Mass spectrometry

The principle of time-of-flight analysis is based on accelerating a set of ions to a detector with the same amount of energy. Ions have the same energy, but a different mass, so that ions reach the detector at different times. The smaller ions will reach the detector faster, because of their greater velocity, while the larger ions take longer time to reach the detector. Thus, the analyzer is called time-of-flight, because the m/z is determined from the ions time of arrival. The arrival time of an ion at the detector is dependent upon the mass charge, and kinetic energy of the ion. In the present work, MALDI-time-of-flight experiments were performed by using a Bruker (Bruker Daltonik, Germany) Biflex ITM linear TOF mass spectrometer with a SCOUT-26-ionization source video system, nitrogen UV laser (337 nm), and a dual channel plate detector. As matrix a saturated solution of α-cyano-4-hydroxy-cinnamic acid (HCCA) in ACN/0.1 % trifluoroacetic acid in water (2:1 v/v) was used. Aproximately 0.8 µl of the sample solution and saturated matrix solution were mixed on the stainless steel MALDI target and dried prior MS measurements. Acquisition of spectra was carried out at an acceleration voltage of 20 kV and a detector voltage of

1.5 kV. An external calibration was performed using the average masses mixtures of singly protonated ion signals of bovine insulin (5734.5 Da), bovine insulin B-chain oxidized (3496.9), human neurotensin (1673.9 Da), human angiotensin I (1297.5 Da), human bradykinin (1061.2) and human angiotensin II (1047.2 Da).

3.8.2 MALDI – FTICR MS

FTICR- Mass spectrometric analysis was performed with a Bruker APEX II FTICR instrument equipped with an actively shielded 7 T superconducting magnet, a cylindrical infinity ICR analyzer cell, and an external Scout 100 fully-automated X-Y target stage MALDI source with pulsed collision gas (Bruker Daltonik, Bremen, Germany) (Figure 3.68). The pulsed nitrogen laser is operated at 337 nm, and ions are directly desorbed into a hexapole ion guide located one mm from the laser target.

The device for pulsing collision gas in direct proximity to the laser target provides cooling of the ions, which have a kinetic energy spread of several electron volts, when produced by the MALDI process. These ions are trapped in the hexapole, where are positive potentials at the laser target and at an extraction plate to help trap ions along the longitudinal axis. After a predefined trapping time, the voltage of the extraction plate is reversed and the trapped ions are extracted for transmission to the ICR cell.

Accumulation of ions from multiple laser shots in the hexapole before mass spectrometric analysis increases the sensitivity. Ions generated by ten laser shots are accumulated in the hexapole for 0.5–1 s at 30 V and extracted at -15 V into the analyzer cell. A 100 mg/mL solution of 2,5-dihydroxybenzoic acid (DHB; Aldrich, Steinheim, Germany) in acetonitrile: 0.1 % TFA in water (2:1) was used as the matrix. Aliquots of 0.5 µL of matrix solution and 0.5 µL of sample solution were mixed on the stainless-steel MALDI sample target and allowed to dry.

Scout 100 MALDI source Superconducting

Scout 100 MALDI source Superconducting

magnet

Figure 3.68. General scheme of Bruker APEX II FT-ICR MS equipped with MALDI source

3.8.3 ESI - Ion Trap MS/MS analysis

LC-MS Analysis was performed on an Esquire 3000+ mass spectrometer (Bruker Daltonik, Bremen, Germany) coupled to an Agilent 1100 binary pump system (Figure 3.69). LC Separations were performed on a 1 mm × 10 cm Discovery Bio Wide Pore 3 µm C18 column (Sigma-Aldrich, Germany) using a flow rate of 50 µl/min.

Peptides were eluted using a linear gradient from 98 % solvent A (0.2 % formic acid in water, (v/v)) and 2 % solvent B (0.2 % formic acid in acetonitrile, (v/v)) to 55 % solvent B over 90 minutes. The electrospray source was operated in the positive ion mode with the following parameters: capillary voltage, 4.0 kV; capillary exit, 120 V.

