TB500 vs Thymosin Beta-4: Not the Same Molecule (2026)

Compound researchOctober 21, 202616 min readVortex Research Editorial Team

Research-use-only context. This article describes the chemical identity, nomenclature and analysis of two related research molecules. Nothing here is guidance for human or veterinary use.

On TB-500 vs thymosin beta-4, the short answer is that the names often point to different molecules. Thymosin beta-4 (Tβ4) is a natural 43-residue, N-terminally acetylated protein of about 4963 g/mol. In the analytical literature, “TB-500” is a synthetic seven-residue peptide, Ac-LKKTETQ (residues 17 to 23 of Tβ4), of about 889 g/mol. In the market the name is used for both, so only a mass measurement shows which one is in a given vial.

TB-500 vs thymosin beta-4 at a glance

Property	Thymosin beta-4 (full protein)	TB-500 as described in anti-doping papers
What it is	Natural protein found in most cell types	Synthetic fragment of Tβ4
Length	43 residues	7 residues
Sequence	Ac-SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES	Ac-LKKTETQ (Tβ4 residues 17-23)
Molecular formula	C212H350N56O78S	C38H68N10O14
Molecular mass	About 4963 g/mol (average)	About 889.0 g/mol (monoisotopic 888.49 Da)
CAS number	77591-33-4 (INN name timbetasin)	885340-08-9 is listed for Ac-LKKTETQ in PubChem
First described	1981, sequence from calf thymus	2012, identified in a product sold as TB-500
Literature	About 1,175 PubMed records; actin biochemistry, cell and animal work	Fewer than 40 PubMed records; mostly doping-control analytics

Source: PubChem CID 16132341 (timbetasin) and CID 62707662 (TB-500); references 1, 8 and 9; PubMed counts from a search run on 21 October 2026.

Research materials: Vortex supplies TB-500 as a lyophilised research reagent: TB-500. The product page lists the full-length thymosin beta-4 identity (CAS 77591-33-4, C212H350N56O78S, 4963.55 g/mol). Lab reports: /coa/.

What thymosin beta-4 is

Thymosin beta-4 was first isolated from calf thymus as part of a mixture called thymosin fraction 5 [3]. In 1981, Low and colleagues reported its full sequence: 43 amino acids with an acetyl group blocking the N-terminus, an isoelectric point of 5.1 and a molecular mass they gave as 4982 [1]. The average mass calculated from the accepted formula today is about 4963. At the time the protein was thought to be a thymic hormone [1, 3].

That view changed in 1991. Safer and colleagues were studying Fx, a small peptide that holds unpolymerised actin in human platelets. They sequenced Fx and found it was identical to thymosin beta-4 [2]. Tβ4 is now understood as the main actin-sequestering peptide inside many eukaryotic cells. It belongs to a family of beta-thymosins, N-terminally acetylated peptides of about 5 kDa and 40 to 44 residues [3]. Its international nonproprietary name is timbetasin.

The protein is modified after it is made. The initiator methionine is removed and the new N-terminus is acetylated. Proteomic work has also found acetylated lysines and phosphorylated threonines, and an enzyme, prolyl oligopeptidase, cuts the N-terminal tetrapeptide Ac-SDKP from it [4]. Each of these forms has its own mass, which matters for anyone reading a mass spectrum of Tβ4.

Tβ4 has no disulfide bonds and little fixed structure on its own. Its actin-binding behaviour is tied to specific short regions of the chain. That fact is the root of the TB-500 naming problem.

Actin binding: the core biochemistry

Actin exists as single subunits (G-actin) and as filaments (F-actin). Tβ4 binds G-actin in a one-to-one complex and stops it from joining filaments [2]. In resting human platelets, at least half of the actin is unpolymerised, and most of that pool is bound to Tβ4 [2].

