MS is one of the most common technologies used in proteomics (see reviews5–7) and refers to an instrumental method for identifying the chemical constitution of a substance by means of the separation of gaseous ions according to their differing mass and charge (Fig 1
). Mass spectrometers can be used for profiling both peptides and small and large proteins. In a typical protein identification workflow, a protein typically is first digested using a proteolytic enzyme (eg, trypsin) that cleaves reproducibly at arginines and lysines. The resulting peptides are then ionized to produce charged (protonized) molecules, and travel through a mass analyzer and then to a mass detector. The two ionization techniques most commonly used are (1) matrix-assisted laser-desorption ionization (MALDI) and (2) electrospray ionization (ESI). With MALDI, proteins and peptides are mixed with an energy-absorbing matrix (eg, cinnamic acid) and then are ionized using a laser. With ESI, a sample is introduced in liquid form, and the application of a very high voltage forms a fine spray through a hypodermic needle to ionize peptides. An electromagnetic field causes the ionized peptides to travel through the mass analyzer to a detector. Mass analyzers include time-of-flight (TOF), ion traps, Fourier transform and quadrupoles or combinations (eg, quadrupole-TOF). In TOF MS, the time that it takes the peptide to reach the detector is converted into a mass/charge ratio and is visualized as a mass spectrum. MALDI is generally paired with TOF analyzers (MALDI-TOF). This is excellent for high-throughput studies and is somewhat forgiving of contaminants such as salts, although only single proteins or very simple mixtures can be analyzed. In tandem MS using an ion trap, peptides of a single mass are “trapped” in an electromagnetic field, fragmented, and the resulting fragments are detected and a second mass spectrum is formed. Similarly, fragmentation can occur in a quadrupole prior to TOF during a tandem MS experiment in a quadrupole TOF. Because the fragmentation of a peptide often results in sequence information, a single, high-quality peptide can often be used to identify a protein. In addition, this information can be used to localize posttranslational modifications. However, long run times make these instruments medium-throughput to low-throughput devices, and ESI is generally not as tolerant of salts as MALDI. Therefore, a typical workflow may include 2-DE followed by MALDI-TOF for high-throughput, single-gel-spot analyses. When additional sensitivity, complex sample analysis, or sequence information is required, ESI-MS/MS can be used.