Glossary of terms

MRM

Multiple reaction monitoring. The application of SRM to multiple fragment ions produced from one or more precursor ions.

MS

Mass spectrometry. A technique for analysing ions based on their mass-to-charge ratio. Mass spectrometers consist of three core components - an ion source, one or more mass analysers and a detector. The ion source generates gaseous ions from an analyte, the mass analyser/s separate ions in space or time and the detector system records the relative abundances of ions resolved by m/z.

MS/MS

Tandem mass spectrometry. The acquisition and analysis of mass spectra collected via more than one stage of mass analysis. Precursor ions are fragmented prior to the second stage of MS analysis.

MS1

Mass spectrometry survey scan. In data-dependent MS/MS methods, mass spectra are collected on precursor ions over a broad range of m/z values. These spectra are known as full scans or survey scans.

MS2

Mass spectrometry fragment scan. A mass spectrum acquired on fragment ions during tandem mass spectrometry.

MSE

A data-independent acquisition (DIA) method for tandem mass spectrometry in which the mass spectrometer alternates between acquiring mass spectra from low energy collisions and high energy collisions to obtain accurate masses of precursor ions and fragment ions, respectively.

MSn

Multistage mass spectrometry, where n is the number of stages of mass analysis. For example, MS/MS is also known as MS2.

Mascot

A search engine that uses mass spectrometry data to identify proteins from peptide sequence databases.

Mass defect

The difference between the exact mass of an atom or molecule and its nominal mass (the sum of the masses of its components).

Mass spectrum

A plot of relative abundance of ions (y axis) against their m/z (x axis).

Middle-down proteomics

A strategy for protein analysis within proteomics that is a hybrid of top-down and bottom-up approaches. Middle-down methods employ restricted proteolysis to generate larger peptides than bottom-up methods.

Molecular function

A gene ontology category containing terms that describe the molecular activities of gene products. For example, catalytic or binding activities.

NHS

N-hydroxy succinimide. NHS esters are commonly used to modify peptides for quantitative proteomics. For example, tandem mass tagging employs reagents containing amine-reactive NHS esters to label peptides with isobaric tags for protein quantification.

NLS

Nuclear localisation signal. A short amino acid sequence that directs proteins to the cell nucleus by enabling their transport through nuclear pores.

NeuCode SILAC

Neutron encoding SILAC. A quantitative proteomics method based on SILAC. NeuCode SILAC exploits the mass defects of stable isotopes to achieve higher multiplexing than SILAC. Peptides with the same number of extra neutrons incorporated into different atoms have subtly different masses due to differences in nuclear binding energies. For example, swapping a 12C atom for a 13C atom along with a 15N atom for a 14N atom produces labels differing by 6 mDa. This method requires high-resolution mass spectrometers to resolve peaks of similar m/z.

OD

Optical density. A measure of how much light is absorbed by a sample when light passes through it.

OOPS

Orthogonal organic phase separation. A method to recover cross-linked protein–RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. It does not require molecular tagging or capture of polyadenylated RNA (see RIC)

PAI

Protein abundance index. A label-free quantitative proteomics method. Protein coverage e.g. number of peptides identified per protein, is used to estimate protein abundance.

PASEF

Parallel accumulation–serial fragmentation. A MS scan mode termed parallel accumulation serial fragmentation (PASEF) that synchronizes TIMS with MS/MS precursor selection

PBS

Phosphate buffered saline. A salt solution commonly that mimics the osmolarity and ion concentrations of human body fluids while providing pH buffering capacity.

PCA

Principal component analysis. A multivariate data analysis technique used to summarize variation in a dataset and identify underlying patterns. A group of correlated variables are transformed into linearly uncorrelated variables, termed principal components, which summarize the variation in the dataset. Condensing complex data into principal components allows one to identify patterns in the dataset by plotting principal component values in 2-dimensional or 3-dimensional plots.

PCP

Protein correlation profiling. A spatial proteomics method for determining the subcellular localisation of proteins. Proteins are partially resolved according to subcellular location via density gradient centrifugation. Fractions are analysed by label-free MS/MS to construct protein relative abundance profiles spanning the density gradient. Subcellular localisation is determined by matching the profiles of proteins with unknown localisation to those of well-characterized marker proteins for specific locations.

PCR

Polymerase Chain Reaction. A molecular biology technique that rapidly amplifies specific DNA sequences through repeated cycles of heating and cooling, creating millions of copies from a small initial amount of DNA. The methods makes use of short synthetic DNA sequences (primers) that define the start and end points of amplification

PM

Plasma membrane. The phospholipid bilayer that surrounds all living cells.

PMF

Peptide Mass Fingerprinting. A mass spectrometry method for protein identification that matches experimentally measured peptide fragment masses with calculated masses from protein sequence databases.