Biochemists have mapped which parts of the chain matter, using cell-free assays:

A 1993 study used the DNase I inhibition assay, which detects G-actin. It confirmed one-to-one sequestration by Tβ4 and found that fragments covering residues 24 to 43 or 13 to 43 did not sequester actin. Tβ4 with its single methionine oxidised to the sulfoxide still did [5].
A 2007 study made Tβ4 variants with single substitutions. A variant missing residues 17 to 23 showed no interaction with G-actin. Variants made in bacteria lacked the natural N-terminal acetyl group, and that changed how stable the actin complex was [6].
Reviews describe the central actin-binding domain as the LKKTET stretch around residues 17 to 23, with the TB-500 heptapeptide adding the following glutamine [7]. Doping-control papers define the TB-500 heptapeptide as residues 17 to 23, LKKTETQ, the segment responsible for actin binding [8, 9].

So the fragment sold as TB-500 is the actin-binding segment of a much larger protein. It is not a shortened copy that keeps every property of the whole. The N-terminal region, the C-terminal region and the post-translational modifications are all absent [4, 5, 6].

Where the name TB-500 comes from, and where it is inconsistent

The clearest published definitions of TB-500 come from anti-doping laboratories. In 2012, a Ghent University group analysed a product sold as TB-500 by high-resolution mass spectrometry. They identified the N-terminally acetylated 17-23 fragment of human Tβ4, Ac-LKKTETQ, and made it by solid-phase peptide synthesis as a reference standard [8]. The same year, the Hong Kong Jockey Club laboratory described a veterinary preparation called TB-500 whose key ingredient was LKKTETQ with an added N-terminal acetyl group, and published an LC-MS method for it in horse urine and plasma [9]. The same laboratory later called N-acetylated LKKTETQ “the active ingredient of TB-500” in a seven-peptide method [10].

The name is not used consistently elsewhere:

A 2023 French analysis of products sold online as TB500 and TB1000 found that their contents were not consistently what earlier descriptions said [11].
Some research papers use “TB-500” as a label for full synthetic Tβ4, without giving a sequence.
Many supplier listings pair the name TB-500 with the full protein’s CAS number (77591-33-4) and mass (about 4963 g/mol). The Vortex product page does the same.
Other listings use the fragment’s identity, Ac-LKKTETQ, at about 889 g/mol.
The fragment itself is sometimes described as residues 17-22 (LKKTET) and sometimes as 17-23 (LKKTETQ) [7, 8].

The two molecules differ more than five-fold in mass. A label alone does not say which one a vial holds. A mass spectrum does.

Two literatures, two molecules

A PubMed search on 21 October 2026 returned about 1,175 records that name thymosin beta-4 in the title or abstract. The same search for TB-500, TB500 or LKKTETQ returned 39 records, several of them unrelated matches. Among the primary papers in that smaller set, the largest group comes from doping-control laboratories in Ghent, Hong Kong, Seoul and France [8, 9, 10, 11, 12, 13].

Most of the widely cited cell and rodent studies of Tβ4 used the full 43-residue protein, not the fragment. Their findings cannot be attributed to Ac-LKKTETQ, and the fragment papers cannot be attributed to the full protein. The cell and molecular studies summarised further down state which molecule each one used. Clinical development of thymosin beta-4, where it has happened, used the full-length protein made to drug-development standards. No human study of Ac-LKKTETQ has been published in PubMed as of October 2026.

The two molecules also break down differently, which is useful analytically because each leaves its own trace:

Molecule	System	Main breakdown products or behaviour reported	Ref.
Ac-LKKTETQ	Horse urine and plasma; in vitro	Parent peptide and metabolites, including Ac-LK	[9, 10]
Full Ac-Tβ4	Enzyme, cell and rat systems	Ac-Tβ1-14 as a common metabolite, proposed as a doping-test marker	[12]
TB500 (with 53 other compounds)	Dried blood spots, serum, plasma	Stable at −20 °C for at least two months; complete degradation in serum at 4 °C and 22 °C; detectable in dried matrices	[13]

Source: abstracts of the cited papers, checked on PubMed. “Molecule” is as stated by the authors.

