Fast Atom Bombardment (FAB)-MS

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Roger Mills
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1 Fast Atom Bombardment (FAB)-MS FAB-MS was developed by Barber and coworkers in the early 1980s and soon became a very powerful technique for desorption ionization of thermally labile molecules. In FAB a beam of fast moving neutral atoms of a noble gas (Ar, Xe), is directed to strike a sample film deposited on a clean metal support: Secondary ions are emitted from the sample layer and then extracted and focused towards the mass spectrometer:

2 Source of fast atoms in FAB The Ar or Xe beam required in FAB is generated within an appropriate atom gun: Ar (or Xe) atoms are ionised by electron ionization within chamber 1 the resulting ions are then focused and accelerated by lenses when arriving in chamber 3, Ar ions exchange charge and energy with Ar neutral atoms 3 Ar ions are separated from accelerated neutral atoms, outgoing from chamber 3, by electrostatic deflectors (4): fast Ar atoms are finally directed outside, towards the sample stage.

3 A Torr Ar (or Xe) pressure is maintained inside chamber 3. Under these conditions a charge exchange without significant loss of momentum occurs in chamber 3 between incoming Ar + ions and Ar atoms. The processes occuring in the different stages of the atom gun are: 1) Ar Ar + + e - 2) Ar + Ar + (fast) 3) Ar + (fast) + Ar Ar (fast) + Ar + Kinetic energies between 3 and 10 kev can be achieved for outcoming atoms. The atom beam is usually directed at a 20 angle with respect to the sample surface:

4 When the sample surface is struck by fast atoms an intense thermal spike is produced and subsequently dissipated towards the surface outer layers through momentum transfers: FAB mechanism Analyte molecules are detached from these layers as a dense gas containing positive and negative ions, as well as neutrals, that can be eventually ionized in the plasma just above the sample surface. Actually, when a dry deposited sample is analyzed by FAB the secondary ion yield decays rapidly, due to surface damage. Much more satisfactory results can be achieved if the sample is dissolved in a liquid matrix.

5 The main requirements for a FAB matrix are: being reasonably involatile (since it must remain liquid under vacuum) having good solvating properties for the analyte Polar compounds with MW lower than 300 Da are usually adopted as matrices for FAB: Occasionally, inorganic or organic acids/bases are added to the matrix to promote protonation/deprotonation.

According to the more recent hypotheses the chemistry leading to ion generation during FAB takes place into special gas cavities, representing the initial interfacial region between the condensed

6 According to the more recent hypotheses the chemistry leading to ion generation during FAB takes place into special gas cavities, representing the initial interfacial region between the condensed phase (i.e. the matrix/analyte mixture) and the gas phase: The cavities are formed in the interfacial region upon sudden heating after fast atom impacts, then desorption occurs and analyte and matrix molecules move towards the so called selvedge region, where ionmolecule reactions can occur. Inorganic salts (even as impurities) can be involved in the process.

7 In the ChemicaI Ionization-like model, the following ion-molecule interactions are hypothesised to explain ion generation during FAB: Proton transfer by any organic ion (FH + ) arising from the matrix Cation/anion attachment from salts dissolved into the sample If the analyte is anionic, partial charge recombination with counterions in the sample or proton abstraction from the matrix (e.g. glycerol, G) may occur:

8 An approach proposed (1997) for the determination of reagent ions, FH +, i.e. ionic species actually involved in ionization of analyte molecules, is based on the acquisition of FAB spectra for pure glycerol samples at increasing times: Peaks related to glycerol clusters are observed, along with the GH + ion (m/z 93): G 2 H +, 185 G 3 H +, 277 G 4 H +, 369 These ions disappear progressively from the FAB spectra as time goes by, due to lower availability of glycerol molecules, required for cluster generation.

9 Relevant ions for the FAB mechanism are likely those related to m/z ratios 31, 45, 57 and 75, because they are predominant when long bombardment times, i.e. thin residual sample thicknesses, are considered. A proposed pattern for their generation is: These ions might be the actual primary ions of FAB, dominant at the interfacial region but attenuated as they pass through the selvedge region, where the following processes may occur:

10 When negative ion FAB is performed deprotonated analyte and matrix molecules are usually observed. If glycerol is used as a matrix a proton transfer can occur: Negative ion FAB spectra obtained at different times for a glycerol sample show further relevant ions, along with [G-H] - (m/z 91):

11 Glycerol clusters with the [G-H] - analysis: ion are observed in the first stage of [2G-H] -, m/z 183 [3G-H] -, m/z 275 [4G-H] -, m/z 367 Further ions, observed at longer bombardment times (m/z 71, 59, 45, 43, 25 and 17) could be the actual reagent ions and have been interpreted with the help of deuterated analogs:

12 Data available on proton affinities (PA) for the cited ions (or structural analogs) indicate that only C 2 H - and OH - can actually react exothermically with glycerol to generate the [G-H] - ion. Other species, like ions with m/z 71, 59 and 43, have slightly higher proton affinities than [G-H] - ion, thus they can deprotonate glycerol only if energy is provided by the environment, which is reasonable, during fast atom bombardment.

