Learn
TB-500
Technical Compound Reference | Orion Peptides Learning Library
TB-500 is a synthetic heptapeptide identified in analytical research as the N-terminally acetylated sequence Ac-LKKTETQ. It corresponds to amino acid residues 17–23 of thymosin beta-4, a naturally occurring 43-residue peptide involved in actin-related cellular processes.
The seven-residue fragment is chemically distinct from the complete thymosin beta-4 molecule.
TB-500 has the molecular formula C₃₈H₆₈N₁₀O₁₄ and an average molecular weight of approximately 889.0 g/mol. Its structure contains two consecutive lysine residues, two threonine residues, one glutamic acid residue, and an N-terminal acetyl group.
These characteristics influence its charge, molecular recognition, fragmentation behavior, and chromatographic analysis.
Published analytical studies have investigated the identity of TB-500 using high-resolution mass spectrometry, peptide synthesis, liquid chromatography, and metabolite identification.
This technical reference focuses on the compound’s amino acid structure, relationship to thymosin beta-4, analytical characterization, molecular properties, and stability considerations.
1. The TB-500 Identity Problem: Fragment or Full-Length Peptide?
The name TB-500 is not always used consistently across research catalogs.
Some references identify TB-500 as a short, N-acetylated fragment of thymosin beta-4.
Other commercial descriptions use TB-500 to refer to the complete 43-amino-acid parent peptide.
These definitions must not be treated as interchangeable.
Two Different Molecular Structures
| Chemical characteristic | TB-500 fragment | Full-length thymosin beta-4 |
|---|---|---|
| Molecular identity | Ac-LKKTETQ | Thymosin beta-4 |
| Amino acid count | 7 | 43 |
| Molecular formula | C₃₈H₆₈N₁₀O₁₄ | C₂₁₂H₃₅₀N₅₆O₇₈S |
| Average molecular weight | Approximately 889.0 g/mol | Approximately 4,963 g/mol |
| CAS number | 885340-08-9 | 77591-33-4 |
| PubChem CID | 62707662 | 16132341 |
| N-terminal modification | Acetylated leucine | Acetylated serine |
| C-terminal residue | Glutamine | Serine |
| Structure | Short linear fragment | Full-length linear peptide |
The difference in molecular size is substantial.
A reference mass of approximately 889 Da describes the N-acetylated heptapeptide.
A reference mass near 4,963 Da describes the complete thymosin beta-4 molecule.
How Was TB-500 Identified?
In 2012, researchers published a study describing the identification of the N-terminally acetylated fragment Ac-LKKTETQ in a material marketed as TB-500.
They used high-performance liquid chromatography combined with high-resolution mass spectrometry.
The researchers also synthesized the same fragment through solid-phase peptide synthesis to support their chemical identification.
This study provides a direct analytical foundation for defining TB-500 as Ac-LKKTETQ.
Primary reference: Esposito S. et al. (2012), Synthesis and Characterization of the N-Terminal Acetylated 17–23 Fragment of Thymosin Beta 4 Identified in TB-500.
https://doi.org/10.1002/dta.1402
2. TB-500 Molecular Profile
The following specifications describe the N-acetylated seven-residue fragment.
| Property | Technical information |
|---|---|
| Compound name | TB-500 |
| Chemical designation | N-acetyl thymosin beta-4 fragment 17–23 |
| Sequence notation | Ac-LKKTETQ-OH |
| Three-letter sequence | Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln-OH |
| CAS number | 885340-08-9 |
| PubChem CID | 62707662 |
| Molecular formula | C₃₈H₆₈N₁₀O₁₄ |
| Average molecular weight | Approximately 889.0 g/mol |
| Monoisotopic mass | Approximately 888.49 Da |
| Peptide length | 7 residues |
| Chemical classification | Synthetic N-acetylated heptapeptide |
| N-terminal group | Acetylated |
| C-terminal group | Free carboxylic acid |
| Cysteine residues | None |
| Disulfide bridges | None |
| Parent peptide | Thymosin beta-4 |
| Structural region | Parent residues 17–23 |
These properties refer to the defined fragment molecule.
The physical form, impurity profile, counterion content, and chromatographic purity of individual research materials are separate characteristics that require batch-specific documentation.
3. Reading the Seven-Amino-Acid Sequence
TB-500 contains a short peptide backbone with seven amino acid residues.