What cell and molecular studies have measured

Beyond identity and analytics, the thymosin beta-4 record includes cell-free binding measurements, a crystal structure and assays in human cells. Each finding below is stated in the system the authors used, together with the molecule they used. These are molecular and cellular measurements. They do not describe outcomes in an organism.

Binding stoichiometry and affinity in vitro

Tβ4 binds G-actin in a one-to-one complex, a stoichiometry reported for Tβ4 purified from platelets and in the DNase I inhibition assay [2, 5]. In 1993, Carlier and colleagues measured the interaction with purified actin in vitro [17]. They reported that Tβ4 had about 50-fold higher affinity for MgATP-actin than for MgADP-actin. Kinetic experiments and calculations showed that this nucleotide dependence gives a route for ADP to release G-actin from Tβ4, and that profilin speeds this release by speeding the dissociation of ATP from actin [17].

Crystal structure of the actin complex

To stabilise Tβ4 on actin for crystallography, Irobi and colleagues fused the C-terminal half of Tβ4 to gelsolin domain 1 in 2004 [18]. The hybrid still sequestered actin monomers, and they solved its complex with actin at 2 Å resolution. The structure showed Tβ4 capping both ends of the actin monomer. It also showed a small overlap between the Tβ4 and profilin binding sites on actin, which the authors proposed mediates the exchange of actin between the two proteins [18]. Tβ4 is the prototype of the WH2 family of actin-binding motifs, so this structure is also a reference point for that family.

Tβ4 and G-actin in isolated human cells

Cassimeris and colleagues measured Tβ4 in human polymorphonuclear leukocytes (neutrophils) in 1992 [19]. By reverse-phase HPLC they found about 169 fg of Tβ4 per cell, which they calculated as a cytoplasmic concentration of about 149 µM. On non-denaturing gels, a large fraction of the G-actin from resting cells ran with the mobility of the G-actin/Tβ4 complex, and that complex fell after chemoattractant stimulation. Pyrenyl-actin polymerisation assays on cell supernatants were consistent with Tβ4 being released as actin polymerised, with no change in its affinity for G-actin [19].

Endothelial cell migration assays

Malinda, Goldstein and Kleinman tested Tβ4 on cultured human umbilical vein endothelial cells (HUVECs) in 1997 [20]. In Boyden chamber assays, HUVEC migration toward Tβ4 was four- to sixfold higher than toward medium alone. In a scratch assay on a HUVEC monolayer, cells moved into the scratched area faster with Tβ4 present. The treated cells also produced more matrix metalloproteinases [20].

In 2003, Philp and colleagues asked which part of the chain carries that activity [21]. They compared naturally occurring Tβ4, proteolytic fragments and synthetic peptides in HUVEC migration assays and in sprouting assays on isolated chick aortic arches. Full Tβ4 and a synthetic seven-residue peptide matching the actin-binding motif showed near-identical activity, while peptides lacking any part of that motif were inactive. The seven-residue peptide blocked HUVEC adhesion to Tβ4, and adding soluble actin inhibited both adhesion and sprouting [21]. The abstract does not state whether the synthetic peptide carried the N-terminal acetyl group that defines TB-500 in the doping-control papers.

Study	Molecule used	System	What was measured
Safer et al., 1991 [2]	Tβ4 purified from platelets (as Fx)	Purified peptide with G-actin, in vitro (non-denaturing gels)	1:1 complex with actin monomers; inhibition of polymerisation
Hannappel and Wartenberg, 1993 [5]	Full Tβ4, its fragments and oxidised Tβ4	DNase I inhibition assay (cell-free)	G-actin sequestration; fragments 24-43 and 13-43 inactive
Carlier et al., 1993 [17]	Tβ4	Purified actin, in vitro	Affinity for MgATP-actin versus MgADP-actin
Irobi et al., 2004 [18]	C-terminal half of Tβ4 fused to gelsolin domain 1	X-ray crystallography	Structure of the actin complex at 2 Å
Cassimeris et al., 1992 [19]	Native Tβ4 inside the cells	Isolated human neutrophils	Tβ4 content per cell; G-actin/Tβ4 complex
Malinda et al., 1997 [20]	Full Tβ4	Cultured HUVECs	Directional migration; metalloproteinase production
Philp et al., 2003 [21]	Full Tβ4, fragments, synthetic seven-residue motif peptide	Cultured HUVECs; chick aortic arch explants	Migration, adhesion and sprouting

Source: abstracts of references 2, 5 and 17 to 21, checked on PubMed. None of these studies reports using Ac-LKKTETQ under the name TB-500.