13 Temporal dependence of analyte signals in FAB-MS The evolution with time of matrix-related ions can influence the temporal dependence of the FAB spectrum for an analyte, as shown in the following for the cardiac glycoside digoxine (MW 780 u):

14 In this case, the quasi-molecular ion of digoxine (m/z 781) and its highmw product ions (m/z 651, 521, 391 and its de-hydrated form, 373) are almost negligible, at any acquisition time. Smaller product ions, like those with m/z 243 and 131, are very relevant already in the first spectra and then undergo a significant decrease, indicating analyte depletion from the matrix. As in experiments involving only glycerol, the [G+H] + (m/z 93) and its cluster ions (m/z 277 and 185) are initially relevant, then fragment ions of glycerol (m/z 43, 45, 57 and 75), generally indicated as F +, become prevailing in the FAB spectrum. The following processes can be generally hypothesized for glycerol/analyte interactions: Considerations related to proton affinities values lead to predict that GH + ions generate more analyte fragments than F + ones.

15 Fast atom gun preparation Practical operation of FAB-MS Before operation, the gas lines (usually Ar or Xe) connected to the fast atom gun need to be flushed out once or twice to remove any air trace. After this operation the inert gas cylinder feeding the gun is usually connected permanently to the gun. Typical gas flows during operation are 0.5 ml/min, whereas accelerating potentials of a few kv are applied. Sample stage (probe tip) preparation The stage on which the matrix/sample mixture is deposited can be made of stainless steel, copper or gold-plated copper. Preliminary cleaning is usually performed with an organic solvent but concentrated HCl can be used to remove persistent remnants of previous samples.

16 Deposition of matrix and analyte solution on the sample stage The matrix is usually deposited onto the sample stage before the sample solution, using a few L (e.g. of glycerol), that need to be properly spread out on the entire area of the stage (to avoid the presence of stage contaminant signals in the FAB spectra). Afterwards, a 1 L aliquot of the analyte solution is added, using a microsyringe, and then mixed with the matrix. Sample stage introduction The sample stage is introduced within the high vacuum region of the spectrometer using a sample probe, passing through a vacuum lock. Solvents adopted for sample dissolution (water or methanol) are quicky evacuated, thus the source pressure will stabilize at an acceptable value after a relatively short time. Afterwards, fast atom gun and extraction voltages can be switched on and the analysis can be started.

17 Intepretation of FAB-MS spectra As already evidenced, the presence of matrix peaks can complicate the interpretation of FAB-MS spectra. A clear example of matrix interference is represented by the FAB positive ion spectrum (with glycerol as matrix) for the pentapeptide methionine enkephalin (MW u): G 3 H + G 4 H + [M+H] + G 5 H + G 6 H + G7H +

18 In case one of the matrix peak has a m/z ratio close to that of the analyte, the matrix has to be changed. When traces of salts, especially Na and K salts, are present in the sample, series of Na or K-adducts with matrix clusters can be observed in the FAB spectrum, thus increasing the risk of spectral interference. In this case the spectrum may exhibit peculiar doublets with a m/z difference of 22 or 38 m/z units, corresponding to substitution of a H + with Na + or K +, respectively. As in FD, deliberate cationization can enhance FAB signals due to analyte adducts with Na + or K +, which can be useful also to recognize if adducts are actually present. When only one dominant peak is present in the FAB spectrum, apart from matrix well known peaks, the dilemma about its nature, e.g. if it corresponds to a [M+H] + or a [M+Na] + ion, can be solved by doping the sample with a K salt, thus introducing a [M+K] + peak. The dominant peak will correspond to [M+H] + or [M+Na] + if the shift observed for the new peak is 38 or 16 m/z units, respectively.

19 Limitations of FAB-MS Besides the risk of spectral interference due to matrix cluster ions, analyte and matrix adducts with alkali cation traces and even matrix-analyte cluster ions, other important limitations need to be considered for FAB-MS: high background below 200 m/z units; upper MW limit at u; difficulty in achieving a precise and accurate quantification: in many cases the analyte signal disappears because the matrix has been desorbed completely so the operator cannot be sure that the analyte has been consumed completely; high competition for ionization in mixtures, with a typical example given by a protein digest: hydrophobic peptides tend to segregate at the upper surface of the matrix/analyte coating, thus can be ionized more efficiently than hydrophilic peptides. In some cases, a single component can totally suppress the signal due to other analytes in the mixture.

20 Fast Ion Bombardment (FIB)-MS Fast ion bombardment was introduced soon after FAB-MS when it was clear that the latter could not ionize (bio)molecules with MW higher than u. The principle of FIB is using a Cs + beam instead of a Xe or Ar one, to hit the sample: Cs + ions are generated by a pellet heated by a filament and then accelerated towards the probe tip by very high potentials, up to 35 kv.

