Methods for generating ions Electron impact EI Chemical ionization CI

Other Methods of forming ions in mass spectrometry

1. Fast atom bombardment High voltage applied + or -

2. Secondary ion mass spectrometry SIMS 3. Plasma desorption

In SIMS the surface of the sample is subjected to bombardment by high energy ions - this leads to the ejection (or sputtering) of both neutral and charged (+/-) species from the surface. The ejected species may include atoms, clusters of atoms and molecular fragments. In traditional SIMS it is only the positive ions that are mass analyzed - this is primarily for practical ease but it does lead to problems with quantifying the compositional data since the positive ions are but a small, non-representative fraction of the total sputtered species. It should be further noted that the displaced ions have to be energy filtered before they are mass analyzed (i.e. only ions with kinetic energies within a limited range are mass analyzed). The most commonly employed incident ions (denoted by I + in the above diagram) used for bombarding the sample are argon ions ( Ar + ) but other ions (e.g. alkali metal ions, Ga + ) are preferred for some applications.

LSIMS liquid secondary ion mass spectrometry A liquid matrix is used using Cs + produced from a heated pellet of cesium aluminum silicate. The matrix is similar to that used in FAB

Solid sample bombarded by 252 Cf source Sample on glycerol bombarded by Xe ions and neutrals Sample on glycerol bombarded by Ar ions and neutrals Gale, P. J. et al. Anal. Chem. 1986, 58, Mw 475

4. Matrix assisted laser desorption ionization

MALDI is based on the bombardment of sample molecules with a laser light to bring about sample ionization. The sample is pre- mixed with a highly absorbing matrix compound for the most consistent and reliable results, and a low concentration of sample to matrix works best. The matrix transforms the laser energy into excitation energy for the sample, which leads to sputtering of analyte and matrix ions from the surface of the mixture. In this way energy transfer is efficient and also the analyte molecules are spared excessive direct energy that may otherwise cause decomposition. Most commercially available MALDI mass spectrometers now have a pulsed nitrogen laser of wavelength 337 nm.

Matrix assisted laser desorption ionisation (MALDI) The sample to be analysed is dissolved in an appropriate volatile solvent, usually with a trace of trifluoroacetic acid if positive ionization is being used, at a concentration of ca. 10 pmol/µL and an aliquot (1-2 µL) of this removed and mixed with an equal volume of a solution containing a vast excess of a matrix. A range of compounds is suitable for use as matrices: sinapinic acid is a common one for protein analysis while alpha-cyano-4-hydroxycinnamic acid is often used for peptide analysis. An aliquot (1-2 µL) of the final solution is applied to the sample target which is allowed to dry prior to insertion into the high vacuum of the mass spectrometer. The laser is fired, the energy arriving at the sample/matrix surface optimized, and data accumulated until a m/e spectrum of reasonable intensity has been amassed. The time-of-flight analyser separates ions according to their mass(m)-to-charge(z) (m/e) ratios by measuring the time it takes for ions to travel through a field free region known as the flight, or drift, tube. The heavier ions are slower than the lighter ones.

Maldi spectrum of a peptide mixture using  -cyano-4- hydroxycinnamic acid (matrix)

5. Field desorption Field ionization: Sample in the gas phase is ionized in the presence of a strong electric filed

Field desorption Sample dipped in solution and allowed to evaporate

field desorption (in mass spectrometry) A term used to describe the formation of ions in the gas phase from a material deposited on a solid surface (known as an ‘emitter’) in the presence of a high electrical field. Intense molecular ions are produced. Ions are produced from a sample place in an intense electric field which causes the electrons to be removed by a “tunneling effect”. Little excess energy is produced during the ionization.

Impurities in the sample and solvent apparently supplied enough Na+ ions for desorption Michael Linscheid, Jay D'Angona, Alma L. Burlingame, Anne Dell, and Clinton E. Ballou, Proc Natl Acad Sci U S A March; 78(3): 1471–1475.

6. Spray ionization techniques

C 20 H 13 N 2 O 3 SNa mw = = 23

( )/10 =

Positive ESI-MS m/z spectrum of the protien hen egg white lysozyme. ( )/13 = ( ) /12 = ( ) /11 = ( ) /10 = ( ) /9 = ( ) /8 = ( )/7 =

Positive ESI-MS m/z spectrum of the protien hen egg white lysozyme. (x+y)/x = (x+1+y) /x+1 = (x+2+y) /x+2 = (x+3+y) /x+3 = … y = mw; x = #H +

C 28 H 37 N 5 O 7 and the calculated monoisotopic molecular weight is Da.

