ESI-ion trap mass spectrometry

Electrospray ionization (ESI) is a soft ionization technique that accomplishes the transfer of ions from solution to the gas phase. The technique is extremely useful for the analysis of large, non-volatile, chargeable molecules such as protein and nucleic acid polymers. For the development of ESI mass spectrometry of biomolecules, John B. Fenn received the Nobel Prize 2002 in Chemistry. ESI-MS is an ionization method which produces gaseous ionized molecules from a solution ^[234] by creating a fine spray of charged droplets in an electric field ^{[235, 236]}. An illustration of the ESI process is shown in Figure 113.

The repulsion between the charges on the surface causes finally intact ions to leave the droplet by a process known as a “Taylor cone” ^[341]. Electrospray ionization is generally leading to the formation of multiply charged molecules.

This is an important feature since the mass spectrometer measures m/z, thus making it possible to analyze large molecules with an instrument of a relatively small mass range. The mass analyzer is responsible for the accuracy, range, and sensitivity of a mass spectrometer. Two most efficient types of mass analyzers used in this work are time-of-flight (TOF) and ion trap. Different analyzers can be used with different desorption methods.

--

-Desolvated macromolecular ion Solvated macromolecular ion

Aerosol of fine charged droplets (microdroplet) Electrospray

10 µm 10 nm 1 nm

Taylor-Cone

Figure 113: ESI mass spectrometry. The formation of the charged droplets in form of highly charged ions takes place in the gas phase. The size of the droplets decreases when reaching the Rayleigh limit. The solution is evaporated into the vacuum system of the ion source. In this example the desolvated ion has a 3+ charge ^[241].

The ion trap mass analyzer is the three dimensional analogue of the linear quadrupole mass filter and is based on the motion of ions in an rf electric field (Figure 114). Ions are subjected to forces applied by an rf field in all three directions. The ion trap consists of three electrodes with hyperbolic surfaces:

two end-cap electrodes at ground potential and between them a ring electrode to which a radio frequency voltage is applied. The device is radial symmetrical and r0 and z0 represent its size.

Figure 114: Geometry of the ion trap mass analyzer, consisting of a ring electrode and two end caps electrodes with special shape.

The ion trap employs an alternating electric field to stabilize and destabilize the passing ions. In the case of ion trap the electric field is used to store ions and to release them in time to be analyzed. The mass separation can be done by several methods. The most important for analytical purposes are: -Stafford’s

mass-selective instability mode scan the fundamental rf-voltage amplitude applied to the ring electrode. Thus, ions of increasing mass-to-charge ratio adopt unstable flight paths and leave the trap towards the detector; -resonance ejection utilizes again the fundamental rf- voltage amplitude to bring the ions into resonance with the supplementary rf- voltage at the end cap electrodes.

Thus they absorb sufficient power to leave the ion trap. In this way the mass range of the ion trap can be extended. The ion trap mass analyzer applied to peptide sequencing generally are combined with an ESI ion source.

A MS/MS experiment starts with ionization 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 stabilized 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 (-NH-CHR-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 localization 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 localizations relative to each other. The nomenclature proposed by Roepstorff which is widely used to describe polypeptide fragmentation in MS is illustrated in Figure 115.

Figure 115: 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 N-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 an, bn, cn and dn (numbered from the N-terminus) while the C-terminal ions are designated xn, yn and zn (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 dn, vn and wn are due to side-chain fragmentations. There are used to distinguish residue pairs such as Leu/Ile and Gln/Lys which have identical masses.

αSyn proteins and peptides were solubilized in 1% formic acid (HCOOH) at a final concentration of 10 ÷ 30 µM and measured through direct infusion with an Esquire 3000 + ion trap device. All MS results were obtained using atmospheric pressure chemical ionization (APCI) in the positive ion mode. Mass spectra were recorded in the full scan mode, scanning from m/z 100 to 3000.

Ion source parameters were 20 psi nebulizer gas and 7 L min^-1 of drying gas with a temperature of 300°C (Table 30).

Table 30: The ESI-ion trap parameters:

Parameter Value Target Mass 1000

Compound stability 100%

Trap drive 80%

Nebulizer 20 psi

Dry gas 7 L min^-1

Dry temp 300°C

Mode positive

Scan 100 - 3000

Average scans 6