POI

Protein of interest. The protein subject(s) that are the focus of an experiment.

PPI

Protein-protein interaction. The physical association of two or more proteins.

PRM

Parallel reaction monitoring. A targeted proteomics method similar to SRM. Specific peptides are selected by m/z in the first stage of a tandem mass spectrometer and fragmented. Unlike SRM, parallel reaction monitoring detects all target fragment ions in one, concerted MS analysis using a high-resolution mass analyser.

PSM

Peptide spectrum match. A peptide hit identified by matching an acquired MS2 spectrum to an MS2 spectrum in a sequence database (for example, containing theoretical MS2 spectra generated by in silico digestion of a proteome).

PTM

Post-translational modification. The diverse processing events applied to proteins following their translation. PTM most commonly refers to the covalent attachment of a functional group to a protein. For example, phosphorylation or acetylation.

PVDF

Polyvinylidene fluoride. A highly non-reactive thermoplastic fluoropolymer often used as a membrane in western blotting.

Peptide mass fingerprinting

A method for protein identification by mass spectrometry in which the molecular masses of unfragmented peptide ions are matched to theoretical peptide masses.

Peptidotypic

Peptidotypic fragment ions are specific to a particular peptide sequence (analogous to proteotypic peptides).

Phosphoproteomics

The branch of proteomics concerned with analysis of phosphorylated proteins and peptides.

Proteoform

The specific molecular form of a protein product arising from a single gene. This distinguishes proteins arising from a particular gene that differ due to genetic variation, alternative splicing and post-translational modification.

Proteome

The complete set of proteins expressed in a cell, tissue or biological system. Proteomes vary with time and biological context.

Proteomics

The large-scale study of proteomes. This term incorporates the large-scale investigation of protein expression, turnover, post-translational modification, localisation and interactions between proteins.

Proteotypic peptides

Peptides whose sequence is unique to a specific protein. Proteotypic peptides are used for protein identification.

QconCAT

A method for absolute quantification of target proteins. A synthetic gene is designed to express heavy isotope labelled concatemers of proteotypic peptides for the protein/s of interest. These labelled polypeptides are used as internal standards for targeted proteomics.

Quantitative proteomics

The systematic analysis of relative or absolute abundance of proteins.

RBP

RNA binding protein

RBPome

The RNA binding proteome. All the proteins in a cell that bind RNA.

RIC

RNA interaction capture. An experimental technique used to identify and study RBPs on a global scale. RIC uses in vivo UV crosslinking, oligo(dT) capture, and proteomics to identify RNA-binding proteomes

RMSD

Root mean square deviation. A statistical measure that quantifies the average difference between predicted and observed values, or between two sets of data points

RNA

Ribonucleic Acid. A fundamental nucleic acid molecule that plays essential roles in gene expression, protein synthesis, and cellular regulation. Unlike DNA, RNA is typically single-stranded, contains ribose sugar instead of deoxyribose and the base uracil rather than thymine.

RPLC

Reversed-phase liquid chromatography. A chromatographic technique for separating mixtures using a non-polar stationary phase and polar mobile phase.

RPM

Revolutions per minute. A unit of the measurement of how many times a complete revolutions a centrifuge rota, for example, makes per minute.

RPPA

Reverse phase protein array. An antibody-based proteomics technology for analysing protein expression or post-translational modification in a large number of samples. Protein lysates are printed onto microarray slides and probed with antibodies to specific proteins.

RT-PCR

Reverse Transcription Polymerase Chain Reaction is a laboratory technique that combines reverse transcription and PCR amplification to detect and quantify RNA molecules, particularly mRNA.

S/N

Signal-to-noise ratio. In mass spectrometry, a measure of instrument sensitivity. The maximum signal intensity of an analyte is divided by noise calculated as the standard deviation or root mean square of the baseline surrounding the signal peak.

SAX

Strong anion exchange. A type of ion exchange chromatography that uses a stationary phase with permanently charged positive groups to separate negatively charged molecules. Quaternary ammonium is the most typically used functional group and elution of proteins and peptides from SAX columns is achieved by increasing salt concentration, changing pH or use of competing anions.

SCX

Strong cation exchange chromatography. A technique for separating mixtures by net surface charge. A negatively charged stationary phase binds positively charged analytes in a mobile phase. SCX can be used to enrich phosphopeptides from a proteolytic digest for phosphoproteomics analysis. At low pH, tryptic peptides typically carry a net charge of +2 or higher. In contrast, phosphorylated peptides carry a lower net charge (0/+1).

SDS

Sodium dodecyl sulphate. An anionic surfactant used to solubilize protein samples.

