Oxidation Products Of Tryptophan Research Articles

Resolving fluorescence spectra of Maillard reaction products formed on bovine serum albumin using parallel factor analysis

Formation of Maillard reaction products (MRPs) is increasingly studied by the use of fluorescence spectroscopy, and most often, by measuring single excitation/emission pairs or use of unresolved spectra. However, due to the matrix complexity and potential co-formation of fluorescent oxidation products on tryptophan and tyrosine residues, this practice will often introduce errors in both identification and quantification. The present study investigates the combination of fluorescence excitation emission matrix (EEM) spectroscopy and parallel factor analysis (PARAFAC) to resolve the EEMs into its underlying fluorescent signals, allowing for better identification and quantification of MRPs. EEMs were recorded on a sample system of bovine serum albumin incubated at 40 °C for up to one week with either glucose, methylglyoxal or glyoxal added. Ten unique PARAFAC components were resolved, and assignment was attempted based on similarity with fluorescence of pure standards of MRPs and oxidation products and reported data from literature. Of the ten fluorescent PARAFAC components, tyrosine and buried and exposed tryptophan were resolved and identified, as well as the formation of specific MRPs (argpyrimidine and Nα-acetyl-Nδ-(5-methyl-4-imidazolon-2-yl)ornithine) and tryptophan oxidation products (kynurenine and dioxindolylalanine). The formation of the PARAFAC resolved protein modifications were qualitatively validated by liquid chromatography-mass spectrometry.

Chemical modification of 5-hydroxytryptophan photoinduced by endogenous sensitizers present in skin

Damage caused to biological targets through sunlight photosensitized reaction is of paramount importance due to their potential effects on human health. In these processes, a chemical alteration of a biomolecule may occur as a result of the initial radiation absorption by another chemical species called photosensitizer. In this respect, pterins are endogenous photosensitizers able of inducing chemical modifications in DNA, proteins and lipids. These molecules are present in many living organisms and play different biological functions. In humans, aromatic pterins (Pt) accumulate in the depigmentation patches on the skin of patients suffering vitiligo. Interestingly, 5-hydroxytryptophan (5-OH-Trp), a common oxidation product of tryptophan acting as a potential endogenous antioxidant, is also present in the skin under oxidative stress conditions, such as those produced by vitiligo. However, the photochemical interaction between Pt and 5-OH-Trp has not been considered yet. With this background, the goal of the present work is to deepen the knowledge of the capability of Pt to photoinduce damage to 5-OH-Trp. By combining different analytical and spectroscopic techniques, we establish that 5-OH-Trp is damaged by Pt through a photosensitized type I process initiated by an electron transfer from the 5-OH-Trp to the Pt triplet excited state that yield the corresponding radical ions. In air-equilibrated aqueous solution four products were identified, two of which correspond to 5-OH-Trp dimers, while the others are a 5-OH-Trp trimer and a dione, in which the 5-OH-Trp has incorporated an oxygen atom. No consumption of Pt was observed in the presence of O2. However, in the absence of O2, further free-radical reactions lead to the reduction of the photosensitizer, and dimers and trimer were the only 5-OH-Trp-derived products detected. The biomedical implications of the generation of this kind of products, in proteins, are discussed.

Oxidation Products of Tryptophan and Proline in Adipokinetic Hormones-Artifacts or Post-Translational Modifications?

Adipokinetic hormones (AKHs) regulate important physiological processes in insects. AKHs are short peptides with blocked termini and Trp in position 8. Often, proline occupies position 6. Few post-translational modifications have been found, including hydroxyproline ([Hyp6]) and kynurenine. Our recent data suggest that the Hyp- and Kyn-containing AKHs occur more often than originally thought and we here investigate if they are natural or artifactual. From crude extracts of the corpora cardiaca (CC) of various insect species, AKHs were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS). Synthetic [Hyp6]-AKHs were tested in an in vivo metabolic assay. Freshly dissected Periplaneta americana and Blaberus atropos CCs (with precautions taken against oxidation) were analyzed. B. atropos CC were placed into a depolarizing saline and the released AKHs were measured. Hyp was detected in several decapeptides from cockroaches. The modified form accompanied the AKH at concentrations below 7%. The [Hyp6]-AKHs of B. atropos were present in fresh CC preparations and were shown to be releasable from the CC ex vivo. Synthetic [Hyp6]-containing peptides tested positively in a hypertrehalosemic bioassay. Hydroxyprolination was also detected for Manto-CC from the termite Kalotermes flavicollis and for Tetsu-AKH of the grasshopper, Tetrix subulata. Oxidized Trp-containing forms of Nicve-AKH were found in species of the burying beetle genus Nicrophorus. Trp oxidation is known to occur easily during sample handling and is likely the reason for the present findings. For hydroxyprolination, however, the experimental evidence suggests endogenous processes.

