Fourier-transform Ion-Cyclotron Resonance mass spectrometry

FT-ICR mass spectrometric analysis was performed with a Bruker APEX II FT-ICR instrument equipped with an actively shielded 7T superconducting magnet (Magnex, Oxford, UK), a cylindrical infinity ICR cell. The ICR cell consists of 3 opposing pair of plates placed in the homogeneous region of the magnet. The analyte ions are trapped inside the cell by a low potential (1V) applied to the trapping plates. In a strong magnetic field along the z-direction the ions are constrained to move in circular orbits in the xy-plane referred as cyclotron motion. The frequency of the ion cyclotron motion is identical for all ions of the same m/z and is independent of their velocity as it can be seen in the cyclotron equation:

ω = zB/m

The excitation is accomplished by applying an alternating voltage to one opposing pair of plates oriented parallel to the electric field. If the frequency of the alternating electric field equals the cyclotron frequency of the ions of a particular m/z, then the

Figure 93: Schematic representation of an ICR cell used in FTMS

The rotating ion packet induces an alternating charge in the detection plates of an ICR cell and an alternating voltage across this conductor, which is amplified and registered as transient or time domain signal. After its acquisition, the time domain data is stored and subsequently Fourier transformed yielding the cyclotron frequency spectrum, which is converted to the mass spectrum. A very good vacuum (10^-9) is required to avoid collisions of the ion packet in the cell with neutral molecules that could lead to a decrease of the radius.

3.9.2.1 MALDI-FT-ICR mass spectrometry

MALDI-FT-ICR mass spectrometry was performed with the Bruker APEX II FT-ICR mass spectrometer using and an external Scout 100 fully automated X-Y target stage MALDI source with pulsed collision gas. A schematic representation of the APEX II FT-ICR mass spectrometer equipped with MALDI source is depicted in the Figure 94.

The MALDI target is a circular steel plate with 25 or 49 sample placement spots. This plate is placed on the end of a cylindrical target manipulation rod and held in place by a magnet. The rod can be inserted into or removed from the mass spectrometric vacuum system. In the inserted position, the target plate is fixed at 1 mm distance from the hexapole ion guide. The pulsed nitrogen laser was operated at 337 nm and ions generated in the MALDI process were cooled with the pulsing collision gas and

B x y

z

Excite

Detect

B x y

z

Excite

Detect

Figure 94: Schematic representation of Bruker APEX II FT-ICR mass spectrometer equipped with MALDI source

Trapping of the ions into the analyzer cell was performed applying a small, symmetric positive voltage (1-1.2 V for positive ions) to the trapping plates. In order to detect the trapped ions, they were excited applying an oscillating electrical field. The image current generated by the coherent motion of the ions in the ICR cell was detected, amplified and digitized yielding a time domain signal or transient. After its acquisition, the time domain data was stored and subsequently Fourier transformed yielding the cyclotron frequency spectrum, which was then converted to the mass spectrum.

A 100 mg ml^-1 solution of 2,5-dihydroxybenzoic acid (DHB) in acetonitrile:0.1%

trifluoroacetic acid in water (2:1) was used as the matrix. 0.5µl of matrix solution and 0.5 µl of sample solution were mixed on the stainless steel MALDI target and allowed to dry in ambient air. External calibration was performed prior to each measurement using the monoisotopic masses of singly protonated ion from the standard peptide mixture (s. Table 17). Acquisition and processing of spectra were performed with XMASS software (Bruker Daltonik, Bremen, Germany).

Scout 100 MALDI source Superconducting

magnet

Scout 100 MALDI source Superconducting

magnet

3.9.2.2 Nano-ESI-FT-ICR mass spectrometry

3.9.2.2.1 Production of gold-coated nanospray capillaries

Nanospray capillaries were manufactured using borosilicate glass capillaries of the type GC120F-10 from Clark Electromedical Instruments (Pangbourne, UK), which were pulled with a microcapillary puller P-97 from Sutter Instrument Co. (Novato, CA, USA) ) in a two step cycle. The parameters for the first step were: heat: 315, pull strength: 100, velocity: 10, cooling time: 200, and for the second step: heat: 295, pull strength: 200, velocity: 30, cooling time: 225. The nanospray capillaries were then coated with gold using a Polaron SC7610 Sputter-Coater (VG Microtech, Uckfield, UK).

3.9.2.2.2 Nano-ESI-FTICR MS analysis

The nanospray capillary was loaded with 0.5–5 µL of sample solution using a GELoader tip or by dipping the capillary tip into the sample solution, in order to avoid plugging due to solvent impurities. The loaded nanospray capillary was fixed in the metal mounting of the x,y,z-nanospray system and then placed in front of the glass capillary entrance of the 7T Bruker Daltonik APEX II FTICR mass spectrometer. The voltage at the capillary tip was manually adjusted to 700–1200 V. The mass spectra were obtained by collecting 32-128 single scans. Experimental conditions: capillary exit voltage (∆CS): 45-70 V; setting of skimmer 1: 10; setting of skimmer 2: 7; RF amplitude: 500; ionisation pulse time: 2500 ms; ionisation delay time: 0.001 s;

excitation sweep pulse: 1.2 ms; excitation sweep attenuation 1: 2.16 dB. Acquisition and processing of spectra were performed with XMASS software (Bruker Daltonik, Bremen, Germany). External calibration was carried out using monoisotopic masses of angiotensin I fragment ions formed by in-source fragmentation (∆CS = 150V).