Glossary of terms

2D-PAGE

Two-dimensional polyacrylamide gel electrophoresis. An electrophoretic technique for separating proteins according to two physicochemical properties - isoelectric point and protein mass.

4-SU

4-Thiouracil. Can be incorporated into RNA in place of uracil. It is often used in studies to determine which proteins bind RNA as it will cross link to proteins at 365 nm UV light with greater efficiency that crosslinking uracil and proteins at 254 nm UV light.

ACN

Acetonitrile (CH₃CN). A widely used mobile phase solvent in reverse phase chromatography and sample clean-up.

ADP

Adenosine Diphosphate. A crucial nucleotide in cellular energy metabolism.

AGPC

Acid guanidium phenol chloroform. Is a phase separation solvent that is used in the classical method for RNA extraction, most commonly known as the TRIzol method or Chomczynski-Sacchi method.

AHA

Azidohomoalanine. A synthetic analogue of methionine, often used in experiments to determine protein synthesis rates and secretion. Proteins that contain this amino acid can be readily isolated via click chemistry.

AMP

Adenosine Monophosphate. A fundamental nucleotide with multiple important roles in cellular biology.

AMP

Ampicillin. A broad-spectrum penicillin (beta lactam) antibiotic widely used in clinical medicine and research. It prevents cross-linking of bacterial peptidoglycan chains, leading to cell lysis

AP-MS

Affinity purification-mass spectrometry. The coupling of affinity purification to MS analysis is used to identify and quantify protein-protein interactions.

APEX

Engineered ascorbate peroxidase. A genetically targetable tag employed for protein labelling. APEX is used to biotinylate proteins nearby a protein of interest. Subsequent affinity purification-mass spectrometry is carried out to analyse protein-protein interactions and map subcellular structure.

AQUA

A strategy for absolute quantification of proteins. Synthetic peptides incorporating a stable isotope labelled amino acid are used as internal standards for targeted proteomics.

ATP

Adenosine Triphosphate. The primary energy currency of cells and released energy when hydrolysed as follows: ATP → ADP + Pi + ~30.5 kJ/mol

Alphafold

AlphaFold is an artificial intelligence system developed by DeepMind (part of Google DeepMind) that predicts protein structures with remarkable accuracy

BCA assay

Bicinchoninic acid assay. A colorimetric assay for quantifying the total concentration of protein in a solution.

BN-PAGE

Blue Native polyacrylamide gel electrophoresis. An electrophoretic technique used to separate protein complexes under native conditions while preserving their structure and interactions that uses Coomassie Brilliant Blue, G-250 due to provide a negative charge to protein complexes via the electrophoresis process.

BPI

Base peak intensity. The base peak is the ion with the highest abundance (intensity) in a mass spectrum. Base peak intensity is typically normalized to 100% (relative abundance) and all other peaks in the spectrum are expressed as percentages relative to the base peak

Base peak chromatogram

A chromatogram in which the signal intensity of the most intense peak (base peak) in a series of mass spectra is plotted against retention time.

BioID

A method for mapping proteins located near a target protein in living cells. This approach works by genetically fusing a modified version of the bacterial enzyme BirA* (a mutant biotin ligase from E. coli) to the protein being studied. The modified enzyme then adds biotin tags to proteins in the immediate vicinity, which can be captured by streptavidin for downstream analysis.

Biological process

A gene ontology category containing terms that describe series of molecular events with a defined beginning and end. For example, metabolic processes.

Bottom-up proteomics

One of the two main strategies for protein analysis within proteomics (contrast with Top-down proteomics). Bottom-up approaches involve the proteolytic digestion of protein samples prior to MS analysis. Peptide ions are fragmented and analysed by tandem mass spectrometry. Peptides are typically identified by comparing the MS2 spectra to theoretical MS2 spectra generated by in silico digestion of a proteome.

C18

Octadecylsilane. Commonly used as the non-polar stationary phase for RPLC.

CHX

Cycloheximide. An antibiotic from Streptomyces griseus that blocks procaryotic translational. A widely used antibiotic in molecular biology protocols. Its effects are reversible upon its removal. It inhibits the translocation step of proteins synthesis through inhibition of peptidyl transferase.

CID

Collision-induced dissociation. A MS fragmentation technique in which precursor ions are accelerated in an electric field and allowed to collide with molecules of an inert gas.

CLIP-seq

Cross-Linking and Immunoprecipitation followed by sequencing: A technique that is used to identify which RNA species bind to an RNA binding protein of interest.

CRAPome

Contaminant repository for affinity purification. A database of common mass spectrometry contaminants.

CRISPR

Clustered regularly interspaced short palindromic repeats. A gene-editing technology that allows precise modification of DNA sequences in living cells.

Cellular compartment

A gene ontology category containing terms that describe gene product location. For example, localisation to subcellular structures or macromolecular complexes.

