ESI-Ion Trap mass spectrometry

3.8.3.1 Direct infusion ESI-Ion Trap mass spectrometer

ESI-Ion Trap mass spectrometry was performed with the Esquire 3000plus Ion Trap mass spectrometer using an API-ESI (Atmospheric Pressure Interface-Electro Spray Ionisation) for generating the ions, focusing and transporting into the ion trap mass spectrometer (Figure 94).

Waste

A solution of sample and solvent is sprayed through a needle into the spray chamber.

This produces droplets of solvent and sample. A heated drying gas is introduced into the spray chamber to help evaporate the solvent from the droplets. In the spray chamber, the droplets are also subjected to a high-voltage electrostatic gradient. The gradient assists in the production of droplets and ions. The droplets evaporate and ions are ejected. The electrostatic gradients guide the ions towards a glass capillary. The pressure differential between the spray chamber and the ion focusing and transport region pushes the ions through the capillary.

The operator controls the high voltage electrostatic gradient, the temperature and flow of the drying gas and the pressure of the nebulizer gas. This control is all done by means of the system software. In the spray chamber the sample in solution is nebulized and ionized through an optimised desolvation process. The sample is introduced into the spray chamber through a nebulizer. The nebulizer spray is directed at a right angle to the sampling capillary. The sampling capillary is shielded by a cylindrical end plate. The spray chamber operates at atmospheric pressure. The drying gas, nitrogen, is used to evaporate solvents in the spray chamber. It enters the spray chamber flowing around and heating a part of the capillary. The gas is typically heated

to 200-250 °C at a flow of 1 L min^-1 to 5 L min^-1. While the drying gas assists in the desolvation process it does not thermally decompose the analyte. Desolvation requires a great deal of energy, so droplet and analyte temperatures remain low.

ESI-MS spectra were acquired in the positive ion mode on an Esquire 3000plus ion trap instrument (Bruker Daltonik, Bremen, Germany), equipped with a syringe pump operated at a flow rate of 1-5 µL min^-1. The peptides were dissolved at a concentration of 0.01 µg µL^-1 in 0.1 % HCOOH. The source parameters employed were as follow:

capillary voltage -3.5 kV, end plate offset 500 V, capillary exit 120 V, nebuliser gas 10 psi, dry gas 7 Lmin^-1.

A MS/MS experiment starts with ionisation and injection of the generated ions into the ion trap. Here the parent ion is isolated and collision-induced dissociation (CID) takes place as a result of collisions in the helium damping gas due to the resonance excitation of the ions. After fragmentation, the CID spectrum of the selected precursor ion is recorded by sequentially ejecting the product ions; only masses higher than 28%

of the parent ion can be stabilised inside the ion trap. Ion trap is about 50 times more sensitive than a triple quadrupole mass spectrometer and the resolution can be improved using a special scan mode. Proteins and peptides are oligomers of repeating (-NHCHR-CO-) units, differing only in the nature of the side chain, R. The amino acid residues are held together by peptide bonds, (-NH-CO-) which have lower bond energies than standard (-N-C-) bonds. When proteins or peptides fragment, peptide bonds are commonly broken releasing mostly intact amino acid residues. In MS experiments, however, there may also be fragmentation at other localisation in addition to peptide bonds resulting in a complex pattern of ions. These are related to each other by incremental m/z differences because only 20 R-groups of known structure are found in proteins and because breakage points have fixed structural localisations relative to each other. The nomenclature proposed by Roepstorff which is widely used to describe polypeptide fragmentation in MS is illustrated in

Figure 95: Peptide cleavage nomenclature proposed by Roepstorff and Fohlman.

Peptide ions fragment at the peptide backbone to produce major series of fragment ions. Fragment ions from N-terminus are called a, b, c, and fragment ions from the C-terminus are called x, y, z ion.

Ions formed from a polypeptide during MS may retain a positive or negative charge either on their C-or terminus. A horizontal line pointing towards the C or N-terminus at the breakage point is used to denote which fragment carries the charge in that particular ion. For the N-terminal ions there are a_n, b_n, c_n and d_n (numbered from the N-terminus) while the C-terminal ions are designated x_n, y_n and z_n (numbered from the C-terminus). The ion represented by a1 represents the first residue in the sequence (minus the CO group) while a₂ represents the first two residues (minus the CO group) and so on. Identical main-chain breakage points are denoted by the pairs a/x, b/y, c/z (each member of a pair referring to a positive charge retained either on the N- or C-terminus, respectively). Breakage points denoted by d_n, v_n and w_n are due to side-chain fragmentations. There are used to distinguish residue pairs such as Leu/Ile and Gln/Lys which have identical masses.

3.8.3.2 Liquid chromatographic/Ion trap mass spectrometric analysis

For LC/MS investigations, an Agilent Technologies (Waldbronn, Germany) HP1100 liquid chromatograph for binary gradient elution (pump model G1312A), including autosampler (G1313A) coupled to an Esquire 3000 ion trap mass spectrometer from Bruker Daltonics (Bremen, Germany) was used. For the LC separation, a binary gradient system consisting of solvent A (0.1 % formic acid in water) and solvent B (80 % acetonitrile, 0.1 % formic acid in water) was employed.

The sample was dissolved in the solvent A. The injection volume was 5 µl. A 150 mm x 4.6 mm x 3 µm Discovery RP-18 column was used for the separation of the peptides.

All LC/MS results were obtained using atmospheric pressure chemical ionisation (APCI) in the positive ion mode. Mass spectra were recorded in the full scan mode, scanning from m/z 100 to 2500. Ion source parameters were 20psi nebulizer gas and 10 Lmin^-1 of drying gas with a temperature of 200°C. MS/MS experiments were carried out in the auto-fragmentation mode.