The analytical consequence: mass spectrometry tells the molecules apart

For TB-500 vs thymosin beta-4, analytical identity is the whole question. The two candidates are easy to separate by mass:

Ac-LKKTETQ has a monoisotopic mass of about 888.49 Da. Electrospray spectra show it mainly as the singly protonated ion near m/z 889.50 and the doubly protonated ion near m/z 445.25.
Full Tβ4 has an average mass of about 4963 Da. Electrospray spectra show a series of multiply charged ions, for example near m/z 993.7 for the 5+ ion and m/z 828.3 for the 6+ ion, that deconvolute to that mass.

Anti-doping laboratories already rely on this. They identified Ac-LKKTETQ in a TB-500 product by high-resolution MS [8]. They detect the fragment and its metabolites in horse samples by LC-MS, with detection limits below 50 pg/mL in plasma [9, 10]. They track full Tβ4 through its own metabolite, Ac-Tβ1-14 [12]. Top-down high-resolution MS of the thymosins also resolves their natural variants: the methionine sulfoxide form, C-terminally truncated forms and lysine-acetylated forms of Tβ4 have all been characterised this way [15].

Low-concentration work has one more trap. A Ghent study of four doping-relevant peptides, TB-500 among them, found that the amount recovered depended on the surface of the tubes and vials used, and that expensive low-bind consumables were not the best choice in every case [14]. Losses to plastic or glass can look like low content if the method does not account for them.

HPLC alone is weaker evidence of identity. An HPLC identity call means a peak eluted where the laboratory’s reference standard eluted. If the reference standard was itself labelled “TB-500”, the identity result inherits the same naming ambiguity. HPLC purity is useful, but it measures how clean the main peak is, not which molecule it is. Our guide to reading a peptide COA covers the difference between identity, purity and net content.

Checking a lot of TB-500

A report for a TB-500 lot should state identity by mass spectrometry with the observed mass, HPLC purity and net content. The observed mass is the line that settles which molecule is present.

Vortex has not published a standalone TB-500 report. The report listed at /coa/ is a partner-facility report for the BPC-157 + TB-500 (10/10 mg) blend, lot PS07-BCTB20. ILS Laboratories in San Diego issued it to Vortex’s wholesale partner facility on the same batch Vortex stocks. It was tested on 08/14/2026 and lists HPLC purity of 99.94% and net content of 21.12 mg (BPC-157 10.56 mg, TB-500 10.56 mg). It also lists identity (by HPLC), heavy metals (ICP-MS), sterility (PCR), endotoxin (USP <85>) and a fentanyl screen. The summary page does not state a mass-spectrometry result or say which TB-500 molecule the identity test referred to. Researchers who need that answer for their work should request mass data for the lot or arrange independent testing. Our post on evaluating a research peptide supplier lists the questions worth asking.

Stability chemistry of Tβ4 and its fragment

Full Tβ4 contains one methionine (position 6), which can oxidise to the sulfoxide, adding 16 Da to the measured mass [5, 15]. It also contains one asparagine (position 26) and three glutamines, which can deamidate, each adding about 1 Da. Ac-LKKTETQ contains no methionine or asparagine but does contain one C-terminal glutamine. These features decide how each molecule looks on a mass spectrum after storage, and they are another reason to check observed mass against the expected value.