ChIP

Chromatin immunoprecipitation (see below)

ChIP-seq

Chromatin Immunoprecipitation followed by sequencing. A method to determine where specific proteins bind to DNA across the entire genome. The method combines chromatin immunoprecipitation, that involves crosslinking DNA binding proteins to DNA, typically using formaldehyde. The protein and the DNA it is bound to are then isolated through immune captures followed by high-throughput DNA sequencing to map protein-DNA interactions at a genome-wide scale.

Co-selection

The isolation and fragmentation of ions of similar m/z to a selected precursor ion due to their coelution during MS/MS. Co-selection negatively impacts upon protein quantification accuracy in MS2-based methods.

CryoEM

Cryo-Electron Microscopy. A structural biology approach that uses electron microscopy to determine the three-dimensional structures of biological macromolecules and complexes at near-atomic resolution. Samples are imaged while frozen in thin ice layers that preserves their native structure.

CryoET

Cryo-Electron Tomography. A form of cryo-electron microscopy (See Cryo-EM above) that generates three-dimensional images of biological specimens by collecting multiple 2D projections from different angles and computationally reconstructing them into 3D volumes.

CyTOF

Cytometry by time of flight. A single-cell analysis technique that combines flow cytometry with mass spectrometry to enable highly multiplexed analysis of individual cells. Cells are first treated with antibodies to target proteins of interest that are conjugated to typically one of a number of available lanthanide atoms typically. The amount of lanthanide ions measured via Inductively Coupled Plasma Mass Spectrometry,(ICP-MS), equates to the amount of the target protein.

DC

Differential centrifugation. A method often used to separate organelles and other subcellular components based on their size, density, and sedimentation rate

DMSO

Dimethyl sulfoxide. A highly polar, organic solvent with the chemical formula (CH₃)₂SO. It's widely used due to its exceptional solvent properties and biological compatibility.

DNA

Deoxyribonucleic Acid. Hereditary material found in nearly all living organisms that stores genetic information.

DTT

Dithiothreitol. A redox reagent commonly used to reduce protein disulphide bonds.

DVP

Deep Visual Proteomics. An approach that integrates AI-powered cellular phenotype imaging with precision laser microdissection of single cells or nuclei, coupled with exceptionally sensitive mass spectrometry analysis. The technique enables scientists to establish direct connections between protein expression levels and intricate cellular structure within their native tissue environments.

Da

Dalton. Unit of molecular mass. 1 Dalton = 1/12th of the mass of a carbon-12 atom.

Data-dependent acquisition (DDA)

A tandem mass spectrometry data collection mode in which precursor ions are selected from survey scans for MS/MS analysis based on predefined rules, such as peak intensity.

Data-independent acquisition (DIA)

A tandem mass spectrometry data collection mode in which all precursor ions in a defined m/z range are selected for MS/MS analysis. For example, SWATH-MS.

Deamidation

A chemical reaction in which an amide group is removed by hydrolysis. During proteomics sample preparation, glutamine and asparagine amino acids can be deamidated to glutamate and aspartate, respectively.

Dimethyl labelling

A stable isotope labelling method for quantitative proteomics which uses a dimethyl group to label peptide primary amines. Quantification is carried out on survey scans.

Discovery proteomics

Discovery or shotgun proteomics strategies are designed to sample and analyse a large number of proteins in a complex mixture using bottom-up proteomics methods. Contrast with Targeted proteomics.

Dynamic exclusion

A mass spectrometry feature that allows the mass spectrometer to stop selecting a precursor ion for fragmentation once it has been selected a specified number of times. This promotes the analysis of less abundant ions.

ECM

Extracellular matrix. a Non-cellular component found outside of cells in multicellular organisms

EDTA

Ethylenediaminetetraacetic acid. A widely used chelating reagents with affinity for Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺.

EGTA

Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid. A chelating agent that has a lower affinity for magnesium, making it more selective for calcium ions.

ELISA

Enzyme-linked immunosorbent assay. A well used technique to measure the amount of a biomolecule in a sample. It employs primary antibodies raised to interact with the target molecule, and secondary antibodies often coupled with enzymes that catalyse a reaction that produces a detectable product, that react with the primary antibody. Fluorescently labelled secondary antibodies are typically now used. The amount of fluorescence measured equates to the amount of the target molecule per sample.

EM

Electron microscopy. An imaging technique that uses a beam of electrons instead of light to create highly detailed images of specimens, achieving much higher magnification and resolution than traditional light microscopy because electrons have much shorter wavelengths than visible light.

ER

Endoplasmic reticulum. An intracellular organelle formed from by extensive network of interconnected tubules and flattened sacs called cisternae. Smooth ER lacks ribosomes and is the location of lipid synthesis amongst other cellular processes and in the site of calcium ion storage and release. Rough ER is studded with ribosomes at its cytosolic interface and is the location of synthesis of membrane and secreted proteins.

