced dissociation (CID) collide with neutral gas molecules (often helium, nitrogen or argon). In the collision some of the kinetic energy is converted into internal energy which results in bond breakage and the fragmentation of the molecular ion into smaller fragments. CID produces fragment ions -primarily b and y type.

Electron capture dissociation (ECD) : 

Electron capture dissociation (ECD) ECD involves the direct introduction of low energy electrons to trapped gas phase ions. ECD produces significantly different types of fragment ions (primarily c and z type) The low fragmentation efficiency. Ion trap & Fourier transform ion cyclotron resonance mass spectrometry.

Electron transfer dissociation (ETD) : 

Electron transfer dissociation (ETD) ETD induces fragmentation of cations (e.g. peptides or proteins) by transferring electrons to them. ETD does not use free electrons but employs radical anions (e.g. anthracene or azobenzene) for this purpose. ETD cleaves randomly along the peptide backbone (so called c and z ions) while side chains and modifications such as phosphorylation are left intact.

Infrared multiphoton dissociation (IRMPD) : 

Infrared multiphoton dissociation (IRMPD) An infrared laser is directed through a the ions. The parent ion becomes excited into more energetic vibrational states until a bond(s) is broken resulting in gas phase fragments of the parent ion. used in Fourier transform ion cyclotron resonance mass spectrometry.

Fragmentation : 

Fragmentation

Slide 93: 

Protein Biomarker Discovery By Mass Spectrometry A specific physical or chemical trait used to indicate the presence or progress of a disease, or the response to treatment

Plasma proteins… : 

Plasma proteins… Novel biomarkers will be found at extremely low concentrations in serum Chart modified from Plasma Proteome Institute http://www.plasmaproteome.org/plasmaframes.htm Novel Biomarkers

Slide 95: 

Plasma proteins…

Protein Biomarker : 

Protein Biomarker iTRAQ - 8 plex isobaric peptide tagging system SILAC - stable isotope labeling of amino acids in cell culture GIST - global internal standard technology, a post digestion peptide level labeling technique ICAT - isotope coded affinity tag-based protein profiling

SILIC : 

SILIC

ICAT : 

ICAT

iTRAQ : 

iTRAQ

iTRAQ : 

iTRAQ

LCMSMS Scan : 

LCMSMS Scan

MALDI TOF/TOF for tissue imaging : 

MALDI TOF/TOF for tissue imaging

Slide 104: 

PTM Specific Scans Precursor Ion Scan Scan complex mixtures for PTM-specific diagnostic fragments PTM Studies in Mass Spectrometry