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TB-500

A product name that analytical work resolved to Ac-LKKTETQ, the N-terminally acetylated 17 to 23 fragment of thymosin beta-4. The fragment, the unacetylated fragment and the full-length protein are three different materials, and the cited papers keep them apart.

For in-vitro research only.Reviewed 2026-09-20

Key findings

  • High resolution mass spectrometry of the TB-500 formulation identified the N-terminally acetylated 17 to 23 fragment of human thymosin beta-4, and the same fragment was then made by solid phase synthesis to confirm the assignment. [1][2]
  • An examination of products sold online as TB500 and TB1000 found their contents were not systematically consistent with the descriptions attached to those names. [3]
  • In work on the full-length protein, a thymosin beta-4 variant missing residues 17 to 23 showed no interaction with G-actin at all, which places the actin binding motif inside exactly the stretch the product name refers to. [4]
  • In two separate assays the parent acetylated fragment was not the active species: human hepatic stellate cell effects tracked the unacetylated 17 to 23 peptide, and scratch closure in fibroblasts tracked the metabolite Ac-LKKTE rather than the parent. [5][6]

Identity and structure

Sequence
Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln, written Ac-LKKTETQ, with artificial acetylation of the N-terminus [1][2]
Position in the parent protein
Residues 17 to 23 of thymosin beta-4, described in the cited work as the region within that protein responsible for actin binding [2][1]
Not the full-length protein
Papers on thymosin beta-4 itself used the full-length protein, a different material from this seven-residue fragment; matching a lot to that literature requires checking which material the paper used [4][1]
Product content
Content of material sold under the TB500 and TB1000 names was found not to be systematically consistent with the descriptions given for those names [3]
Form as supplied
Sterile lyophilized powder

Mechanism as studied

Work on the full-length protein locates the relevant function in this stretch of sequence. Thymosin beta-4 sequesters G-actin in a one to one complex, single residue substitutions in the N-terminal helix and in the putative actin binding motif change the stability of that complex, and a variant lacking residues 17 to 23 does not interact with G-actin at all. [4]

Cross-linking of the full-length protein to actin places three contact sites along actin subdomains 1 to 3, including a lysine within the 17 to 23 stretch cross-linked to the actin N-terminus. The authors describe the complex as flexible rather than rigid, so the fragment corresponds to one contact region and not the whole interface. [7]

Where the fragment itself has been compared against other parts of the protein, the 17 to 23 peptide and not the 1 to 15 peptide blocked Akt phosphorylation at both sites and the downstream PRAS40 phosphorylation in human hepatic stellate cells. That work used the unacetylated form of the fragment, so its acetylation state differs from the product. [5]

Research findings

Analytical
System
The TB-500 formulation, plus the same fragment prepared independently by solid phase peptide synthesis
Measured
Identification of the peptide present in the formulation by high performance liquid chromatography with high resolution mass spectrometry on an Orbitrap Exactive instrument, and an analytical strategy for plasma and urine on a triple quadrupole instrument
Reported
The N-terminally acetylated 17 to 23 fragment of human thymosin beta-4, Ac-LKKTETQ, was detected and identified in TB-500, and the synthesized fragment supported that identification. [1]
Analytical
System
Three products offered for sale online under the names TB500, TB1000 and SGF1000
Measured
Composition of each product by an extensive analysis of its contents
Reported
The contents of the TB500 and TB1000 products were not systematically consistent with their former descriptions, and SGF1000 was found to be mainly sheep extracellular matrix and blood proteins with the purported growth promoters present only at a very diluted signal. [3]
Analytical
System
Equine urine and plasma, with candidate metabolites first identified from in-vitro studies
Measured
Simultaneous detection of N-acetylated LKKTETQ and its metabolites by solid phase extraction on ion exchange cartridges followed by liquid chromatography mass spectrometry, identified by retention time and product ion abundance
Reported
N-acetylated LKKTETQ was detected and confirmed at 0.02 ng/mL in equine plasma and 0.01 ng/mL in equine urine, and the parent peptide and its metabolites were confirmed in equine urine and plasma collected after exposure. [2]
In vitro
System
Commercial proteolytic enzymes, human blood serum, human liver and kidney microsomes and liver S9 fraction, with this fragment studied alongside growth hormone releasing peptides and desmopressin
Measured
Biotransformation of each peptide in each incubation system and the diversity of metabolites produced
Reported
High metabolic activity was seen for this fragment with human kidney microsomes and liver S9 fraction, peptides degraded through cleavage of all bonds regardless of protective modifications in the primary structure, fewer metabolites came from human serum, and the commercial proteases gave a poor metabolite pattern of nonspecific hydrolysis products. [8]
In vitro
System
Human serum and enzyme incubation systems with synthesized authentic standards, urine from rats given the fragment, and fibroblast cultures
Measured
Simultaneous quantification of the parent fragment and its metabolites by UHPLC Orbitrap tandem mass spectrometry, plus cytotoxicity and scratch closure of the parent and each metabolite in fibroblasts
Reported
Ac-LK was the metabolite present at highest concentration in the early intervals and Ac-LKK persisted longest. Neither the parent nor its metabolites were cytotoxic, and only Ac-LKKTE showed a significant scratch closure effect against control, leading the authors to attribute previously reported activity to that metabolite rather than to the parent. [6]
In vitro
System
Early passage human hepatic stellate cell cultures stimulated with PDGF-BB, comparing the unacetylated 17 to 23 peptide LKKTETQ against the 1 to 15 peptide of the same protein
Measured
PDGF beta receptor, alpha smooth muscle actin and collagen 1 by RT-PCR and Western blot, Akt phosphorylation at T308 and S473, PRAS40 and mTOR phosphorylation, and proliferation and migration assays
Reported
The 17 to 23 peptide but not the 1 to 15 peptide blocked the PDGF-BB driven rise in PDGF beta receptor, alpha smooth muscle actin and collagen 1, blunted Akt phosphorylation at both sites, reduced PRAS40 phosphorylation, and lowered proliferation and migration. The 1 to 15 peptide blocked Akt phosphorylation only at S473 and changed neither proliferation nor migration. [5]
In vitro
System
Full-length thymosin beta-4 and E. coli generated variants of the full-length protein, none of them N-terminally acetylated, in complex with G-actin
Measured
Stability of the complex formed with G-actin for each variant, and G-actin sequestering activity, including a variant lacking residues 17 to 23
Reported
The complex with nonacetylated full-length protein was more stable than with the naturally acetylated protein, complexes with two lysine substituted variants were 15 times less stable, sequestering activity tracked complex stability except for one variant where it was attenuated, and the variant missing residues 17 to 23 showed no interaction with G-actin. [4]
Analytical
System
The complex of actin with full-length thymosin beta-4, stabilised by chemical and enzymatic cross-linking
Measured
Location of cross-linking sites after SDS-PAGE separation, tryptic in-gel digestion and high resolution HPLC electrospray mass spectrometry, plus molecular modelling of the resulting conformation
Reported
Three contact sites were identified after chemical cross-linking, including K18 or K19 of the protein to the actin N-terminal residues, and three more after enzymatic cross-linking. The cross-linked fragments appeared alongside their non-cross-linked counterparts, so the sites were not formed in parallel, and the complex was described as flexible and localised along actin subdomains 1 to 3. [7]
Order TB-500 with the certificate for the lot that ships