Nitrogen was used as both nebulizing (20 psi) and drying gas (9 L/min). The desolvation temperature was 300^oC. For MS/MS analysis, the instrument was operated in the data-dependent mode such that a full scan spectrum was obtained followed by X MS/MS spectra obtained for the X largest ions observed in the proceeding full scan spectrum with an abundance above a specified threshold. Each MS was the sum of 6 microscans, and 20 microscans were collected for each MS/MS scan, for a maximal accumulation time of 200 ms. The intensity threshold triggering ion selection was 2 × 10⁴. For MS/MS acquisition, the SmartFrag function of the ion trap was used, which ramps the fragmentation amplitude from 30 % to 200 % of the

preset value (1.10 V). Eluting peptides were analysed in the data dependent MS/MS mode over a 300-1200 m/z range. All peptides were selected for MS/MS analysis by collision-induced dissociation. Mass spectra were evaluated using the DataAnalysis 3.3 software package (Bruker Daltonics). MS/MS spectra were searched with SwissProt database using Mascot search algorithm allowing one missed cleavage site; Carbamidomethyl was taken as fixed modification and oxidized methionine as variable modification.

Figure 3.69. General scheme of the Bruker Esquire 3000 ion trap mass spectrometer

3.8.4 Linear ion trap (Orbitrap) mass spectrometry

Measurements were performed in collaboration with Dr. Andreas Marquardt, Proteomics Facility, University of Konstanz. Peptide mixtures were analyzed reversed phase liquid chromatography nanospray tandem mass spectrometry (LC-MS/MS) using an LTQ-Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) and an Eksigent nano-HPLC (Eksigent, Dublin, USA). The dimensions of the reversed-phase LC column were 3 µm, 100 Å pore size C18 resin in a 75 µm i.d. × 15 cm long piece of fused silica capillary (Dionex). Separation was performed at a flow rate of 0.3 µl/min. using 0.1% formic acid (v/v, solvent A) and 100%

acetonitril/0.1% formic acid (v/v, solvent B). The column was pre-equilibrated with 10% (v/v) solvent B, and peptides were eluted using a linear gradient from 10 to 40%

(v/v) of solvent B in 65 min., then to 80% B in an additional 5 min. Mass spectrometric analysis was performed on a LTQ-Orbitrap mass spectrometer equipped with a nanoelectrospray ion source (Proxeon Biosystems). Data were aquired in a data-dependent mode using Xcalibur (Thermo Finnigan, San Jose, CA, USA) software. The precursor ion scan MS spectra (m/z 380-1,800) were aquired with resolution of R = 30000 at m/z 400 (number of accumulating ions 5 x 10⁶). The five most intense ions were isolated and fragmented using 3x10⁴ accumulated ions.

Dynamic exclusion was allowed. The lock mass option using ions generated in the electrospray process from ambient air (protonated Bis (2-ethylhexyl) phlatate, m/z 391.284284) was used for internal calibration in real time. The Mascot Server 2.2 (Matrix Science, London, UK) software was used for Mascot database searching. Up to one missed cleavage was allowed, mass tolerance for protein identification was 3 ppm for MS and 0.5 Da for MS/MS. Identification of modified or non-specifically cleaved peptides was obtained by error tolerant search of all significant protein hits.

Determination of ion intensities was performed with Xcalibur software from extracted MS ion chromatograms (peak area) of the accurate peptide m/z values ±5ppm. The LTQ-Orbitrap mass spectrometer was operated in a data dependent mode in which each full MS scan (30,000 resolving power) was followed by five MS/MS scans where the five most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35 % in the LTQ ion trap. Sequence information for each peptide can then be gained by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), where a peptide ion is selected and collided with an inert gas. The m/z of the resulting fragment ions is then measured. Normally, the low-energy collision-induced cleavage of the peptide backbone occurs at the amide bond giving rise to y- and b-type ions that contain the C- and N-terminal part of the peptide, respectively.

CID fragmentation of multiply charged ions can also give rise to multiply charged fragment ions and it is not uncommon for multiply charged ions to lose one or several N-terminal amino acid residues as neutral fragments [232]. The nomenclature for the peptide fragmentation [233] is illustrated in Figure 3.70.

Figure 3.70. Collision induced dissociation (CID) peptide fragment ion nomenclature. (A) The indices of the amino terminal containing a-, b-, and c-ions denote the number of residues counted from the N-terminus, while the indices of the carboxy terminal x-, y-, z-ions are counted from the C-terminus. (B) Linear b-ions are unstable and cyclize to form an oxazolone structure involving the carbonyl group of the adjacent residue

3.9 Bioinformatic tools for proteome analysis