Mass Spectrometry THE MAIN USE OF MS IN ORG CHEM IS: DETERMINE THE MOLECULAR MASS OF ORGANIC COMPOUNDS DETERMINE THE MOLECULAR FORMULA OF ORGANIC COMPOUNDS

HOW DO WE ACHIEVE THIS? PERSUADE THE MOLECULE TO ENTER THE VAPOR PHASE (CAN BE DIFFICULT) PRODUCE IONS FROM THE MOLECULES THAT ENTER THE GAS PHASE SEPARATE THE IONS ACCORDING TO THEIR MASS-TO-CHARGE RATIOS (m/z)) MEASURE AND RECORD THESE IONS

What is Mass Spec? Analytical tool measuring molecular weight (MW) of sample Only femtomolar concentrations required Within an accuracy of 0.01% of total weight of sample and within 5 ppm for small organic molecules For a Mr of 40 kDa, there is a 4 Da error This means it can detect amino acid substitutions / post-translational modifications

What sort of info is returned? Structural information can be generated Particularly using tandem mass spectrometers Fragment sample & analyse products Useful for peptide & oligonucleotide sequencing Plus identification of individual compounds in complex mixtures

Simplified Schematic The analyser, detector and ionisation source are under high vacuum to allow unhindered movement of ions Operation is under complete data system control

m/z spectrum example Leucine Enkephalin, Pentapeptide, YGGFL University of Mons-Hainaut Leucine Enkephalin, Pentapeptide, YGGFL Expected Mr = 555.3 Da, Calculated Mr = 555.1 Da (dominant ions at 556.1)

Mass Spectrometry Ion source Mass analyzer Detector The general operation of a mass spectrometer is: create gas-phase ions separate the ions in space or time based on their mass-to-charge ratio measure the quantity of ions of each mass-to-charge ratio The ion separation power of a mass spectrometer is described by the resolution, which is defined as: R = m / delta m, where m is the ion mass and delta m is the difference in mass between two resolvable peaks in a mass spectrum. E.g., a mass spectrometer with a resolution of 1000 can resolve an ion with a m/e of 100.0 from an ion with an m/e of 100.1. Ion source Mass analyzer Detector (EI=Electron Impact; CI=Chemical Ionization; SIMS=Secondary Ions Mass Spec; FAB=Fast Atom Bombardment; LDMS=Laser Desorption Mass Spec; PDMS=Plasma Desorption Mass Spec; TS=Thermospray; AS=Aerospray; ESMS=Electrospray Mass Spec). (EB=Electrostatic-Magnetic; ion trap; Q=Quadrupole; TOF=Time of Flight.)

Sample Introduction & Ionisation Direct into ionisation source or via chromatography for component separation (HPLC, GC, capillary electrophoresis) Ionisation can be positively charged (for proteins) or negatively charged (for saccharides and oligonucleotides)

Ionisation methods Atmospheric Pressure Chemical Ionisation (APCI) Chemical Ionisation (CI) Electron Impact (EI) Electrospray Ionisation (ESI) Fast Atom Bombardment (FAB) Field Desorption / Field Ionisation (FD/FI) Matrix Assisted Laser Desorption Ionisation (MALDI) Thermospray Ionisation

WHY MALDI? Less Sensitive to Salts Lower PRACTICAL detection limits Easier to interpret spectra (less multiple charges) Quick and easy Higher mass detection Higher Throughput (>1000 samples per hour)

Matrix Needs to be involatile (most are solids at room temperature) Needs to absorb the laser wavelength that you are using. (most cases 337 nm) Preferably dissolves in same solvent as the sample Typically, the matrices are acidic.

HOT and Cold Matrices DHB is a cold matrix, the samples are not as likely to be fragmented, may not ionize some molecules. Alpha-cyano dihydroxybenzoic acid is considered a hot matrix. More likely to fragment the molecules. Can produce multiply charged proteins.

Time-of-Flight Mass Spectrometry (TOF-MS) A time-of-flight mass spectrometer uses the differences in transit time through a drift region to separate ions of different masses. It operates in a pulsed mode so ions must be produced or extracted in pulses. An electric field accelerates all ions into a field-free drift region with a kinetic energy of qV, where q is the ion charge and V is the applied voltage. the kinetic energy of an ion leaving the ion source is:  The ion velocity, v, is the length of the flight path, L , divided by the flight time, t:   

- Protein identification strategies Maldi-Tof MS Small proteins Unknown proteins

Parent ions MS/MS Spectrum CID Non-dissociated Parent ions + + + + F L G K + + F L G K b3 y1 F L G K + + Parent ions + + F L G K F L G K CID b2 y2 + F L G K + + + + F L G K F L G K b1 y3 Non-dissociated Parent ions Daughter ions y1 y2 y3 b1 b2 b3 K G L F MS/MS Spectrum Relative Intensity L F G K m/z