MS instrumentation used in proteomics. The fundamental principle of MS analysis involves the conversion of the subject molecules to either cations or anions in the ion source, separation according to their mass/charge (m/z) ratios in the mass analyzer, and subsequent detection. Several configurations of mass spectrometers that combine ES and MALDI with a variety of mass analyzers (linear quadrupole mass filter [Q], time-of-flight [ToF], quadrupole ion trap, and Fourier transform ion cyclotron resonance [FTICR] instrument) are routinely used. (A) In MALDI, the sample is embedded in a large excess of a matrix that has a strong absorption at the laser wavelength. Following the laser irradiation of the sample surface, the matrix accumulates a large amount of energy that is thought to initiate the proton transfer between the matrix and the analyte compound to form ions. The ions observed during MALDI MS are mainly singly charged. This results in simple spectra (because the m/z ratio is what is being measured) even in the case of analysis of mixtures but can be a disadvantage for peptide sequencing, which requires the achievement of peptide fragmentation, a process that preferentially occurs with multicharged peptides. The concurrent and independent development of ToF mass analyzer and its compatibility with this ionization method resulted in the rapid development of MALDI-ToF as a routine analytical mass spectrometer.⁹²  Other configurations, however, have also been successfully applied for the analysis of peptides and proteins, such as MALDI-ion trap,⁵⁰  MALDI-QqToF⁵²  (where q represents a quadrupole ion decomposition region), and MALDI-ToF-ToF.⁵¹  This type of ionization method is also used in SELDI-ToF MS. (B) ES is a continuous nebulization process that produces ions directly from solution and facilitates interfacing of LC with MS. A sample is passed with flow rates of 1 to 10 μL/min through a capillary held at high potential relative to ground and counter electrode. The strong electric field obtained induces charge accumulation at the liquid surface situated at the end of the capillary and leads to the formation of a mist of highly charged droplets. The ES process results in the formation of multiply charged ions. This is one of the principal advantages of this method because it allows the analysis of ions from high-molecular-mass molecules, such as proteins and peptides, using mass spectrometers of limited m/z ratio range. (C) The quadrupole analyzer is frequently termed a mass filter because it transmits only ions within a narrow m/z range. The analyzer uses the stability of the trajectory to separate these ions according to their m/z ratio. The stability of the oscillating trajectories of the ions is based on the joint application of direct current and radio-frequency voltages on 4 parallel cylindrical metal rods. (D) Unlike the scanning devices such as quadrupoles, ToF analyzers separate ions temporally rather than spatially. The ions are rapidly accelerated into a “field-free” drift region also called a “flight tube,” and their separation is achieved by measuring the difference in transit time from the ion source to the detector. The reflectron, which consists of a series of rings or grids that act as an ion mirror, compensates for the initial kinetic energy distributions of ions. (E) The ion trap analyzer captures the ions, which collide with the helium “bath” gas and start to oscillate in a predicted motion. Ion trap can be used as a “tandem-in-time” instrument as selection, fragmentation, and analysis of ions take place in the same space. Fourier transform ion cyclotron resonance mass spectrometer (FTICR; not illustrated) is also a trapping device, in this case by using strong magnetic fields, and offers great opportunities for investigating protein interactions and PTMs, with high sensitivity, mass accuracy, and resolution.⁹³