SDS-PAGE

Sodium dodecyl sulphate polyacrylamide gel electrophoresis. An electrophoretic technique for separating proteins according to their molecular mass. Proteins are denatured by the anionic detergent sodium dodecyl sulphate (SDS). SDS binds to polypeptides in proportion to their molecular mass, imparting a uniform charge density (negative charge) to polypeptides. Samples are separated in a polyacrylamide gel matrix by applying an electric field. Polypeptides migrate towards the anode as a linear function of their molecular mass. Mass-resolved protein samples are commonly analysed by Western blotting or non-specific protein staining.

SEM

Scanning Electron Microscopy. An electron beam is scanned across the surface of the sample and secondary electrons emitted from the surface create detailed 3D-like images of the surface composition. SEM has slight lower resolution than TEM (see below) typically 1-10 nanometres. It excels at showing surface details and can examine thicker specimens.

SILAC

Stable isotope labelling by amino acids in cell culture (SILAC) is a MS-based quantitative proteomics technique in which proteins are labelled via the incorporation of stable isotopic forms of amino acids during cell growth.

SIMAC

Sequential elution from IMAC. A phosphoproteomics strategy for separating monophosphorylated peptides from multiply phosphorylated peptides. Monophosphorylated and multiply phosphorylated peptides are eluted from IMAC under acidic or basic conditions, respectively. Monophosphorylated peptides are subsequently enriched from unphosphorylated peptides using TiO2 chromatography.

SNP

Single nucleotide polymorphism

SPS-MS3

Synchronous precursor selection-MS3. A tandem mass spectrometry method for data acquisition, which uses isolation waveforms with multiple frequency notches to isolate and fragment multiple MS2 product ions simultaneously. SPS-MS3 is employed for quantitation via isobaric labelling methods, such as TMT. SPS-MS3 reduces ratio distortion caused by co-selection, while concomitantly increasing the number of reporter ions for quantitation in MS3 spectra over the standard MS3 method.

SRM

Selected reaction monitoring. A targeted proteomics method typically performed on a triple quadrupole mass spectrometer. A specific peptide precursor ion is selected by m/z in the first mass analyser, fragmented in a collision cell and a specific fragment ion selected by m/z in the second mass analyser. These precursor/fragment ion pairs, termed transitions, are used to selectively monitor target proteins.

SRMAtlas

A database of targeted proteomics assays (SRM/MRM).

STRING

Search tool for the retrieval of interacting genes/proteins. A database of known and predicted protein-protein interactions.

SUMO

Small ubiquitin-like modifier. A family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. This process is called SUMOylation. Unlike ubiquitination, the addition of SUMO to a target protein does not lead to its degradation.

SVM

Support vector machine. A robust supervised learning method designed for classification and regression problems that operates by identifying the best decision boundary (hyperplane) to distinguish between different categories of data.

SWATH-MS

In this data-independent acquisition MS method, all precursor ions in a predefined m/z window are selected for MS/MS concurrently. The mass spectrometer repeatedly cycles through consecutive precursor isolation windows to acquire convoluted fragment ion spectra for all detectable analytes in a wide m/z range.

Secretome

The entire set of proteins secreted by a cell, tissue or organism.

Shotgun proteomics

See Discovery proteomics.

Soft ionization

Ionization techniques that produce gas-phase ions with minimal fragmentation of the analyte. Examples include ESI and MALDI.

Spectral counting

A semiquantitative label-free proteomics method in which the total number of MS2 spectra derived from a protein is used as a measure of protein abundance.

Static modification

A type of modification that can be specified when carrying out peptide identification by sequence database searching. Static or fixed modifications are applied to every instance of the specified amino acid or terminus and do not increase the computational complexity of a search.

Subproteome

A subdivision of a proteome. For example, the protein complement of an organelle.

TAP

Tandem affinity purification. A common method for affinity purification that utilizes a dual tag to sequentially purify a fusion protein. In the original method, a TAP tag is fused to the C-terminus of a protein. This tag consists of a calmodulin-binding peptide, TEV protease cleavage site and Protein A (N-terminal to C-terminal). The TAP-tagged protein is affinity purified using an IgG matrix, cleaved with TEV protease and further purified using calmodulin beads.

TCA

Trichloroacetic acid. Used for protein precipitation.

TCEP

Tris-(2-carboxyethyl)phosphine. A reducing agent commonly used to reduce protein disulphide bonds.

TEAB

Triethylammonium bicarbonate buffer, a commonly used reagent in proteomics and mass spectrometry applications, buffering solutions around pH 8.0-8.5

TEM

Transmission Electron Microscopy. An electron beam is directed through exceptionally thin sample sections, usually under 100 nanometres in thickness. As electrons pass through the specimen, they form an image that is captured by a digital sensor. TEM offers remarkable resolution capabilities, reaching approximately 0.05 nanometres, which enables imaging of subcellular components.