The sequence is:
Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln-OH
In one-letter notation:
Ac-LKKTETQ-OH
The letters correspond to the following residues.
| Position | Amino acid | Symbol |
|---|---|---|
| 1 | Leucine | L |
| 2 | Lysine | K |
| 3 | Lysine | K |
| 4 | Threonine | T |
| 5 | Glutamic acid | E |
| 6 | Threonine | T |
| 7 | Glutamine | Q |
The sequence includes several chemical groups that influence molecular charge, polarity, and analytical detection.
Leucine
Leucine is a hydrophobic amino acid with a branched aliphatic side chain.
It occupies the first position in TB-500.
The leucine residue carries the N-terminal acetyl modification.
Lysine
The fragment contains two consecutive lysine residues.
Lysine has a basic side-chain amino group that can carry a positive charge under many aqueous conditions.
The two lysines are important to the fragment’s acid-base behavior.
Threonine
Two threonine residues occur in the sequence.
Threonine contains a hydroxyl group that contributes to its polarity.
Glutamic Acid
Glutamic acid contains a carboxyl-containing side chain.
It can carry a negative charge depending on solution pH.
Glutamine
Glutamine is the C-terminal amino acid.
Its side chain contains an amide functional group.
The peptide ends with a free C-terminal carboxylic acid.
4. The Importance of N-Terminal Acetylation
One of the defining chemical features of TB-500 is its N-terminal acetyl group.
Acetylation changes the functional group at the beginning of the peptide chain.
In the unmodified fragment, the first leucine residue has a free N-terminal amino group.
In TB-500, that group is acetylated.
What Is N-Terminal Acetylation?
N-terminal acetylation introduces an acetyl group, commonly represented as CH₃CO–, onto the terminal amino group.
The modification changes the molecular formula and removes the free N-terminal amine’s usual basic character.
How Much Mass Does Acetylation Add?
The net addition of an acetyl group increases the molecular mass by approximately 42 Da.
This produces a clear difference between acetylated and unacetylated versions of the same peptide sequence.
Acetylated vs Unacetylated Fragment
| Feature | Ac-LKKTETQ | LKKTETQ |
|---|---|---|
| N-terminal group | Acetylated | Free amino group |
| Peptide length | 7 | 7 |
| Approximate molecular weight | 889.0 g/mol | 847.0 g/mol |
| Chemical identity | TB-500 reference fragment | Unacetylated related fragment |
| Identical structures? | No | No |
This distinction is important when interpreting mass spectrometry or chemical database records.
Does Acetylation Guarantee Greater Stability?
No.
N-terminal acetylation can influence interactions with some enzymes and alter a peptide’s chemical properties.
However, it does not provide universal protection against degradation.
The stability of TB-500 depends on its complete sequence, chemical form, and environmental conditions.
5. The LKKTETQ Motif Within Thymosin Beta-4
The TB-500 sequence originates from the central region of thymosin beta-4.
Thymosin beta-4 contains 43 amino acid residues.
It has been studied extensively for its association with actin, a structural protein involved in cellular organization and movement.
What Is Actin?
Actin is a protein found in eukaryotic cells.
It exists in forms commonly described as G-actin and F-actin.
G-actin refers to monomeric actin.
F-actin refers to filamentous structures formed through actin polymerization.
The balance between these forms is important in cytoskeletal organization.
What Is the Actin-Binding Region?
The LKKTET region of thymosin beta-4 is associated with an important actin-interaction motif.
The TB-500 sequence includes that motif together with the following glutamine residue.
However, the presence of a recognizable motif does not mean that the isolated fragment has all the properties of the complete parent peptide.
Why Is the Full-Length Molecule Different?
A 43-residue peptide contains additional regions that may contribute to its folding, molecular contacts, and binding properties.
Removing those regions changes the molecular environment around the retained sequence.
Therefore, actin-binding findings from full-length thymosin beta-4 cannot automatically establish the binding affinity or functional behavior of TB-500.
Is TB-500 a Confirmed Equivalent of Thymosin Beta-4?
No.
The fragment and parent molecule share a short sequence region, but they differ substantially in overall structure.
Their chemical identity, experimental behavior, and analytical characteristics must be evaluated separately.
6. What Published Laboratory Research Has Examined
TB-500 has been the subject of several analytical studies investigating molecular identification and peptide metabolites.
These studies are especially relevant to the technical characterization of the fragment.
The 2012 Structural Identification Study
Esposito and colleagues used high-resolution mass spectrometry to characterize the substance identified as TB-500.
The investigation established that the material contained the N-acetylated thymosin beta-4 17–23 fragment.
A Second 2012 Analytical Investigation
Ho and colleagues developed liquid chromatography-mass spectrometry methods for detecting N-acetylated LKKTETQ and related metabolites in experimental samples.
