Table 1.

Glossary of MS terminology.

Term	Explanation
Biomarker	A characteristic that is used as an indicator of normal biological processes, pathogenic processes or responses to a therapeutic intervention.
Mass spectrometry (MS)	The basic principle underlying MS is the identification of peptides or proteins based on separation by mass and charge. Peptides or proteins are ionised, accelerated and usually deflected by a strong electromagnetic field, such that they reach a detector at different times based on their mass and charge. Detection of a peptide or protein is recorded as a peak, and used to determine the peptide sequence and identity. This may also be termed peptide mass fingerprinting, or peptide sequencing.
Types of Ionisation; ESI, MALDI and SELDI.	Introduction into the MS of sample containing proteins or peptides of interest requires ionisation. Two key methods include 1. electron spray ionisation 'ESI' and 2. laser desorption ionisation (matrix-associated ‘MALDI’ and surface-enhanced ‘SELDI’). MS involves different combinations of ionisation and mass analysis, see below.
Tandem MS; MS2 and MS3	MS may also involve the fragmentation of peptide or protein ions, such that a precursor peak is recorded along with fragment peaks (MS2) or fragments of fragments (MS3). This improves selectivity, identification and detection.
Types of Mass Analysers; Quadrupole, ToF and Orbitrap	Mass analysers separate peptide or protein ions within the MS. Two key methods include 1. quadrupole, four metal rods with alternating electromagnetic fields, and 2. time of flight, based on time alone. 3. Orbitrap, based on ionised ions rotate around a central rod at high voltage and vacuum. MS may involve a single or combination of methods.
Top-down vs. Bottom-up	Prior to MS, samples may be digested with an enzyme (usually trypsin) into peptide fragments. This is termed bottom-up MS. In contrast, if there is analysis of intact proteins or it is termed top-down MS. Bottom-up is more common, however there are instances when top-down is required, for example for analysis of proteins in their native structure.
Unbiased vs Targeted (e.g. PRM, SRM, MRM)	MS performed to identify specific pre-selected proteins or peptides is termed targeted, whereas discovery MS performed to identify any/ novel proteins or peptides is termed unbiased or shotgun 'you don't need to know what you are looking for'. Targeted MS is usually more sensitive than unbiased, and also allows high-throughput, e.g. parallel reaction monitoring (PRM), selected reaction monitoring (SRM) and multiple reaction monitoring (MRM).
Dynamic range	The range in concentrations of proteins present in a sample. This may vary by many orders of magnitude, leading to difficulties in identifying low abundant proteins of interest.
Sample preparation	Steps involved in preparing a sample prior to introduction in the MS. This usually involves disassembly of the 3D structure with denaturation (unfolding; e.g. urea), reduction (breaking disulphide bonds in the secondary structure e.g. dithiothreitol) and alkylation (capping free thiol chains to prevent reformation of disulphide bonds). The protein sheets are then digested into smaller (e.g. 9 amino acid chain) peptide fragments.
Depletion	Removal of high abundant proteins (e.g. albumin and IgG), to improve MS identification of low abundant proteins of interest. Typically involves immunodepletion, i.e. antibody methods.
Pooling	Combining several samples in a single analysis.
Labelling	Addition of isobaric mass tags to peptides during sample preparation, e.g. iTRAQ or TMT, to enable the analysis of multiple samples in a single MS run. This allows relative quantitation of peptides or proteins, reduces the time and obviates bias due to inter-run variation.
Separation and Fractionation; Liquid-chromatography (LC)	In order to deal with the high linear dynamic range in clinical samples, proteins are separated by various methods based on different properties of the proteins such as their mass, charge or hydrophobicity. Multidimensional separation refers to multiple methods being performed on the same sample, and orthogonal when the methods are based on different properties. These methods may be prior to digestion, such as by gel-electrophoresis, after which the gel is cut up and prepared to be loaded on the MS. There are various methods coupled with the mass spectrometry machine, e.g. liquid chromatography. e.g. strong cation exchange, high pH or low pH reverse-phase and C18 analytical column.
Functional analysis or Clustering	Identified proteins are investigated using programs that analyse the subcellular location, biochemical pathways and functions. Clustering refers to the mapping of proteins by their reported functions, to examine whether multiple proteins are involved in similar pathways, as this may strengthen evidence for their roles.