Quantification of hydrolyzed peptides and proteins by amino acid fluorescence

Journal of Peptide Science : an Official Publication of the European Peptide Society
Martina D AllenspachChristian Steuer

Abstract

Reliable quantification of peptides and proteins is essential for drug discovery. We report the successful development and validation of an accurate and broadly applicable high performance liquid chromatography hyphenated to fluorescence detector procedure for the quantitative determination of the aromatic amino acids tyrosine, phenylalanine, and tryptophan, without relying on derivatization chemistry. Using ion-pair chromatography, fluorescent amino acids were clearly separated within 10 minutes. The hydrolysis of peptides was performed under acidic and heated conditions to yield the monomeric building blocks. Various protecting agents were tested to ensure tryptophan stability. The presented analytical method accurately (>95%) quantifies all fluorescent residues. The power of the method was confirmed by correct quantification of protein reference standard to 98.6% over all fluorescence traces. The method allowed us to identify pre-analytical differences between the nominal and actual concentrations of 12 peptide solutions. Salt formation, weighing errors, and other pre-analytical pitfalls resulted in noteworthy differences of up to 85% between the indicated and actual concentration of peptide solutions, subsequently leading t...Continue Reading

References

Jul 1, 1987·Journal of the American Academy of Dermatology·C W Lober
Dec 1, 1971·Australian Journal of Biological Sciences·A S InglisC M Roxburgh
Jan 23, 1999·Journal of Chromatography. a·M Fountoulakis, H W Lahm
Nov 16, 2001·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·F Wu, E Tanoue
Nov 20, 2003·Bioconjugate Chemistry·Nicole MarméJürgen Wolfrum
Dec 6, 2006·Journal of Pharmaceutical and Biomedical Analysis·Michael J LittleSteven R LaPlante
Nov 27, 2007·Journal of Peptide Science : an Official Publication of the European Peptide Society·Stéphane RouxNicolas Fay
Sep 2, 2009·Anais Da Academia Brasileira De Ciências·Adriana K CarmonaLuiz Juliano
Jul 20, 2012·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Suraj SaraswatDragan Isailovic
Dec 3, 2014·Drug Discovery Today·Keld Fosgerau, Torsten Hoffmann
Feb 18, 2016·Analytical and Bioanalytical Chemistry·George W Preston, David H Phillips
Jun 2, 2017·Accounts of Chemical Research·Katja ZerbeJohn A Robinson
Jul 25, 2017·Journal of Medicinal Chemistry·Antoine HenninotJohn M Nuss
Mar 28, 2018·Journal of Medicinal Chemistry·Aline D de AraujoDavid P Fairlie

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.