Tandem mass spectrometry

Structure information by specific fragmentation of gas-phase ions of biomolecules can be obtained by several MS/MS techniques. Tandem MS consists of activation of a primary precursor ion by addition of excess internal energy to this ion, dissociation, followed by mass analysis of the product ion.

FTICR, Ion trap and Triple quadrupole MS, provides multistage MSⁿ, simultaneous detection of a wide mass range of ions, and offers different excitation and fragmentation methods (CID, SORI, IRMPD, ECD). The choice of a protein fragmentation path depends on the purpose of the fragmentation and the already known characteristics of the protein. Fragmentation pattern for a given protein is highly reproducible and specific.

The types of fragment ions observed in an MS/MS spectrum depend on many factors including primary structure, internal energy, how the energy was introduced, and charge state. A generally accepted nomenclature for fragment ions is the system proposed by Roepstorff and Fohlman [225]. Fragments can only be detected if they carry at least one charge. The cleavage pattern of a bond in the peptide chain backbone can occur in either of three types of bonds Cα-C, C-N, or N-Cα, which yields six types of fragments that are labelled an, bn, cn, when the positive charge is kept on the N-terminal part and xn, yn, zn, when the positive charge is kept on the C-terminus part [226].

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 ions. The ions obtained by IRMPD and CID are mostly b and y ions and are marked with a red square.

3.9.3.1 FT-ICR MS/MS analysis

Once ions have been formed (by either MALDI or ESI ionization methods) and trapped in the analyzer cell, isolation of a precursor ion was achieved by ejecting from the cell all ions of higher and lower masses through the application of suitable excitation pulses with the appropriate frequencies and amplitudes. Parameters used for isolation of precursor ions were: correlated sweep attenuation: 8-10 dB, ejection safety belt: 500 -1000 Hz.

Infrared multiphoton dissociation (IRMPD) provides a greater selectivity than collision induced dissociation and appears to dissociate more stable ions [227]. With this

firing a relatively high powered IR laser through the center of the cell and, slowly increasing the dissociation ladder. For a typically applied CO2 (IR) laser (10.6 µm), the steps in energy are 0.12 eV. The laser power was set to 50% and the laser irradiation time to 50-200 msec.

3.9.3.2 nano-ESI-triple-quadrupole-linear ion trap MS/MS analysis

MS/MS analysis was conducted on an Applied Biosystems/MDS SCIEX QTRAP^TM, a hybrid triple-quadrupole – ion trap mass spectrometer equipped with a Flow NanoSpray^TM source. This instrument is based on a triple quadrupole ion path and is capable of all conventional tandem quadrupole scan modes as well as several high sensitivity ion trap mass spectrometer scans using the final quadrupole as a linear ion trap (LIT) MS. The enhanced scan modes (Enhanced Product Ion, Enhanced Resolution and Enhanced MS) use the “trap” capabilities of the instrument to improve sensitivity, resolution, and mass accuracy.

Enhanced Product Ion (EPI) scan is performed when a precursor ion that is resolved through the first quadrupole (Q1) collides with nitrogen gas in the quadrupole collision cell (Q2). The product ions are transferred to Q3 where they are trapped by the Q2-Q3 interquad lens and an exit lens after Q3. After the Q3 LIT has been filled, the ions are resonantly ejected through the exit lens to the ion detector. Scan speeds available are 250, 1000 and 4000 Da/s. Best resolutions are achieved at 250 Da/s scan speeds. A schematic representation of the hybrid triple-quadrupole-linear ion trap mass spectrometer working in the EPI scan mode is shown in Figure 96.

Figure 96: Schematic representation of the hybrid triple-quadrupole ion trap mass spectrometer working in Enhanced Product Ion (EPI) scan mode.

Enhanced Resolution (ER) scan is a narrow range scan used to determine charge states and accurate monoisotopic masses of precursor ions for MS/MS. In Enhanced Resolution scan mode, selected ions are filtered through Q1 and passed through Q2 without fragmentation. They are then trapped in the Q3 LIT, scanned slowly out of the trap (250 Da/s scan speed) and detected. In Enhanced MS (EMS) scan mode the ions are passed through Q1 and Q2 without fragmentation, trapped in the Q3 LIT for a specified time and then scanned out and detected.

Gold-coated nanospray capillaries were manufactured as described before. The nanospray capillary was loaded with 4 µL of desalted sample solution using a GELoader tip. The loaded capillary was fixed in the metal mounting of the NanoSpray source and then pushed forward into the ion source region. The voltage at the capillary tip was manually adjusted to 900 V. Automated MS/MS analysis was performed utilizing the Information Dependent Acquisition (IDA) feature of Analyst®

software 1.3.2. For each cycle of IDA experiment, a survey spectrum was first acquired using Enhanced MS, and then m/z values of three most abundant ion peaks were automatically selected and measured in Enhanced Resolution scan mode (to get the charge state and accurate monoisotopic mass). MS/MS analysis of these three ions was then performed using Enhanced Product Ion scan mode. After each cycle, the analysed m/z values were automatically added to a dynamic exclusion list.

Precursor ion selection

Ion accumulation Fragmentation Trap & Scan

N₂CAD Gas

Linear Ion Trap 3x10^-5Torr

Q1 Q2 Q3

Q0

Precursor ion selection

Ion accumulation Fragmentation Trap & Scan

N₂CAD Gas

Linear Ion Trap 3x10^-5Torr Linear Ion Trap

3x10^-5Torr

Q1 Q2 Q3

Q0

3.9.3.3 ESI -Ion Trap MS/MS mass analysis

The API-ESI (Atmospheric Pressure Interface-ElectroSpray Ionization) generates ions, focuses and transports them into the ion trap mass analyser. The spray chamber is outside the vacuum manifold. The ion transport and focusing components are located inside the vacuum manifold. 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.

ESI-MS and MS/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 3 uL/min. The peptides were dissolved at a

Capillary

Figure 97: Schematic representation of the Bruker Esquire 3000 ion trap mass spectrometer

MS/MS experiments were isolated using an m/z window of 2 Da, and the fragmentation amplitude was optimized within the range 0.5-1.3 V, depending on the fragmentation efficiency of each individual peptide precursor ion.