TB-500: the thymosin beta-4 fragment and what the research covers

TB-500 is the name the research market uses for one well-defined synthetic peptide: Ac-LKKTETQ, seven amino acids with an acetyl group capping the N-terminus. The sequence is copied from residues 17 to 23 of thymosin β4 (Tβ4), a 43-residue protein, and in 2012 doping-control chemists confirmed that this acetylated heptapeptide is what a TB-500 product contains. This guide reads the sequence residue by residue, places it inside the parent protein, separates what the actin literature learned about the protein from what it learned about the fragment, explains how analytical laboratories confirm the molecule, and covers US supply and status. We sell it as TB-500.

Research use only. TB-500 is supplied strictly for in-vitro laboratory research. It is not for human or veterinary use and has not been evaluated by the FDA.

TB-500 at a glance

Reading the sequence

Seven residues is short enough to read one at a time:

Three things follow from that list.

The formula adds up. Seven amino acids joined by six peptide bonds, with six waters lost, give C₃₆H₆₆N₁₀O₁₃ for LKKTETQ; the acetyl cap adds C₂H₂O, which yields the C₃₈H₆₈N₁₀O₁₄ on the specification. Without the cap the peptide would weigh about 42 mass units less, near 847 g/mol, a number worth recognizing on a spectrum.

The net charge is close to zero at neutral pH. The two lysines carry positive charges, while the glutamate and the free C-terminal carboxylate carry negative ones. The acetyl group removes the N-terminal amine that would otherwise add a third positive charge. The result is a small, very polar, water-soluble molecule.

There is little for oxygen or light to attack. The sequence contains no methionine, cysteine, tryptophan, histidine or tyrosine, the residues most prone to oxidation, and none of the aromatic residues where photo-oxidation mainly starts. Its C-terminal glutamine can in principle deamidate, though glutamine does so far more slowly than asparagine. None of this makes a solution immune to time; it does mean temperature is the main variable to control.

Where the seven residues sit in thymosin β4

Low, Hu and Goldstein sequenced thymosin β4 from calf thymus in 1981 and reported 43 residues, an acetylated N-terminus and an isoelectric point of 5.1. A decade later Safer and colleagues showed that Fx, the peptide holding much of the unpolymerized actin in resting human platelets, has the identical sequence. Written out, with the TB-500 stretch in bold:

Ac-SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES

Two points stand out. First, TB-500 comes from the middle of the chain, so inside Tβ4 leucine-17 has no free amine: it is joined to lysine-16 by a peptide bond. The synthetic fragment's acetyl cap leaves its own N-terminus uncharged as well. Second, a different named fragment sits at the very start of the protein: the N-terminal tetrapeptide Ac-SDKP, which Huff and colleagues' review describes as possibly generated from Tβ4. Ac-SDKP and TB-500 share a parent and nothing else; they are separate molecules with separate literatures.

Thymosin β4 and actin: what the protein does

Actin, the protein that builds a cell's filament network, exists as single subunits (G-actin) and as polymerized filaments (F-actin). The β-thymosins are the main peptides that hold G-actin in reserve:

• One-to-one binding. Safer's group showed that purified Tβ4 binds G-actin in a 1:1 complex and inhibits its polymerization. Huff and colleagues' review describes β-thymosins as present at high concentration in almost every cell and acting as actin buffers, holding monomers back from filaments while keeping a pool on hand.
• Folding on contact. Using NMR, Domanski and colleagues found Tβ4 mostly unstructured in solution; on binding G-actin it folds completely, into a central extended region flanked by an N-terminal and a C-terminal helix.
• Capping both ends. Irobi and colleagues solved a 2 Å crystal structure of actin bound to a hybrid of gelsolin domain 1 and the C-terminal half of Tβ4. It shows Tβ4 sequestering the monomer by capping both of its ends, and places Tβ4 as the prototype of the WH2 family of actin-binding motifs.

What part do residues 17-23 play?

This is the question that decides how far protein results can be read across to TB-500. Van Troys and colleagues mapped the actin-binding site of full-length Tβ4 using synthetic variants and found two separate structural pieces: an N-terminal segment (residues 1-16), which has to form an α-helix to bind and contacts actin through a patch of hydrophobic residues, and a hexapeptide motif at residues 17-22, the LKKTET stretch. Electrostatic contacts made by lysine-18 in the motif, and by lysine-14 in the helix, appeared important.

