Activity-based proteomics, or activity-based protein profiling (ABPP) is a functional

proteomic Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...

technology that uses chemical probes that react with mechanistically related classes of enzymes.

Description

The basic unit of ABPP is the probe, which typically consists of two elements: a reactive group (RG, sometimes called a "warhead") and a tag. Additionally, some probes may contain a binding group which enhances selectivity. The reactive group usually contains a specially designed

electrophile In chemistry, an electrophile is a chemical species that forms bonds with nucleophiles by accepting an electron pair. Because electrophiles accept electrons, they are Lewis acids. Most electrophiles are positively charged, have an atom that carrie ...

that becomes

covalently A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atom ...

-linked to a

nucleophilic In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are ...

residue in the

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate ( binding site) ...

of an active

enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...

. An enzyme that is inhibited or post-translationally modified will not react with an activity-based probe. The tag may be either a reporter such as a

fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...

or an affinity label such as

biotin Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', bo ...

or an

alkyne \ce \ce Acetylene \ce \ce \ce Propyne \ce \ce \ce \ce 1-Butyne In organic chemistry, an alkyne is an unsaturated hydrocarbon containing at least one carbon—carbon triple bond. The simplest acyclic alkynes with only one triple bond and n ...

azide In chemistry, azide is a linear, polyatomic anion with the formula and structure . It is the conjugate base of hydrazoic acid . Organic azides are organic compounds with the formula , containing the azide functional group. The dominant appli ...

for use with the

Huisgen 1,3-dipolar cycloaddition The azide-alkyne Huisgen cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole. Rolf Huisgen was the first to understand the scope of this organic reaction. American chemist Kar ...

(also known as

click chemistry In chemical synthesis, click chemistry is a class of biocompatible small molecule reactions commonly used in bioconjugation, allowing the joining of substrates of choice with specific biomolecules. Click chemistry is not a single specific reactio ...

Advantages

A major advantage of ABPP is the ability to monitor the availability of the enzyme active site directly, rather than being limited to protein or mRNA abundance. With classes of enzymes such as the

serine hydrolase Serine hydrolases are one of the largest known enzyme classes comprising approximately ~200 enzymes or 1% of the genes in the human proteome. A defining characteristic of these enzymes is the presence of a particular serine at the active site, which ...

s and

metalloprotease A metalloproteinase, or metalloprotease, is any protease enzyme whose catalytic mechanism involves a metal. An example is ADAM12 which plays a significant role in the fusion of muscle cells during embryo development, in a process known as myo ...

s that often interact with endogenous inhibitors or that exist as inactive

zymogen In biochemistry, a zymogen (), also called a proenzyme (), is an inactive precursor of an enzyme. A zymogen requires a biochemical change (such as a hydrolysis reaction revealing the active site, or changing the configuration to reveal the activ ...

s, this technique offers a valuable advantage over traditional techniques that rely on abundance rather than activity.

Multidimensional protein identification technology

In recent years ABPP has been combined with

tandem mass spectrometry Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A com ...

enabling the identification of hundreds of active enzymes from a single sample. This technique, known as ''ABPP-MudPIT'' (multidimensional protein identification technology) is especially useful for profiling inhibitor selectivity as the potency of an inhibitor can be tested against hundreds of targets simultaneously. ABPP were first reported in the 1990s in the study of proteases.

References

{{DEFAULTSORT:Activity-Based Proteomics Proteomics Genomics Protein methods Mass spectrometry

Description

Advantages

Multidimensional protein identification technology

See also

References