Handling for in-vitro work

Container surfaces in cited work
Recovery of this peptide from laboratory surfaces varied with the consumable used, and the authors report that low-bind glassware and plasticware were not advantageous in every case; selection should follow the physicochemical properties of the peptide [9]
Solvent in cited work
Dissolved in aqueous incubation media and serum in the cited metabolism experiments [8]
Storage
Lyophilized at -20 °C, dark and dry; reconstituted aliquots kept cold and used promptly

Open questions

  • The acetylated fragment sold under this name, the unacetylated fragment used in the hepatic stellate cell work, and the full-length protein are three different materials, and the cited work does not establish that a result for one carries to another.
  • One cited metabolism paper attributes scratch closure to the metabolite Ac-LKKTE rather than to the parent fragment, which leaves the active species in fibroblast assays unsettled.
  • The analytical work that assigned the sequence examined particular product samples, and another cited paper found product content inconsistent with the name, so a supplied lot needs its own identity test.
  • No cited paper characterises the acetylated fragment in complex with actin; the structural contact data come from the full-length protein.

Clinical and veterinary literature attached to this name concerns finished preparations rather than a research material, and is out of scope for this profile.

Lot records

Check the record for the exact material you order. A published paper and a batch certificate answer different questions.

Read a certificate of analysis ↗

References

  1. Esposito S, Deventer K, Goeman J, et al. Synthesis and characterization of the N-terminal acetylated 17-23 fragment of thymosin beta 4 identified in TB-500, a product suspected to possess doping potential. Drug testing and analysis. 2012.

    PubMed 22962027 · doi:10.1002/dta.1402

  2. Ho EN, Kwok WH, Lau MY, et al. Doping control analysis of TB-500, a synthetic version of an active region of thymosin β₄, in equine urine and plasma by liquid chromatography-mass spectrometry. Journal of chromatography. A. 2012.

    PubMed 23084823 · doi:10.1016/j.chroma.2012.09.043

  3. Delcourt V, Garcia P, Chabot B, et al. TB500/TB1000 and SGF1000: A scientific approach for a better understanding of misbranded and adulterated drugs. Drug testing and analysis. 2023.

    PubMed 36482504 · doi:10.1002/dta.3421

  4. Zoubek RE, Hannappel E Influence of the N terminus and the actin-binding motif of thymosin beta4 on its interaction with G-actin. Annals of the New York Academy of Sciences. 2007.

    PubMed 17495251 · doi:10.1196/annals.1415.026

  5. Shah R, Reyes-Gordillo K, Rojkind M Thymosin β4 inhibits PDGF-BB induced activation, proliferation, and migration of human hepatic stellate cells via its actin-binding domain. Expert opinion on biological therapy. 2018.

    PubMed 30063851 · doi:10.1080/14712598.2018.1478961

  6. Rahaman KA, Muresan AR, Min H, et al. Simultaneous quantification of TB-500 and its metabolites in in-vitro experiments and rats by UHPLC-Q-Exactive orbitrap MS/MS and their screening by wound healing activities in-vitro. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 2024.

    PubMed 38382158 · doi:10.1016/j.jchromb.2024.124033

  7. Knop J, App C, Horn AH, et al. High-resolution HPLC-ESI-MS characterization of the contact sites of the actin-thymosin β(4) complex by chemical and enzymatic cross-linking. Biochemistry. 2013.

    PubMed 23924371 · doi:10.1021/bi400664k

  8. Zvereva I, Semenistaya E, Krotov G, et al. Comparison of various in vitro model systems of the metabolism of synthetic doping peptides: Proteolytic enzymes, human blood serum, liver and kidney microsomes and liver S9 fraction. Journal of proteomics. 2016.

    PubMed 27569051 · doi:10.1016/j.jprot.2016.08.016

  9. Judák P, Van Eenoo P, Deventer K Adsorption effects of the doping relevant peptides Insulin Lispro, Synachten, TB-500 and GHRP 5. Analytical biochemistry. 2017.

    PubMed 28887173 · doi:10.1016/j.ab.2017.09.003

Publication records fetched from PubMed on 2026-09-20. Profile text reviewed 2026-09-20.