Mass Spectroscopy

Mass Spectroscopy

1. Laser Ionisation

   • A laser pulase ablates material from the surface of a sample, and creates a microplasma that ionises some of the sample constituents. The laser pulase accomplishes both vaporisation and ionisation of the sample
   • Matrix-Assisted Laser Desorption Ionisation (MALDI)
     • A LIMS method of vaporising and ionising large biological molecules (eg. protein) Soft technique - isolates sample molecules in a chemical environment which enhances the probability of ionisation without fragmentation Generates spectra that have just a singly charged ion
     • Working Principle
       • A large excess of organic matrix is mixed with a small quantity of analyte where the matrix absorbs (majority of) photon energy from the laser beam and transfers into excitation energy of the solid system and dilutes the analyte molecules, so that their intermolecular forces are reduced and their aggregation is minimised. The matrix must absorb at the laser wavelength so that the matrix transfers some of its energy to the analyte.
       • Short pulses of light focused on to the sample spot. UV radiation is absorbed by the matrix (neutral) where the matrix is then dissociated. There is a phase change of the matrix to super-compressed gas where both the analytes and matrix are charged. Matrix expand at supersonic velocity in which analytes is trapped in the matrix and eventually leads to a explosion (sample vaporise). The ions formed are accelerated by a high voltage supply and then allowed to drift down a flight tube where they separate according to mass. Arrival at the end of the flight tube us detected and recorded by a high speed recording device
     • Positive mode -> ions of M+H Negative mode -> ions of M-H
     • Compared to ESI: more robust (tolerates salts and nonvolatile components); easier to use and maintain and capable of high throughput; requires 10microL of 1picomol/microL sample (very small vol req); sensitive
     • Applications
       • monitor and optimise enzymatic digests, characterise proteins or used for quality control for peptide synthesis
       • A method of N-terminal and C-terminal protein/peptide sequencing (analyse molecules with different size and get the sequencing information)
       • Rapid conformation of post translational modifications and the quantitation of drugs and chelators conjugated to monoclonal antiborides
       • Determination of bacteria in food
     • Precautions
       • MALDI samples free of SDS and not radioactive
       • Best to remove salts and detergents and dissolve sample in suitable solvent (eg. 0.1% TFA/water) which will not degrade the spectrum
         • Too much salt => salt intensity so large that it effectively suppresses out the sample signal, giving no sample spectrum
         • Trifluoroacetic acid (TFA) can cause ion suppression if used as ion pairing additive in mobile phase - analyte cannot be ionised easily after ion pairing
       • If can't remove contaminants, samples should be in a higher concentration / dilute sample such that the contaminants will have little effect on spectrum => higher signal to noise ratio
   • Electrospray Ionisation (ESI)
     • Particularly useful for large biological molecules that are difficult to vaporise or ionise
     • Working Principle
       • A dilute polymer solution containing the sample is forced through a needle held at high potential at atmospheric pressure. The high potential on the needle produces solvent droplets that are highly charged. The droplets shrink until the surface charge of the droplets exceeds the Rayleigh limit of instability, then the droplets burst into smaller droplets through a series of 'coulombic' explosions until the droplets disappear, leaving highly charged analyte molecules
         • Samples dissolved in polar, volatile buffer
         • Strong voltage (3-4kV) applied at tip along with flow of nebulizing gas causes the sample to aerosolize. Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)
         • Rayleigh limit: limiting droplet size of which self-fragmentation will occur whereby the repulsive forces between like charges in an electrolytic solution overcome the cohesive forces of solvent
         • Coulomb Explosion: mechanism for coupling electronic excitation energy into atomic motion
     • Functional groups that readily accept H+ (eg. amide and amino groups -> Proteins) -> Positive mode -> add formic acid -> ions of M+H Functional groups that readily lose a proton (eg, carboxylic acid ad hydroxyls -> DNA) -> Negative mode -> add ammonia -> ions of M-H
     • Advantages
       • Very sensitive technique, requires less than a picomole of analyte
       • Can be modified to "nanospray" system with flow < 1 microL/min
         • very little sample solution is required to yield useful mass spectral information for lengthy experiments
       • Drastically increases the upper limit for m/z measurement, making it amenable to a large variety of mass spectrometers
       • the ability to directly couple liquid chromatographic methods such as SEC or HPLC to MS
     • Multiply Charged Ions
       • Consider a positive ion with charge z1 whose mass-to-charge ratio is measured as being m1 Th (Thomson), issued from a molecular ion with mass M Da to which z1 protons have been added. We then have z1 m1= M + z1 mp where mp is the mass of the proton.
       • An ion separated from the first one by (j−1) peaks, in increasing order of mass-to-charge ratio, has a measured ratio of m2Th and a number of charges z1–j, so that m2 (z1− j ) = M + (z1− j )mp These two equations lead to z1= j (m2 − mp)/(m2− m1) and M = z1(m1− mp) In the case of negative multiply charged ions, analogous equations lead to z1= j (m2 + mp)/(m2− m1) and M = z1(m1+ mp) Multiply Charged Ions