Role of Essential Amino Acids in Age-Induced Bone Loss

Age-induced osteoporosis is a global problem. Essential amino acids (EAAs) work as an energy source and a molecular pathway modulator in bone, but their functions have not been systematically reviewed in aging bone. This study aimed to discuss the contribution of EAAs on aging bone from in vitro, in vivo, and human investigations. In aged people with osteoporosis, serum EAAs were detected changing up and down, without a well-established conclusion. The supply of EAAs in aged people either rescued or did not affect bone mineral density (BMD) and bone volume. In most signaling studies, EAAs were proven to increase bone mass. Lysine, threonine, methionine, tryptophan, and isoleucine can increase osteoblast proliferation, activation, and differentiation, and decrease osteoclast activity. Oxidized L-tryptophan promotes bone marrow stem cells (BMSCs) differentiating into osteoblasts. However, the oxidation product of tryptophan called kynurenine increases osteoclast activity, and enhances the differentiation of adipocytes from BMSCs. Taken together, in terms of bone minerals and volume, more views consider EAAs to have a positive effect on aging bone, but the function of EAAs in bone metabolism has not been fully demonstrated and more studies are needed in this area in the future.

Chemical Stability of Proteins in Foods: Oxidation and the Maillard Reaction.

Protein is a major nutrient present in foods along with carbohydrates and lipids. Food proteins undergo a wide range of modifications during food production, processing, and storage. In this review, we discuss two major reactions, oxidation and the Maillard reaction, involved in chemical modifications of food proteins. Protein oxidation in foods is initiated by metal-, enzyme-, or light-induced processes. Food protein oxidation results in the loss of thiol groups and the formation of protein carbonyls and specific oxidation products of cysteine, tyrosine, tryptophan, phenylalanine, and methionine residues, such as disulfides, dityrosine, kynurenine, m-tyrosine, and methionine sulfoxide. The Maillard reaction involves the reaction of nucleophilic amino acid residues with reducing sugars, which yields numerous heterogeneous compounds such as α-dicarbonyls, furans, Strecker aldehydes, advanced glycation end-products, and melanoidins. Both protein oxidation and the Maillard reaction result in the loss of essential amino acids but may positively or negatively impact food structure and flavor.

Molecular features toward high photo-CIDNP hyperpolariztion explored through the oxidocyclization of tryptophan.

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) is a promising solution to the inherent lack of sensitivity in NMR spectroscopy. It is particularly interesting in biological systems since it operates in water, at room temperature, and it can be repeated if the bleaching of the system can be controlled. However, the photo-CIDNP signal enhancement is well below those of other hyperpolarization techniques. While DNP, PHIP, and SABRE reach polarization enhancements of 103 to 104-fold, photo-CIDNP enhancement is typically only one order of magnitude for 1H and two orders of magnitude for 13C in the amino-acids tryptophan and tyrosine. Here we report on a photo-oxidation product of tryptophan that is strongly photo-CIDNP active under continuous wave light irradiation. In conjunction with the dye Atto Thio 12, a 1H signal enhancement of 120-fold was observed on a 600 MHz spectrometer, while at 200 MHz the enhancement was 380-fold. These enhancements in signal to noise correspond to a reduction in measurement time of 14 400-fold and 144 400-fold, respectively. The enhancement for 13C is estimated to be over 1200-fold at 600 MHz which corresponds to an impressive measurement time reduction of 1 440 000-fold. This photo-CIDNP active oxidation product of tryptophan has been identified to be 3α-hydroxypyrroloindole. The reasons for its improved signal enhancement compared to tryptophan have been further investigated.

Light exposure accelerates oxidative protein polymerization in beef stored in high oxygen atmosphere

Protein oxidation of beef patties stored in high oxygen modified atmosphere packaging for 9 days was investigated. Meat was either stored in the dark, under light, or in the dark with addition of FeCl2/H2O2/myoglobin (forced oxidation). SDS–PAGE analysis showed high degree of protein polymerization for meat exposed to light, compared to the other samples. Light exposure induced reducible (disulfide) and non-reducible cross-links, while mainly disulfides were formed in meat stored in the dark. Light exposure was responsible for 58% loss of free thiols (Cys residues). No significant loss of other amino acid residues was observed and none of the most common oxidation products of tryptophan, tyrosine, and phenylalanine were detected. Intrinsic fluorescence measurements of tryptophan showed 27% loss in samples exposed to light, which was ascribed to loss of protein solubility via protein polymerization rather than tryptophan oxidation. Protein carbonyls were mainly detected in forced oxidized samples at Day 0.