Ala A-NH.CH.(CH 3 ).CO-71.0 Arg R -NH.CH.[(CH2) 3.NH.C(NH).NH 2 ]CO Asn N -NH.CH.(CH 2 CONH 2 ).CO Asp D -NH.CH.(CH 2 COOH).CO Cys C -NH.CH.(CH 2 SH).CO Gln Q -NH.CH.(CH 2 CH 2 CONH 2 ).CO Glu E -NH.CH.(CH 2 CH 2 COOH).CO129.0 Gly G -NH.CH 2.CO-57.0 His H -NH.CH.(CH 2 C 3 H 3 N 2 ).CO Ile I -NH.CH.[CH.(CH 3 )CH 2.CH 3 ].CO Leu-NH.CH.[CH 2 CH(CH 3 ) 2 ].CO Lys K -NH.CH.[(CH 2 )4NH 2 ].CO MetM -NH.CH.[(CH2) 2.SCH 3 ].CO Phe F -NH.CH.(CH 2 Ph).CO Pro P -NH.(CH2) 3.CH.CO-97.1 Ser S -NH.CH.(CH 2 OH).CO-87.0 Thr T -NH.CH.[CH(OH)CH 3 ).CO Trp W -NH.CH.[CH 2.C 8 H 6 N].CO Tyr Y -NH.CH.[(CH 2 ).C 6 H 4.OH].CO Val V -NH.CH.[CH(CH 3 ) 2 ].CO-99.1

= (0.99) 99 (0.01)=0.37 Remember Pascal triangle 200* =201.6

MW = 1142 – 2H + =

Other Methods for Generating Ions 1.Fast atom bombardment 2. MALDI matrix assisted laser desorption ionization MS 3. Spray ionization techniques 4. MS –MS techniques

8.1 Tandem mass spectrometry Tandem mass spectrometry (MS-MS) is used to produce structural information about a compound by fragmenting specific sample ions inside the mass spectrometer and identifying the resulting fragment ions. This information can then be pieced together to generate structural information regarding the intact molecule. Tandem mass spectrometry also enables specific compounds to be detected in complex mixtures on account of their specific and characteristic fragmentation patterns. A tandem mass spectrometer is a mass spectrometer that has more than one analyser, in practice usually two. The two analysers are separated by a collision cell into which an inert gas (e.g. argon, xenon) is admitted to collide with the selected sample ions and bring about their fragmentation. The analysers can be of the same or of different types.

Product or daughter ion scanning: the first analyser is used to select user-specified sample ions arising from a particular component; usually the molecular-related (i.e. (M+H)+ or (M-H)-) ions. These chosen ions pass into the collision cell, are bombarded by the gas molecules which cause fragment ions to be formed, and these fragment ions are analysed i.e. separated according to their mass to charge ratios, by the second analyser. All the fragment ions arise directly from the precursor ions specified in the experiment, and thus produce a fingerprint pattern specific to the compound under investigation. This technique is frequently used for identifying protein structure

There are three different types of bonds that can fragment along the amino acid backbone: the NH-CH, CH-CO, and CO-NH bonds. Each bond breakage gives rise to two species, one neutral and the other one charged, and only the charged species is monitored by the mass spectrometer. The charge can stay on either of the two fragments depending on the chemistry and relative proton affinity of the two species. Hence there are six possible fragment ions for each amino acid residue and these are labeled as in the diagram, with the a, b, and c" ions having the charge retained on the N-terminal fragment, and the x, y", and z ions having the charge retained on the C-terminal fragment. The most common cleavage sites are at the CO-NH bonds which give rise to the b and/or the y" ions. The mass difference between two adjacent b ions, or y"; ions, is indicative of a particular amino acid residue

Ala A-NH.CH.(CH 3 ).CO-71.0 Arg R -NH.CH.[(CH 2 ) 3.NH.C(NH).NH 2 ]CO Asn N -NH.CH.(CH 2 CONH 2 ).CO Asp D -NH.CH.(CH 2 COOH).CO Cys C -NH.CH.(CH 2 SH).CO Gln Q -NH.CH.(CH 2 CH 2 CONH 2 ).CO Glu E -NH.CH.(CH 2 CH 2 COOH).CO129.0 Gly G -NH.CH 2.CO-57.0 His H -NH.CH.(CH 2 C 3 H 3 N 2 ).CO-137. Ileu I -NH.CH.[CH.(CH 3 )CH 2.CH 3 ].CO LeuL-NH.CH.[CH 2 CH(CH 3 ) 2 ].CO LysK -NH.CH.[(CH 2 ) 4 NH 2 ].CO MetM -NH.CH.[(CH 2 ) 2.SCH 3 ].CO Phe F -NH.CH.(CH 2 Ph).CO Pro P -NH.(CH 2 ) 3.CH.CO-97.1 Ser S -NH.CH.(CH 2 OH).CO-87.0 ThrT -NH.CH.[CH(OH)CH 3 ).CO Trp W -NH.CH.[CH 2.C 8 H 6 N].CO Tyr Y -NH.CH.[(CH2).C 6 H 4.OH].CO Val V -NH.CH.[CH(CH 3 ) 2 ].CO-99.1

Determining the sequence of gramicidin S by MSMS C 60 H 92 N 12 O 10 MW 1140 Amino acid analysis: Val, Orn, Leu, Phe, Pro Val 99 Orn 114 Leu 113 Phe 147 Pro 97 Total = * How many degrees of unsaturation? C=O; 8 Ph; pro 2:

C60H9 2N12O 10 C 60 H 92 N 12 O 10 MW 1140 orn-val-pro val-pro-orn val orn-pro val-pro-phe-leu-orn ׀ orn-leu-phe-pro-val Ignoring N and C terminals, how many orn-val-pro isomers are there? orn-leu-phe orn-phe-leu phe-orn-leu

Monitoring a specific ion current vs total ion current for following a reaction in complex mixtures.