Doping-control papers describe the 17-23 stretch as the actin-binding site of Tβ4, and it is one of the two. TB-500 carries the motif but not the N-terminal helix that, in the whole protein, also takes part in binding. What the isolated fragment does on its own is therefore a question for experiments run with the fragment, not something to infer from protein data.

Which molecule did the paper use?

Much of what is written about TB-500 draws on research into full-length thymosin β4, the protein surveyed in Goldstein and colleagues' 2012 review. Before leaning on a citation, check its methods for which molecule was actually used, at what concentration and in which model. Mass is the quickest way to tell the candidates apart:

A product sold as "thymosin beta-4" at research-peptide prices deserves a second look. A full-length 43-residue protein and a seven-residue fragment are different materials, and the mass on the certificate is what settles which one is in the vial.

How analytical laboratories identify TB-500

The clearest published work on TB-500 as such is analytical, and it comes from anti-doping laboratories.

• Ghent, 2012. Esposito and colleagues at a doping-control laboratory detected and identified Ac-LKKTETQ in a TB-500 product by liquid chromatography with high-resolution (Orbitrap) mass spectrometry, made the peptide by solid-phase synthesis as a reference standard, and proposed an LC-MS/MS approach for finding it in plasma and urine.
• Hong Kong, 2012. Ho and colleagues at a racing laboratory built an LC-MS method for the acetylated peptide and its breakdown products in equine urine and plasma, after first identifying candidate metabolites in vitro, with detection limits in the hundredths of a nanogram per milliliter.

The same logic serves a buyer. On a mass spectrum, Ac-LKKTETQ should give a singly protonated ion near m/z 889.5 and, because its two lysines readily pick up protons, a doubly protonated ion near m/z 445.3. Dominant ions near 847.5 and 424.3 point to the unacetylated peptide; a protein-sized mass points to full-length Tβ4.

What a TB-500 certificate should show

1. Identity by mass spectrometry at the expected mass for Ac-LKKTETQ, not a generic "conforms".
2. Purity by HPLC, given as a figure for that lot with the method named.
3. The molecule named precisely: fragment or full-length protein, acetylated or not.
4. Format and concentration for the material as supplied.
5. A batch number and a date that match the vial in hand.

Ours are published by batch in the COA library, and the Certificate of Analysis guide explains how to read one.

TB-500 next to BPC-157, and in blends

TB-500 is most often discussed alongside BPC-157, a synthetic 15-residue peptide. The two are chemically unrelated: different parent sequences, different lengths, nothing shared. We supply the pair together as the Wolverine Stack; with GHK-Cu added it becomes the Glow Stack, and the Klow Stack adds KPV as well. A blend suits a study that calls for the combination and limits one that does not, because no component can be varied alone, which is why TB-500 is also sold as a single vial. The BPC-157 vs TB-500 guide compares the two compounds directly.

Format, storage and US supply

Most TB-500 listed in the US is lyophilized powder. Ours ships pre-mixed as a solution, so the dissolving step, with its solvent and pH choices, happens once under controlled conditions instead of at each bench; the pre-mixed vs lyophilized guide sets out the trade-offs. As a solution it needs 2-8 °C, a sealed vial, protection from light and no freezing.

Orders are dispatched cold-chain from the warehouse that serves the United States, with tracked delivery across the country next business day, priced in US dollars and payable by Visa, Mastercard or Apple Pay. Strengths and batch details are on the TB-500 product page.

Status: the FDA and WADA

• FDA. TB-500 is not an FDA-approved drug and has not been evaluated by the FDA. It is supplied for in-vitro laboratory research only, not for human or veterinary use.
• WADA. The World Anti-Doping Agency's Prohibited List names thymosin β4 and its derivatives, with TB-500 given as an example, among the growth factors and modulators prohibited at all times. BPC-157 is on the List as well. The List is revised every year, so the current edition is the authority, not any supplier's page.

What this page does not cover

We do not publish dosing or administration information for TB-500. Concentrations in published work are specific to each model and endpoint, and the primary literature is the place to find them.

Related reading

• What is BPC-157? · BPC-157 vs TB-500 · What is the Wolverine Stack? · What is KLOW peptide? · Pre-mixed vs lyophilized peptides