ERLIC

Electrostatic repulsion hydrophilic interaction chromatography. A type of hydrophilic interaction chromatography (HILIC) on which ion-exchange chromatography is superimposed by using an ionic column surface chemistry. ERLIC is employed for phosphopeptide enrichment.

ESI

Electrospray ionization. A 'soft' ionization technique for mass spectrometry that produces gaseous ions by applying an electric field to a liquid analyte eluting from a narrow capillary tube.

ETD

Electron-transfer dissociation. A MS fragmentation technique in which electron transfer from radical anions to multiply-charged precursor cations induces fragmentation of the cations.

Expression proteomics

The large-scale analysis of the relative or absolute abundances of proteins. For example, to identify proteins that are differentially expressed in response to a drug.

FACS

Fluorescent activated cell sorting. A technique used to sort and analyse cells based on their fluorescent properties.

FAIMS

Field Asymmetric Waveform Ion Mobility Spectrometry: An atmospheric pressure ion mobility technique that separates gas-phase ions by their behaviour in strong and weak electric fields and is easily integrated with electrospray ionization systems.

FASP

Filter-aided sample preparation. A method for preparing peptide samples for proteomics analysis. Samples are solubilized in sodium dodecyl sulphate, which is subsequently exchanged for urea using an ultrafiltration device.

FDR

False discovery rate. In proteomics, FDR is a statistical metric for the rate of false positives in peptide spectral matches (PSMs) accepted for protein identification. This can be estimated by adding decoy sequences to a database for sequence database searching.

FISH

Fluorescence In Situ Hybridization imaging. A molecular biology technique used to detect and localize specific DNA or RNA sequences within cells and tissues. FISH makes use of fluorescently labelled DNA or RNA probes that are complementary to target sequences.

FWER

Family-wise error rate. A statistical concept that addresses the problem of multiple comparisons in hypothesis testing. FWER is the probability of making at least one Type I error (false positive) when performing multiple statistical tests simultaneously within a family of hypotheses.

GWAS

Genome-wide association studies. Large-scale studies that examine genetic variation across the entire genome to identify associations with diseases, traits, or other phenotypes.

GeLC-MS/MS

A strategy for MS analysis of complex protein mixtures. Proteins are separated by SDS-PAGE, digested in-gel and analysed by liquid chromatography-tandem mass spectrometry.

Gene ontology (GO)

Hierarchical, defined terms for gene product properties. GO terms are categorised into three domains - cellular component, biological process and molecular function.

Gorilla

Gene ontology enrichment analysis and visualization tool. A tool for identifying enriched GO terms.

HCA

Hierarchical cluster analysis:HCAA statistical technique for arranging objects into groups based on similarity. Objects are either sequentially split (divisive) or combined (agglomerative) into groups as the hierarchy progresses along a dendrogram. This method may be used to visualize protein expression data.

HCD

Higher-energy collisional dissociation. A CID technique in a hybrid linear ion trap–orbitrap mass spectrometer in which fragmentation occurs externally to the ion trap in a dedicated collision cell. Consequently, HCD fragmentation does not suffer from loss of low m/z fragment ions.

HEPES

(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) . A zwitterionic biological buffer widely used in cell culture and biochemical research with a pKa of 7.5.

HILIC

Hydrophilic interaction chromatography. A chromatographic technique for separating mixtures using a hydrophilic stationary phase and a hydrophobic mobile phase. Solutes are eluted from the column in order of increasing polarity. HILIC can be used for the enrichment of post-translationally modified peptides, such as phosphorylated and glycosylated peptides.

HPA

The Human Protein Atlas: An antibody-based proteomics resource containing expression and localisation profiles of human proteins in different tissues, cancer types and cell lines.

HPLC

High-performance liquid chromatography. The chromatographic separation of compounds in solution at high pressure, in narrow columns packed with small sorbent particles. This provides higher resolution than traditional low-pressure liquid chromatography.

Heat map

A diagram used to display data values represented as colours.

ICAT

Isotope-coded affinity tag. Thio-reactive isotopic tagging reagents for MS-based protein quantification of cysteine-containing peptides.

IDR

Intrinsically disordered region. segments of proteins that lack stable secondary or tertiary structure under physiological conditions, yet remain functionally important.

IEC

Ion exchange chromatography. A separation technique that isolates molecules based on their net charge and charge density. It uses a stationary phase containing fixed ionic groups that interact with oppositely charged molecules in the mobile phase. There are two main types, a) cation exchange uses negatively charged groups to bind positively charged molecules b) anion exchange (see SAX below) that employs positively charged groups that capture negatively charged species.

IEF

Isoelectric focussing. An electrophoretic technique that separates proteins based on their isoelectric point (pI) - the pH at which a protein carries no net electrical charge.