The study provided information about mass spectrometric fragmentation and analytical detection.
The 2024 Metabolite Characterization Study
A later study published in the Journal of Chromatography B investigated TB-500 and its metabolites using UHPLC coupled with high-resolution Orbitrap mass spectrometry.
The researchers identified several shorter peptide fragments associated with experimental metabolism.
They also compared selected peptide-related responses in cell-based experiments.
Importantly, the findings did not demonstrate that the parent TB-500 molecule reproduces every activity attributed to full-length thymosin beta-4.
These studies show that the identity of a peptide and the identity of its metabolites must be considered separately.
7. TB-500 Fragmentation and Metabolite Identification
Peptides may undergo cleavage into smaller molecular fragments.
Analytical researchers investigate these changes to characterize molecular stability and identify related species.
What Is Peptide Cleavage?
Peptide cleavage involves breaking a peptide bond.
A seven-residue peptide contains six backbone peptide bonds.
Cleavage at different positions can create fragments with different sequences and molecular masses.
Examples of Studied TB-500-Related Fragments
Research has investigated shorter fragments including:
- Ac-LK
- Ac-LKK
- Ac-LKKTE
These structures contain the N-terminal acetyl modification but have fewer amino acid residues than the original heptapeptide.
Why Is Metabolite Identification Important?
A smaller fragment can have different chemical properties from its parent molecule.
Differences may include molecular mass, charge distribution, chromatographic retention, and interactions with experimental systems.
A signal detected during mass spectrometry should therefore be assigned to the correct molecular species.
Does a Metabolite Have the Same Molecular Activity?
Not necessarily.
Molecular activity cannot be inferred solely from sequence overlap.
Each fragment requires its own experimental characterization.
8. Physicochemical Properties of TB-500
TB-500 combines polar, charged, and hydrophobic amino acid residues within a short peptide chain.
These features influence its behavior in different analytical environments.
Charge Distribution
The two lysine side chains contain basic groups.
The glutamic acid side chain and the C-terminal carboxyl group contain acidic functional groups.
The net charge depends on the protonation state of these groups.
Why Does pH Matter?
Changes in pH alter the charge states of ionizable functional groups.
This can influence solubility, molecular interactions, and chromatographic behavior.
Hydrophobicity
Leucine contributes a hydrophobic side chain.
The remaining residues provide several polar functional groups.
The overall behavior depends on the entire molecule rather than a single residue.
Does TB-500 Contain a Disulfide Bond?
No.
TB-500 contains no cysteine residues.
It therefore does not have a native cysteine-to-cysteine disulfide linkage.
Does TB-500 Absorb Ultraviolet Light?
The fragment does not contain tryptophan, tyrosine, or phenylalanine, which are major aromatic contributors to peptide absorbance at 280 nm.
Its UV absorbance near 280 nm is therefore relatively weak.
Analytical methods may instead use lower wavelengths associated with the peptide backbone or other appropriate detection techniques.
9. How TB-500 Is Identified Through Mass Spectrometry
Mass spectrometry is particularly useful for distinguishing TB-500 from full-length thymosin beta-4.
The molecular masses differ by several thousand daltons.
Intact Molecular Mass
The expected average molecular weight of the acetylated fragment is approximately 889.0 g/mol.
A mass spectrometric result consistent with this reference supports the identification of the short peptide.
Monoisotopic Mass
TB-500 has a monoisotopic neutral mass of approximately 888.49 Da.
This differs slightly from the average molecular weight because of isotopic composition.
What Does a Mass Spectrometer Measure?
Mass spectrometry measures mass-to-charge ratios.
A peptide may form ions with different charge states.
The observed signal must be interpreted according to the ion type and charge.
Can Mass Spectrometry Distinguish the Fragment From Thymosin Beta-4?
Yes, suitable mass spectrometric measurements can readily distinguish their substantially different molecular masses.
However, molecular mass alone does not establish every aspect of amino acid sequence.
What Is Tandem Mass Spectrometry?
Tandem mass spectrometry, or MS/MS, examines fragment ions produced from a selected precursor ion.
The resulting pattern can provide information about peptide sequence.
For TB-500, fragmentation data can support identification of the LKKTETQ backbone and its N-terminal acetyl modification.
10. TB-500 HPLC Purity and Batch Analysis
High-performance liquid chromatography is commonly used to separate synthetic peptide components.
For TB-500, reversed-phase HPLC can help characterize the principal peptide peak and certain impurities.
What Does a Chromatogram Show?
A chromatogram displays detector response over the course of chromatographic separation.
Different components may appear at different retention times.
The interpretation depends on the method and detector.