Lyophilised thymosin material can persist for decades. In 2018, a sample of thymosin fraction 5 that had been stored lyophilised at room temperature for 37 years was re-analysed by high-resolution MS. The authors detected more than 100 monoisotopic masses, including intact Tβ4 alongside truncated forms of Tβ4 and other thymosins [16]. Intact protein survived, but so did a long tail of breakdown products, which only a mass measurement reveals.

The Vortex product page lists −20 °C away from light for unopened lyophilised powder. Our post on lyophilised peptide storage and stability covers moisture, light and temperature. TB-500 is also sold in a blend with BPC-157; for that compound see what BPC-157 is. For the published record on the two together, see BPC-157 and TB-500: what research exists on the pair.

Open questions

Which molecule is in products sold as TB-500? Only lot-level mass data can answer this.
Do products labelled TB-500 contain the acetylated or the free N-terminal form of LKKTETQ? The two differ by 42 Da.
What is the forced-degradation profile of Ac-LKKTETQ as a lyophilised powder? Published stability data cover biological matrices only [13].

Research materials from Vortex Research. Lyophilised research peptides, with lot reports from independent labs published for each tested batch.

Browse the catalogue → · View lab reports → · More from the research library →

Frequently asked questions

Is TB-500 the same as thymosin beta-4?

Not in the analytical literature. Thymosin beta-4 is a 43-residue protein of about 4963 g/mol. Anti-doping studies define TB-500 as Ac-LKKTETQ, a seven-residue fragment of about 889 g/mol. Some suppliers and papers still use “TB-500” for the full protein.

What is the sequence and mass of TB-500?

In the anti-doping literature, TB-500 is Ac-LKKTETQ: residues 17 to 23 of thymosin beta-4 with an acetylated N-terminus. Its formula is C38H68N10O14, its average mass is about 889.0 g/mol and its monoisotopic mass is about 888.49 Da.

Where does the name TB-500 come from?

TB-500 is a market name, not a scientific one. Doping-control laboratories in Ghent and Hong Kong defined it by analysing products sold under that name in 2012 and identifying Ac-LKKTETQ as the key ingredient. Product labels still apply the name to both the fragment and the full protein.

Did cell studies of thymosin beta-4 use TB-500?

Mostly not. The cell-free binding studies and the endothelial cell migration studies used the full 43-residue protein, and the crystal structure used half of it fused to another protein. A 2003 study in human umbilical vein endothelial cells compared full Tβ4 with a synthetic seven-residue actin-binding peptide and reported near-identical migration activity, but its abstract does not say whether that peptide was acetylated like TB-500.

How can a lab tell which molecule is in a vial labelled TB-500?

Mass spectrometry. Ac-LKKTETQ has a monoisotopic mass of about 888.49 Da, while full Tβ4 has an average mass of about 4963 Da. An HPLC identity result only shows that a peak matched a reference standard, so it inherits any ambiguity in how that standard was named.

How this article was prepared

Written by the Vortex Research Editorial Team. Sources were found through PubMed searches for thymosin beta-4, Tβ4, TB-500, TB500 and LKKTETQ, and through review articles. Literature counts come from title-and-abstract searches run on 21 October 2026. Every PMID below was checked against PubMed for title, journal and year, and each citation was matched to the sentence it supports. For each study we recorded which molecule the authors said they used. This article covers identity, nomenclature, chemistry, analysis and a set of cell and molecular measurements; it does not summarise the rodent pharmacology. Product facts come from the live Vortex product and /coa/ pages; chemical identities come from PubChem.

References

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Safer D, Elzinga M, Nachmias VT. Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable. J Biol Chem. 1991;266(7):4029-4032. PMID: 1999398.
Mannherz HG, Hannappel E. The beta-thymosins: intracellular and extracellular activities of a versatile actin binding protein family. Cell Motil Cytoskeleton. 2009;66(10):839-851. PMID: 19405116. doi:10.1002/cm.20371
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Carlier MF, Jean C, Rieger KJ, Lenfant M, Pantaloni D. Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics. Proc Natl Acad Sci U S A. 1993;90(11):5034-5038. PMID: 8506348. doi:10.1073/pnas.90.11.5034
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