IMAC

Immobilized metal affinity chromatography. A technique for enriching phosphopeptides or histidine-containing peptides/proteins. This is based on the coordination of phosphate groups or histidine residues to immobilized metal ions. The choice of metal ion and chelating ligand depends upon the application. Fe3+, Ga3+ or Ti4+ are commonly used for phosphopeptide enrichment.

IMS

Imaging mass spectrometry. A method that combines mass spectrometry with spatial information to create molecular maps, which involves ionizing molecules directly from the surface of tissue sections or other surfaces. Common ionisation methods used are MALDI-IMS (Matrix-Assisted Laser Desorption/Ionization), DESI-IMS (Desorption Electrospray Ionization) and SIMS-IMS (Secondary Ion Mass Spectrometry).

IMS

Ion mobility separation. A method that separates gas phase ions based on their interaction with a collision gas and their masses.

In-gel digestion

The digestion of protein samples within a polyacrylamide matrix, following protein separation by polyacrylamide gel electrophoresis. This sample preparation method permits efficient solubilization of proteins using a sodium dodecyl sulphate-containing buffer, since low molecular weight components are removed during gel electrophoresis.

In-solution digestion

A sample preparation method in which proteins are solubilized and proteolytically digested in a solution (contrast with In-gel digestion). In-solution digestion methods are typically detergent-free and rely on high concentrations of chaotropes, such as urea, to denature and solubilize proteins.

IntAct

A database of molecular interactions containing data with direct experimental evidence.

Interactomics

The study of interactions of proteins, typically with other proteins.

Iodoacetamide (IAA)

An alkylating agent commonly used to covalently bind the reduced thiol of cysteine residues and prevent disulphide bond formation.

Isobaric tagging

A quantitative proteomics technique in which peptides are chemically labelled with tags of the same nominal mass. Fragmentation during tandem mass spectrometry releases reporter ions of a m/z characteristic to each tag. By differentially labelling samples with isobaric tagging reagents, one can quantify the relative abundances of proteins between samples. Isobaric tags include TMT and iTRAQ.

Isoelectric focusing

An electrophoretic technique for separating molecules by their isoelectric point (pI). For example, proteins can be separated by isoelectric point in 2D-PAGE. This involves the application of an electric field to samples in a fixed pH gradient.

Isotopologues

Molecules with the same mass number, but different isotope compositions.

KD

Knock down. Refers to the partial reduction of gene expression or protein levels, typically through RNA interference (RNAi) or other post-transcriptional mechanisms

KEGG

Kyoto encyclopaedia of genes and genomes. A database of molecular functions (of genes and proteins) integrated into networks, for example signalling pathways.

KO

Knock out. Inactivating or "knocking out" a specific gene to study its function

LC-MS/MS

Liquid chromatography tandem mass spectrometry. The coupling of a liquid chromatography system to a tandem mass spectrometer is used to reduce sample complexity before MS analysis.

LCM

Laser capture microdissection. A method for isolating specific regions/cells from tissue sections viewed under microscopy.

LFQ

Label-free quantification: Any MS method for protein quantification that does not rely on stable isotope labelling. Label-free methods include summing precursor ion signal intensity and spectral counting.

LOPIT

Localisation of organelle proteins by isotope tagging. A proteomics method for the high-throughput characterization of protein subcellular localisation. Proteins are partially resolved according to subcellular location using density gradient centrifugation. Fractions are proteolytically digested and differentially labelled with isobaric tags. Relative abundances of proteins between fractions are determined using reporter ion profiles acquired via tandem mass spectrometry. Subcellular localisation is calculated using classification algorithms that assign proteins based on the profiles of organelle marker proteins.

Lys-C

Protease that hydrolyses peptide bonds at the C-terminal side of lysine residues. Used for sample preparation in bottom-up methods. Unlike trypsin, Lys-C cleaves Lys-Pro bonds.

MALDI

Matrix-assisted laser desorption/ionization. A 'soft' ionization technique for mass spectrometry that produces gaseous ions by pulsed laser irradiation of an analyte embedded in an organic matrix.

MAR

Missing at random. A classification of missing data where the probability that a value is missing depends on observed variables in the dataset, for example failure to measure proteins/peptides stochastically across a set of samples.

MCMC

Markov-chain Monte-Carlo. A class of computational algorithms used to sample from complex probability distributions by constructing a Markov chain that has the desired distribution as its equilibrium distribution.

MNAR

Missing not at random. A category of missing data where the probability that a value is missing depends on the unobserved value itself, for example, lack of a measurement of a protein or peptide simply because it does not exist in a particular sample.

MOAC

Metal oxide affinity chromatography. A chromatographic technique for enriching phosphopeptides based on the affinity of phosphate groups for metal oxides. For example, TiO2.