TIMS

Trapped ion mobility spectrometry. TIMS is a form of IMS that inverts the separation principle of classical drift tube ion mobility. Ions entering the TIMS analyser are positioned in an electrical field by the drag of a gas flow. Lowering the electrical force releases ions from the TIMS device separated by their ion mobility, while the IMS resolution is proportional to the user-defined ramp time.

TMT

Tandem mass tag. Amine-reactive isobaric tagging reagents for protein quantification by tandem mass spectrometry.

TPP

Thermal Protein Profiling. A mass-spectrometry method often used to study drug-protein interactions and the thermal stability of proteins across a temperature range, TPP is based on the principle that proteins denature when subjected to heat treatment and become insoluble. Proteins change their thermal stability upon interactions with small molecules and other biomolecules. When a drug binds to its target proteins it typically stabilizes it makes it more resistant to heat induced denaturation. By measuring proteins left in solution across a temperature range and how this changes upon drug binding indicates which proteins are stabilized by a particular drug.

Targeted proteomics

Targeted proteomics methods analyse predefined subsets of proteins at high sensitivity using mass spectrometry. Examples include selected reaction monitoring (SRM). Contrast with Discovery proteomics methods.

Th

Thomson. A unit of mass-to-charge ratio used in mass spectrometry, named after J.J. Thomson who discovered the electron.

TiO2

Titanium dioxide. Used for phosphopeptide enrichment by metal oxide affinity chromatography (MOAC).

Top-down proteomics

One of the two main strategies for protein analysis within proteomics (contrast with Bottom-up proteomics). Intact proteins are ionized and analysed by tandem mass spectrometry. Fragment ions are used for protein identification.

Total ion current (TIC)

The summed signal intensity of all ions in a mass spectrum or in a specified m/z range of a mass spectrum.

Total ion current chromatogram

A chromatogram in which the total ion current in a series of mass spectra is plotted against retention time.

Trypsin

Protease that hydrolyses peptide bonds at the C-terminal side of lysine and arginine residues. Used for sample preparation in bottom-up methods. May be used in combination with alternative proteases, such as Lys-C, to increase sequence coverage.

UV

Ultraviolet light. Electromagnetic radiation with wavelengths shorter than visible light but longer than X-rays, typically ranging from about 10 to 400 nanometres.

Variable modification

A type of modification that can be specified when carrying out peptide identification by sequence database searching. Variable or dynamic modifications are those which are sometimes present on a specific amino acid or terminus. For example, methionine oxidation, which causes a mass shift of 16 Da for a peptide containing a single oxidized methionine. Variable modifications increase the computational complexity of a search.

Western blotting

A technique for protein analysis. Protein samples separated by gel electrophoresis are transferred onto the surface of a membrane. The membrane is probed with a primary antibody that recognizes one or more epitopes specific to a particular protein. Primary antibodies are detected with species-specific secondary antibodies. Most commonly, secondary antibodies are conjugated to horseradish peroxidase (HRP), which generates luminescence when incubated with a cleavable chemiluminescence reagent. This signal is proportional to the amount of protein and is typically detected with photographic film or a CCD camera.

XIC

Extracted ion chromatogram. A chromatogram in which the signal intensity of one or more specified m/z values are plotted against retention time.

emPAI

Exponentially modified protein abundance index. A label-free quantitative proteomics method. Protein coverage is determined using MS2 spectra for peptide identification and used to estimate protein abundance.

iBAQ

Intensity-based absolute quantification. Equivalent to PAI but uses peptide intensities instead of SC

iPAC

Interactomes by parallel affinity capture. An affinity purification-mass spectrometry method which uses dual tags for the affinity purification of a protein of interest in two independent experiments. Genuine binding partners are identified by their consistent identification in both affinity purification experiments.

iTRAQ

Isobaric tag for relative and absolute quantitation. Amine-reactive isobaric tagging reagents for protein quantification by tandem mass spectrometry.

k-NN

k-nearest neighbours. A straightforward and effective machine learning approach that can handle both classification and regression problems.

kDa

kilo Dalton (see Da - Dalton)

m/z

Mass-to-charge ratio. The quantity formed by dividing the mass number of an ion by its charge number.

ppm

Parts per million. A unit of measurement used to express very small concentrations or ratios. It represents the number of units of a substance present in one million units of a sample.

siRNA

siRNA (small interfering RNA). Short, double-stranded RNA segments that function as key regulators of gene expression via RNA interference (RNAi). These molecules precisely bind to matching mRNA sequences and trigger their breakdown, thereby blocking protein production from targeted genes.

t-SNE

t-distributed stochastic neighbour embedding. A machine learning technique used for dimensionality reduction and data visualization. It is often employed to visualize high-dimensional data reducing it to 2D or 3D representations while preserving local structure and relationships between data points.