Is HPLC Purity the Same as Chemical Identity?
No.
HPLC purity describes the sample’s chromatographic profile under specified conditions.
Chemical identity requires evidence that the observed substance corresponds to the intended molecule.
A combination of HPLC and mass spectrometry provides complementary information.
Can High HPLC Purity Exclude All Impurities?
No.
A high chromatographic purity result does not establish the absence of every possible impurity.
Substances may differ in detection response, separation behavior, or compatibility with the analytical method.
What About Salt Forms?
Counterions such as acetate may be associated with synthetic peptide material.
They influence sample composition and may not be fully represented by a conventional peptide HPLC purity measurement.
The chemical form should therefore be documented where possible.
11. Storage Stability and Chemical Degradation
TB-500 stability depends on its molecular structure and the conditions surrounding the material.
The short peptide backbone does not guarantee stability under every environment.
Potential Chemical Changes
Researchers may investigate:
Peptide bond hydrolysis: Cleavage of the peptide backbone under suitable chemical conditions.
Deamidation: Changes involving susceptible amide-containing residues, including glutamine.
N-terminal modification changes: Alterations involving the acetylated terminal group under relevant conditions.
Aggregation or association: Physical interactions between molecules that may affect analytical behavior.
Temperature and Moisture
Temperature and moisture can influence chemical reaction rates and physical stability.
The magnitude of these effects depends on the formulation and storage environment.
Solid-State vs Solution-State Material
Lyophilized material may behave differently from the same peptide in solution.
Freeze-drying reduces water content but does not establish a universal shelf life.
How Is Stability Evaluated?
Analytical studies may use chromatography and mass spectrometry to track changes in molecular composition over time.
The observation period and conditions should be documented.
Storage recommendations should be based on actual stability data for the specified material.
12. What Should a TB-500 Certificate of Analysis Show?
A Certificate of Analysis records the analytical findings associated with an identified sample or batch.
For TB-500, one of its most important functions is distinguishing the seven-residue fragment from full-length thymosin beta-4.
| COA field | What researchers should verify |
|---|---|
| Compound name | Whether TB-500 refers to the fragment or full-length molecule |
| Amino acid sequence | Ac-LKKTETQ for the fragment |
| CAS number | Identifier corresponding to the correct chemical structure |
| Expected mass | Approximately 889 Da for the acetylated fragment |
| Observed MS result | Consistency with the claimed molecular identity |
| HPLC data | Chromatographic separation and purity |
| Chemical form | Free peptide or associated salt form |
| Batch number | Traceability to the specific material |
| Analytical method | How identity and purity were measured |
| Testing date | When analysis was performed |
| Laboratory information | Organization responsible for analysis |
Why Is Sequence Documentation Essential?
TB-500 is a commercial name that has been applied inconsistently.
A COA reporting only TB-500 without a sequence or suitable identity result may not clearly establish which substance was tested.
Is a Molecular Weight of 4,963 Da Correct for TB-500?
It is correct for full-length thymosin beta-4, not for the seven-residue Ac-LKKTETQ fragment.
Does One Purity Result Verify Every Batch?
No.
Analytical results belong to the material tested.
Different batches require their own traceable documentation.
13. TB-500 in Multi-Peptide Formulations
TB-500 is also included in commercially named peptide blends.
These formulations contain additional molecular components.
TB-500 and BPC-157
Some mixtures contain TB-500 and BPC-157.
BPC-157 is a distinct synthetic 15-residue peptide.
The two compounds differ in sequence, molecular weight, chemical properties, and research history.
GLOW Blend
GLOW commonly combines GHK-Cu, BPC-157, and TB-500.
KLOW Blend
KLOW contains GHK-Cu, BPC-157, TB-500, and KPV.
Why Must TB-500 Identity Be Verified in a Blend?
A multi-peptide mixture contains several compounds with different molecular properties.
The exact identity of TB-500 affects the expected molecular masses, chromatographic separation, and component assay.
The fragment and full-length thymosin beta-4 cannot be substituted for one another in analytical calculations.
A formulation containing several peptide components also requires component-specific characterization rather than one universal molecular-weight value.
14. Frequently Asked Technical Questions About TB-500
What is TB-500?
TB-500 is commonly identified as the synthetic N-terminally acetylated thymosin beta-4 fragment Ac-LKKTETQ.
How many amino acids are in TB-500?
The Ac-LKKTETQ fragment contains seven amino acid residues.
What is the TB-500 sequence?
Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln-OH.
What is the one-letter sequence?
Ac-LKKTETQ-OH.
What is the molecular formula of TB-500?
