LC, HPLC, RPLC
Liquid chromatography (LC), more specifically referred to as high-performance liquid chromatography (HPLC) or reversed-phase liquid chromatography (RPLC) is used as a 'front-end' chromatographic fractionation method in mass spectrometry-based proteomics. This process, often called LC-MS, separates peptides according their hydrophobicity before they are introduced to the mass spectrometer, typically resulting in much higher detection rates than does the direct analysis of protein samples by MS.
ESI
ESI, or electrospray ionization, is a 'soft' ionization technique used in mass spectrometry for producing charged ions from proteins, peptides and other nonvolatile macromolecules (also see MALDI). It is considered 'soft' because fragmentation during the ionization process is minimal. Electrospraying involves using high voltage to charge the liquid containing the analyte, which forces the liquid through a small capillary tube to form an aerosol mist of charged droplets. The solvent evaporates, leaving behind multiply charged molecular ions that move to the mass analyzer for detection. Because it is liquid-based, ESI is easily coupled to front-end fractionation by liquid chromatography (known as LC-MS), and as such, ESI is the most widely used ionization technique for proteomic studies. John Fenn was honored with part of the 2002 Nobel Prize in Chemistry for his development of ESI for the ionization of proteins.
MALDI
MALDI, or matrix-assisted laser desorption/ionization, is another 'soft' ionization technique (also see ESI) for ionizing nonvolatile biological macromolecules intact. Peptides or proteins are mixed with a matrix (often a weak organic acid), deposited on a special plate and irradiated with a pulsed laser. As the matrix absorbs energy, the mixture is heated and expands, leading to the ionization of the analyte and its transfer into the gas phase. MALDI is most often combined with a TOF mass analyzer, and unlike ESI, it cannot be easily coupled with liquid chromatography. Some interesting biological applications such as the proteomic analysis of intact tissues are facilitated by MALDI. Koichi Tanaka was honored with part of the 2002 Nobel Prize in Chemistry for showing that proteins can be ionized by MALDI.
MS1
MS1 refers to the initial mass-to-charge-ratio (m/z) spectrum collected for all components in a sample. Molecular ions can be selected for further analysis by fragmentation (see MS/MS).
MS/MS
In MS/MS, also called tandem MS or MS2, selected molecular (precursor) ions from the MS1 stage are dissociated into fragments (see CID, CAD, ECD and ETD). The fragment ion masses are measured, providing further information about the molecular structure of the precursor ion. A second mass analyzer is often required, hence the term 'tandem MS'.
MS/MS/MS, MS3, MSn
MS/MS/MS, also called MS3 or sometimes MSn, subjects the fragment ions themselves to further fragmentation steps. More fragmentation gives even more information about the molecular structure of the precursor ion, but at a cost of reduced sensitivity as well as increased analysis time. MS/MS/MS can typically only be performed on abundant peptides.
CID, CAD
Collision-induced dissociation (CID), also known as collisionally activated dissociation (CAD), is the most common fragmentation method used in tandem MS. Molecular ions are collided with inert gas molecules, causing the ions to fragment into smaller pieces. When peptides fragment at amide bonds, the resulting 'ladder' of ions can be used to deduce the amino acid sequence.
ECD
Electron capture dissociation (ECD) is a newer fragmentation method that uses low-energy electrons to fragment molecular ions. ECD results mainly in peptide backbone fragmentation (also see ETD), leaving many post-translational modifications intact. As such, it is a useful method for looking at whole proteins or large peptide fragments by 'top-down' mass spectrometry. It is performed mainly on Fourier-transform mass spectrometers (see FTMS).
ETD
Electron transfer dissociation (ETD) is a recently developed ion excitation method that uses free radical anions to fragment molecular ions. Like ECD, ETD results mainly in peptide backbone fragmentation, leaving many post-translational modifications intact, and as such is useful in 'top-down' approaches. Unlike ECD, however, ETD may be used on more common and cheaper instruments such as quadrupole ion trap instruments, though it is not yet widely available.
