Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry

ABSTRACT

Objectives: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances.

Methods: A top–down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5.

Results: We detected more than 100 monoisotopic masses corresponding to thymosin β4 and truncated forms of ubiquitin, prothymosin α, thymosin β4, and thymosin β9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3.

Conclusion: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.

KEYWORDS:

• Top-down proteomics
• α-Thymosins
• β-Thymosins
• SH3 domain-binding
• glutamic acid-rich-like protein 3
• thymosin fraction 5

Author contributions

The three authors contributed equally to this work. The scientific background and experience of all three authors have been equally important for this work. Specifically, E. Hannappel initiated the study and wrote the manuscript; F. Iavarone performed the experiments and interpreted data together with the other two authors; M. Castagnola supervised the experiments.

Declaration of interest

E. Hannappel is a non-paid member of the Scientific Advisory Board of RegeneRx Biopharmaceuticals, Inc. and does not own any stock. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Additional information

Funding

This paper has been published as part of a supplement issue covering the proceedings of the Fifth International Symposium on Thymosins in Health and Disease and is funded by SciClone Pharmaceuticals.