C₃₈H₆₈N₁₀O₁₄ for the N-acetylated fragment.
What is the molecular weight?
Approximately 889.0 g/mol.
What is the monoisotopic mass?
Approximately 888.49 Da.
What is the CAS number of TB-500?
885340-08-9 is associated with the N-acetylated fragment.
What is the PubChem CID?
Is TB-500 identical to thymosin beta-4?
No. The defined fragment contains seven residues, while thymosin beta-4 contains 43.
What region of thymosin beta-4 does TB-500 represent?
Residues 17–23.
Is TB-500 acetylated?
Yes. Its N-terminal leucine carries an acetyl group.
Why is N-terminal acetylation important?
It changes the chemical identity, charge-related properties, and molecular mass of the peptide.
Is LKKTETQ the same as Ac-LKKTETQ?
No. The acetylated version contains an additional chemical group.
Is TB-500 a cyclic peptide?
No. It is a linear heptapeptide.
Does TB-500 contain a disulfide bridge?
No. It contains no cysteine residues.
Is TB-500 a GHRH analog?
No. It is derived from a region of thymosin beta-4.
Is full-length thymosin beta-4 research directly applicable to TB-500?
Not automatically. The two molecules require separate experimental characterization.
Can HPLC confirm TB-500 purity?
HPLC can provide chromatographic purity information under specified conditions.
Does HPLC establish exact molecular identity?
Not by itself. Complementary methods such as mass spectrometry may be needed.
Why is mass spectrometry useful for TB-500?
It helps distinguish the approximately 889 Da fragment from the approximately 4,963 Da parent peptide.
Can TB-500 form metabolites?
Analytical studies have identified shorter peptide-related molecular species in experimental systems.
Does TB-500 have a universal laboratory shelf life?
No. Shelf-life information must be supported by material-specific stability data.
What should a TB-500 COA include?
It should identify the exact peptide sequence and report suitable mass spectrometry, chromatography, and batch-specific information.
Is TB-500 FDA-approved?
No. The TB-500 fragment is not an FDA-approved drug.
15. Technical Research References
1. TB-500 Structural Identification
Esposito S. et al. (2012)
Synthesis and Characterization of the N-Terminal Acetylated 17–23 Fragment of Thymosin Beta 4 Identified in TB-500, a Compound Suspected to Possess Doping Potential.
Journal: Drug Testing and Analysis.
Research relevance: Identity confirmation, chemical synthesis, chromatography, and high-resolution mass spectrometry.
https://doi.org/10.1002/dta.1402
2. Chromatography and TB-500 Metabolite Analysis
Ho E.N.M. et al. (2012)
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: Journal of Chromatography A.
Research relevance: Analytical detection, LC-MS characterization, and peptide metabolite identification.
https://doi.org/10.1016/j.chroma.2012.09.043
3. Advanced TB-500 Metabolite Characterization
Rahaman K.A. et al. (2024)
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: Journal of Chromatography B.
Research relevance: High-resolution mass spectrometry, molecular fragments, metabolite quantification, and analytical method validation.
4. TB-500 Chemical Database
PubChem: TB-500
Reference information: Acetylated fragment structure, molecular formula, mass, and chemical identifiers.
5. Thymosin Beta-4 Parent Structure
PubChem: Thymosin Beta-4
Reference information: Full-length parent peptide and molecular properties.
6. FDA Chemical Identity and Regulatory Information
FDA: Certain Bulk Drug Substances for Use in Compounding That May Present Significant Safety Risks
Research relevance: Chemical identification and regulatory concerns involving thymosin beta-4 fragment LKKTETQ.
Conclusion
TB-500 is a chemically defined seven-residue peptide identified in published analytical research as Ac-LKKTETQ.
Its structure corresponds to residues 17–23 of thymosin beta-4, but it is distinct from the complete 43-residue parent peptide.
The N-terminal acetyl group, two lysine residues, and short linear backbone contribute to the fragment’s molecular identity and physicochemical properties.
Published research has established methods for identifying TB-500 through chromatography, mass spectrometry, and metabolite analysis.
For technical documentation, the most important distinction is between the seven-residue fragment and full-length thymosin beta-4.
Their sequences, molecular formulas, and theoretical masses must be reported separately.
Accurate characterization also requires distinguishing the defined reference structure from purity, content, counterion composition, and stability measurements obtained for a specific research batch.
Technical research notice: This resource is intended for chemical and analytical research education. It does not provide dosing, administration, reconstitution, or medical-use guidance. TB-500 is not FDA-approved, and research-material characterization does not establish suitability for human or veterinary use.
