Quantification of HPLC-separated peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry

Saraswat, Suraj; Snyder, b James P.; Isailović, Dragan

doi:10.1016/j.jchromb.2012.06.018

Cited by 12 publications

(10 citation statements)

References 27 publications

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“…Usually, CEPs have a strong preference for hydrophobic caseins and Lactococcus CEPs have been classified into several types and subtypes depending on their substrate specificity for αS1-, β-, and κ-caseins ( Kunji et al, 1996 ). Casein hydrolysis can give rise mainly to opioid/anti-opioid peptides, antithrombotic and antihypertensive, immunomodulatory, mineral-binding and antimicrobial peptides ( Thakur et al, 2012 ) easily detectable by means of chromatographic techniques after microbial food digestion ( Saraswat et al, 2012 ).…”

Section: Encrypted Bioactive Peptidesmentioning

confidence: 99%

Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

2016

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Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain functional food. The detailed knowledge of the modulation of human physiology, exploiting the health-promoting properties of fermented food, is an open field of investigation that will constitute the next challenge.

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Section: Encrypted Bioactive Peptidesmentioning

confidence: 99%

Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

2016

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“…However, when there are no reference standards available for quantification, other approaches have to be applied. Philips and Preston suggested to quantify intact peptides by their intrinsic fluorescence . The phenomenon of fluorescence quenching interferes with the quantification of non‐hydrolyzed peptides and, therefore, limits the general applicability of this method .…”

Section: Introductionmentioning

confidence: 99%

“…6,7 To date, purity testing and quantification of peptides and proteins have usually been performed by UV absorption, by middle infrared absorption and by mass spectrometry against an appropriate reference standard. [11][12][13] The amide bond is monitored at λ = 214 nm, phenylalanine (Phe) at 260 nm, tyrosine (Tyr) at 278 nm, and tryptophan [15][16][17][18] The phenomenon of fluorescence quenching interferes with the quantification of non-hydrolyzed peptides and, therefore, limits the general applicability of this method. 14,[19][20][21][22] Another method for quantification of peptides is amino acid (AA) analysis by UV absorption with or without derivatization.…”

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confidence: 99%

Quantification of hydrolyzed peptides and proteins by amino acid fluorescence

Allenspach¹,

Fuchs²,

Doriot³

et al. 2018

Journal of Peptide Science

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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 to false positive or negative interpretation of activity data. Finally, only one solution is needed to perform quantification as well as UV-purity tests and can further be used as stock solution for activity testing.

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“…This value is in the range of LLOQ (2–300 ng/mL) published in other studies for the determination of various pharmaceutical peptides in biological matrices using HPLC with spectrofluorimetric detection (either intrinsic fluorescence or after a derivatization step; Allen, Stafford, & Nocerini, ; Boppana & Miller‐Stein, ; Chepyala, Tsai, Sun, Lin, & Kuo, ; Hinton et al, ; Lawless, Hopkins, & Anwer, ; Mendoza et al, ). Comparable values of LLOQ (from 5 to 40 ng/mL) have also been reported for peptides quantified in biological fluids with HPLC/MS methods (Gil et al, ; Jiang et al, ; Saraswat et al, ).…”

Section: Resultsmentioning

confidence: 74%

“…Recently, HPLC in combination with intrinsic fluorescence detection has been explored for the highly selective and highly sensitive quantitation of peptides and proteins. Intrinsic fluorescence detection (based on native fluorescence of tryptophan, tyrosine or phenyl alanine) is a label‐free methodology for quantitation of peptides and proteins, which provides similar sensitivity, better linearity and repeatability compared with MS detection (Russell et al, ; Saraswat, Snyder, & Isailovic, ). When possible, exploitation of tryptophan fluorescence is usually preferred owing to its high quantum yield, but successful use of tyrosine fluorescence has been reported (Saraswat et al, ; Zhdanova et al, ).…”

Section: Introductionmentioning

confidence: 99%

LR12‐peptide quantitation in whole blood by RP‐HPLC and intrinsic fluorescence detection: Validation and pharmacokinetic study

Parent

Boudier

Maincent

et al. 2017

Biomedical Chromatography

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A simple, sensitive, selective and robust HPLC method based on intrinsic fluorescence detection was developed for the quantitation of a dodecapeptide (designated as LR12), inhibitor of Triggering Receptor Expressed on Myeloid cells-1, in rat whole blood. Sample treatment was optimized using protein precipitation and solid-phase extraction. Chromatographic separation was carried out in a gradient mode using a core-shell C column (150 × 4.6 mm, 3.6 μm) with mobile phases of acetonitrile and water containing trifluoroacetic acid at 1.0 mL/min. The method was validated using methodology described by the US Food and Drug Administration guidelines for bioanalytical methods. Linearity was demonstrated within the 50-500 ng/mL range and the lower limit of quantitation was 50 ng/mL. Finally, a preliminary pharmacokinetic study after intraperitoneal injection of LR12 in rats was conducted to evaluate both LR12 monomer and its corresponding disulfide dimer, the main product of degradation. Beyond the fact that this paper describes the first fully validated method for LR12 analysis in blood samples, the approach followed here to optimize pre-analytical steps could be beneficial to develop HPLC and/or MS methods for other pharmaceutical peptides.

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Quantification of HPLC-separated peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry

Cited by 12 publications

References 27 publications

Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

Quantification of hydrolyzed peptides and proteins by amino acid fluorescence

LR12‐peptide quantitation in whole blood by RP‐HPLC and intrinsic fluorescence detection: Validation and pharmacokinetic study

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