TOF
Time-of-flight (TOF) instruments are the simplest and least expensive mass analyzers. Ions are accelerated to a uniform kinetic energy, then introduced into a field-free tube. The velocity at which they travel to reach the detector is proportional to the reciprocal square root of m/z. TOF instruments are typically very fast and cover a very wide m/z range, but are less sensitive than ion trap instruments. TOF instruments can operate in tandem mode, with a second mass analyzer monitoring fragment ions (TOF/TOF).
Quadrupole
The quadrupole mass analyzer uses four parallel rods with fixed direct current and alternating radio-frequency potentials to selectively focus ions for transmission through the rods according to their m/z ratio. Triple quadrupole and quadrupole-quadrupole-TOF (QqTOF) instruments are particularly well suited for MS/MS because they permit efficient ion selection by a quadrupole, fragmentation within the collision cell, and fragment ion detection by quadrupole or TOF mass analyzers. The mass accuracy of QqTOF instruments is typically higher than that of triple quadrupoles.
Ion trap
Ion trap instruments isolate ions within a three-dimensional (quadrupole ion trap) or rectangular (linear ion trap) storage cell surrounded by electrodes of opposite polarity. The radio-frequency and direct-current potentials are varied to eject ions of varying m/z from the storage cell to a detector, allowing mass detection as well as ion selection and fragmentation to be carried out by the same cell. Detection sensitivity can be enhanced by filling the trap for longer periods. Fragmentation within quadrupole ion traps is usually efficient, but low-mass ions are not efficiently trapped.
Orbitrap
The Orbitrap is a new, high-resolution mass analyzer that electrostatically maintains ions in orbit around a central electrode. Ion masses are measured from the frequencies within the image current generated by the orbiting ions. Orbitraps have high mass accuracy, high sensitivity, and good dynamic range detection capabilities, and as such have become very popular tools for proteomics.
FTMS
Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS, or just FTMS) maintains ions moving in circles within a static magnetic field. Ion masses are measured by detecting their cyclotron frequencies within the image current; the frequency of ion cycling is inversely proportional to m/z ratio. Fragmentation can be carried out by several ion excitation methods. Like the Orbitrap, FTMS instruments have very high resolution, though their sensitivity is not as high as that of ion trap instruments.
ICAT
Isotope-coded affinity tagging (ICAT) uses isotopic labeling to enable relative quantitative comparisons between two proteomic samples. The ICAT tag contains a cysteine-reactive group, an isotopically encoded linker, and a biotin tag to facilitate affinity purification. One sample is thus labeled with a 'heavy' ICAT tag (isotopically encoded), while the other is labeled with a 'light' ICAT tag (not isotopically encoded). The samples are combined and digested, and the tagged peptides are captured with (strept)avidin affinity purification. The captured peptides are then subjected to MS analysis; peptides that show differential abundance between the two samples may be further analyzed by MS/MS.
iTRAQ
Like ICAT, isobaric tagging for relative and absolute quantification (iTRAQ) uses isotopic labeling to enable relative quantitative comparisons. Up to four different proteomic samples can be labeled using four different isobaric tags. The iTRAQ tags derivatize N-terminal amines and lysine residues and therefore label more peptides than the ICAT tag, which only labels the less common cysteine residues. During MS/MS, the iTRAQ tags are cleaved to produce fragments of different molecular masses (from m/z 114-117) that report the relative abundances of peptides from each sample.
SILAC
Stable isotope labeling with amino acids in cell culture (SILAC) is a method to metabolically label proteins for relative quantitative comparison. One cell population is fed amino acids of normal isotopic composition; the other cell population is fed amino acids labeled with heavier isotopes. The heavy amino acids are incorporated into newly synthesized proteins, eventually completely replacing the cells' proteins, such that labeling efficiency is near 100%. The cell populations are then mixed together and digested for MS analysis to determine differential protein abundances.
