A basic mass spectrometer contains an inlet that introduces digested or whole proteins to the mass spectrometer, where they are ionized at an ion source. In the case of MALDI spectrometers, a soft ionization technique is used in which a plate or matrix bearing the proteins is hit by a laser so they may move into the gas phase without fragmentation. Macromolecules may not withstand the heat and may not be amenable to MALDI-based protein mass spectrometry. In ESI-based protein mass spectrometers, the ion source is an electrospray (5,6).
Through the mass spectrometer, the flow of charged molecules is captured by ion detectors that provide feedback to an instrument control capable of adjusting the kinetic energy (MALDI instruments), and frequency and/or voltage (ESI instruments), and measuring the m/z ratio of the molecule.
ESI-based Protein Mass Spectrometry
In ESI-based protein mass spectrometry, the electrospray promotes ion flow toward mass analyzers. Mass analyzers may contain rods (quadrupole mass filter) or ring electrodes (ion trap) surrounding the flow of ions. The polarized rods or electrodes will attract peptide cations of a certain mass-to-charge (m/z) ratio to essentially ‘trap’ them; the ions that continually flow through will be detected by a sensor called the ion detector, where the ions will be measured based on their mass, charge and the time at which they have been recorded. An example of a mass analyzer is the quadrupole (5,6).
Figure 2. Infographic of an ESI-based mass spectrometer.
While ESI-based MS instruments initially contained one mass analyzer and one ion detector, nowadays, ion cells are typically flanked by the ion source and the ion sensor and sandwiched between electrodes. The latter can modulate the frequency and voltage to ‘select’ a window for the desired m/z ratio and then redirect the flow of ions for additional fragmentation. An example of an ion cell that can act as either, or both a mass analyzer and an ion detector is the ion trap. The ion source, and mass analyzers and ion detectors are all kept under vacuum.
As peptides travel between mass analyzers and ion detectors, collisions can fragment these peptides further via high-energy collision dissociation (HCD), or electron-transfer high-energy collision dissociation (EThcD). All of the internal systems feed into the instrument control. Essentially, at any of these points, data can be detected for the user may select a specific range of ions and record the spectra of these fragments for data analysis.
A mass spectrometer that comprises different sequential cells with mass analyzer and ion detector capabilities is referred to as a tandem mass spectrometer. Mass spectrometers used to only be able to house a mass analyzer and an ion detector. However, mass spectrometry technology has evolved to such a sophisticated level that current tandem mass spectrometers house many more components than original instruments. Whereas they used to be huge, with the advent of the benchtop mass spectrometer, these instruments can now conveniently fit in one corner of the laboratory (5).