Since the first description of a mass spectrometry (MS) experiment in the early 1900s by Thomson [1], MS-based approaches have come a long way from being narrowly applied in analytical chemistry to a key device for clinical testing. The description in the late 1980s of two different soft ionization methods named Matrix-Assisted Laser Desorption/Ionization (MALDI) [2, 3] and ElectroSpray Ionization (ESI) [4] (Fig. 61.2) significantly accelerated MS-based interrogation of large biologically important molecules. The principle of operation of MALDI-based proteomics involves the co-crystallization of a protein/peptide sample on a designated matrix medium (Fig. 61.2a). The vaporization and ionization of the protein/peptide sample is achieved by pulse-irradiation of the surface with a laser. This process generates ions that are accelerated into the mass analyzer for analysis, filtering, and detection. By comparison, the ESI process involves the transfer of ions from solution to gaseous phase at atmospheric pressure (Fig. 61.2b). The application of a very high voltage across a sample solution at very low flow rates in a capillary needle causes ions to accumulate at the narrow tip of the needle where they are then drawn out as the sample solution is pushed out of the needle creating charged droplets that are accelerated in an electric field. Via the Coulomb explosion, large charged droplets are sequentially blown apart to form smaller droplets upon reaching a certain limit leaving only the discrete peptide sample ions to enter the mass analyzer.

After ionization the sample reaches the mass analyzer which separates ions by their mass-to-charge (m/z) ratios. Ion motion in the mass analyzer can be manipulated by electric or magnetic fields to direct ions to a detector which registers the number of ions at each individual m/z value. Different types of mass analyzers are available: time-of-flight (TOF), ion trap, and quadrupoles. These mass analyzers differ considerably in sensitivity, resolution, mass accuracy, and the possibility to obtain fragment peptide ions which results in mass spectra with high content of information (MS/MS spectra). The choice of the combination of the ion source with the mass analyzer and the detector depends on the specific application.

Mass spectrometry-based proteomic analyses can be divided into top-down or bottom-up processes. In top-down proteomics, intact proteins or polypeptides are directly analyzed by mass spectrometry. On the other hand, bottom-up proteomics entails digestion of complex protein mixtures into peptides using a proteolytic enzyme (typically trypsin). Resulting peptides are separated by liquid chromatography (LC) and analyzed by tandem mass spectrometry (MS/MS). Proteins are identified by matching experimental spectra with those from theoretical spectra of translated genomic databases generated by in silico cleavage using specific enzymes (Fig. 61.3).

Quantitative measurement of protein concentrations is one of the key advancements in MS-based proteomics. Global quantitation of protein levels can be achieved by stable isotope labeling of proteins/peptides, use of heavy peptides as standards, and label-free quantitation (Fig. 61.4). Isotope labels can be introduced metabolically (i.e., in vivo), chemically, or enzymatically (i.e., in vitro). Since the metabolic labeling (stable isotope labeling with amino acids in cell culture, [SILAC]) requires maintaining cells in culture for several passages (usually six or seven passages) in the presence of stable isotopes of amino acids (heavy Arg, Lys, Leu, or Ile), its direct application to clinically relevant scenarios is less likely. The in vitro methods for labeling include isotope-coded affinity tagging (ICAT) and isobaric tags for relative and absolute quantitation (iTRAQ). These methods may be adaptable to clinical scenarios since cell viability is not required for labeling. In the original ICAT labeling method, cysteine residues were specifically derivatized with a reagent containing either zero or eight deuterium atoms as well as a biotin group for affinity purification of cysteine-derivatized peptides and subsequent mass spectrometry analysis. The iTRAQ method is another in vitro labeling reagent which targets the N-terminus amino acid of peptides with a varying mass. The AQUA (absolute quantification) quantitative approach uses isotope-labeled synthetic peptides as standards spiked at a known amount into a sample preparation. The quantitation is achieved by comparing the mass spectrometric signal of the synthetic peptide to the endogenous peptide in the sample. Label-free quantitation methods are gaining in popularity and are increasingly being employed as an alternative to label-based approaches. These techniques do not use isotopic labeling, but instead directly compare signal intensities across different mass spectrometry runs using either the signal intensity of peptide precursors or the number of fragment spectra identifying peptides of a given protein.