21 In its typical configuration FIB-MS is very similar to Secondary Ion Mass Spectrometry (SIMS), although applied to a liquid, instead of a solid, sample; the alternative name Liquid SIMS (LSIMS) has been then given to the technique. Although FAB and FIB mass spectra are almost identical when relative low (< 1000 u) MW analytes are considered, the higher energy of Cs + ions, compared to that of Xe or Ar fast atoms, ensures much higher sensitivities with FIB-MS. In the case of high MW compounds the difference is more striking. Good S/N mass spectra have been obtained by FIB-MS even for molecules like trypsinogen (MW u).

Continuous Flow Fast Atom Bombardment Continuous Flow Fast Atom Bombardment was developed in the middle 1980s to introduce samples in a flow of solvent directed towards a special probe for FAB (or

22 Continuous Flow Fast Atom Bombardment Continuous Flow Fast Atom Bombardment was developed in the middle 1980s to introduce samples in a flow of solvent directed towards a special probe for FAB (or FIB)-MS analysis. Two general configurations are used for CF-FAB, both adopting a special hollow direct insertion probe: Frit FAB (Ito et alt., 1985) In this case the analyte/matrixcarrying solvent flows in a fused silica tubing (~40 m internal diameter) contained in a stainless steel capillary passing through the probe. At the probe tip the silica tubing delivers the liquid through a stainless steel frit, thus a thin analyte-matrix layer is formed on the frit.

23 Continuous flow (dynamic)-fab (Caprioli et alt., 1986) The main difference with respect to Frit-FAB is that the silica tubing delivers the analyte/matrix solution directly on the probe tip, through a small hole. Copper tips were adopted only initially and then replaced by goldcoated copper tips. In both cases maximum flows of a few L/min can ensure the proper balance between delivery and vacuum/thermal desorption of solvent/analyte/matrix at the probe tip.

24 In a further design reported for CF-FAB an absorbant pad is placed just below the end of the liquid-delivering fused silica capillary: The pad absorbs excess liquid at the surface of the probe tip, thus making the sample/analyte/matrix coating thinner.

25 Operating modes for CF-FAB Three operating modes are usually adopted for CF-FAB: a) continuous sampling of the compound or reaction mixture of interest (the liquid is drawn simply by the vacuum at the opposite end of the silica capillary) b) consecutive, discrete injections of different samples through a valve (like in Flow Injection Analysis, FIA); c) coupling to a separation technique, like HPLC or Capillary Electrophoresis (CE)

26 Practical operation of CF-FAB One of the most critical points in CF-FAB is obtaining a stable flow at the probe tip, in order to obtain a steady ion beam from the source. The main parameters to check are: flow rate (usually lower than 10 L/min) flow fluctuations (a pulse-free solvent delivery pump should be used) composition of solvent-matrix mixture (usually a water/methanol or acetonitrile/glycerol 0.9 : 0.9 : 0.2 v/v/v mixture is adopted) silica capillary length (usually 1 m) and protrusion from the probe tip (1-2 mm)

The optimal combination between temperature and flow rate can be determined by reporting the signal stability

27 probe tip temperature: differently from conventional FAB-MS, gentle heat is required in CF-FAB to remove excess solvent. The optimal combination between temperature and flow rate can be determined by reporting the signal stability as a function of the two parameters: A B Even small changes of one of the two parameters, if leading to cross the border of the stability region, can lead to evident modifications of the CF-FAB signal stability: A B

28 Reaction monitoring by continuous sampling-fab Continuous sampling enables the FAB-MS monitoring of reaction mixtures in real time. A typical application is following the evolution of enzymatic digests of peptides or proteins, like Substance P (a neuropeptide) digestion by carboxypeptidase Y, an enzyme that hydrolyses the peptide bond at the COOH terminus: 1217 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met (MW u)

29 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met (MW u) The sequential appearance of smaller peptides arising from carbopeptidase Y-catalyzed hydrolysis provides important information on the digestion kinetics.

30 HPLC-FAB-MS Continuous flow-fab was adopted as one of the first LC-MS interfaces before ESI became the prevailing approach. The typical FAB matrix, glycerol, can be either added already to the HPLC mobile phase (it has almost the same eluotropic strength as methanol), although at percent values not higher than 5 % (v/v), or introduced as post-column reagent, using suitable T-junctions: Further T-shaped splitters need to be usually introduced before and/or after the HPLC colum to adapt high LC flow rates (hundreds of L/min) to those suitable for CF-FAB.

31 Microbore (i.d. 1 mm) or capillary (i.d. 0.3 mm) HPLC columns can be adapted more easily to CF-FAB-MS, as their typical working flows are and 5 L/min, respectively: The post-column introduction of FAB matrix by a syringe pump is evidenced in the figure. The need for matrix can lead to an increase of flow rates, thus a subsequent Split Tee is required.

Applications of HPLC-FAB-MS One of the most interesting applications reported for HPLC-FAB-MS is the separation and MS analysis of peptides arising from protein enzymatic digestion.

32 Applications of HPLC-FAB-MS One of the most interesting applications reported for HPLC-FAB-MS is the separation and MS analysis of peptides arising from protein enzymatic digestion. The TIC trace and a composite trace, obtained by overlap of extracted Ion Chromatograms (XIC) for selected peptides, relevant to the digest of horse heart cytochrome C with trypsine, are shown as an example: TIC XIC

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