Characterisation and quantification of protein oxidative modifications and amino acid racemisation in powdered infant milk formula

Modification of proteins in infant milk formula (IF) is of major concern to the dairy industry and consumers. Thermal treatment is required for microbiological safety, but heat, light, metal-ions and other factors may induce oxidative damage, and be a health risk. In this study protein modifications in IFs were quantified. IFs contained both reducible (disulphide) and non-reducible (di-tyrosine, lanthionine, lysinoalanine) protein cross-links. Dehydroalanine and the cross-linked species lanthionine and lysinoalanine were detected. Protein carbonyls were detected predominantly on high molecular mass materials. Oxidation products of phenylalanine (m-tyrosine), tryptophan (N-formylkynurenine, kynurenine, 3-hydroxykynurenine), tyrosine (di-tyrosine) and methionine (methionine sulphoxide) were detected, consistent with amino acid modification. Higher levels of most of the markers of protein modification were present in the hydrolysed protein brand, when compared to the conventional IF samples, indicative of increased damage during additional processing. Significant levels of racemised (D-) amino acids were present. These data indicate that amino acids in proteins in IFs are modified to a significant extent during manufacture, with hydrolysed IF being particularly prone.

Pork proteins oxidative modifications under the influence of varied time-temperature thermal treatments: A chemical and redox proteomics assessment

The impact of thermal processing on meat proteins oxidation was investigated. Cooking treatments included 58 °C for either 72 min or 17 h (mimicking low temperature-long time sous vide cooking), 80, 98 and 160 °C for 72 min (mimicking common cooked meat products, stewing and roasting, respectively) and 118 °C for 8 min (autoclaving). Tryptophan degradation, fluorescent oxidation products, free thiol content and α-aminoadipic and γ-glutamic semialdehydes were tracked. For all of them, there was a consistent trend to increased levels of oxidative damage with higher cooking temperatures and longer cooking times, although the extent varied from one indicator to another. Through proteomics, peptide oxidative modifications like carbonylation, malonaldehyde adducts and hydroxykynurenin (tryptophan oxidation products) were also detected at residue level. Our findings indicate that protein oxidation is dependent upon the heat treatment, which point out to a different effect on the nutritional quality of proteins in meat products.

Tryptophan and Cysteine Oxidation Products Dominate in α-Lactalbumin-Derived Peptides Analyzed with LC-MSn.

α-Lactalbumin (α-La), a major milk whey protein, is comprised of several amino acids prone to metal-catalyzed oxidation (MCO) typical in processing and during storage of foods. New tools are needed for the detection of characteristic oxidation products especially from tryptophan and cysteine that often remain unrecognized when using the traditional methods of carbonyl formation monitoring. In this study, the oxidative changes in α-La were investigated through tryptic digestion and collection of 3 descriptive peptides fitted into a metal-catalyzed oxidation (Fenton reaction) model. The peptide samples were oxidized at +37 °C for 14 d and explored with liquid chromatography-quadrupole ion trap-mass spectrometer (LC-MSn ). The fractionated α-La peptides were valyl-glycyl-isoleucyl-asparaginyl-tyrosyl-tryptophyl-leucyl-alanyl-histidyl-lysine (VGINYWLAHK), leucyl-aspartyl-glutaminyl-tryptophyl-leucyl-cysteinyl-glutamyl-lysine (LDQWLCEK), and tryptophyl+16 -leucyl-alanyl-histidyl-lysyl-alanyl-leucyl-cysteine (W+16 LAHKALC). Oxidation of several amino acids, such as cysteine, histidine, lysine, and tryptophan was observed. In the peptide LDQWLCEK, cysteine was rapidly trioxidized to sulfonic acid, followed by other amino acid side chains as secondary oxidation sites. Tryptophan oxidation was more pronounced in the peptides W+16 LAHKALC and VGINYWLAHK, and also formation of the harmful N-formylkynurenine was observed. As a conclusion, several stable and promising oxidation markers are proposed for α-La, which could be implemented in the evaluation of quality and safety of dairy protein-containing products.

Kinetics and Mechanism of Silver(I)-Catalyzed Oxidation of Tryptophan by Platinum(IV) in Perchlorate Solutions

The kinetics of oxidation of tryptophan by platinum(IV) was investigated in aqueous perchlorate solutions in the presence of silver(I) catalyst at a constant ionic strength of 2.0 mol dm-3 and at 25°C. The progress of the reaction was followed spectrophotometrically. The reaction did not proceed in the absence of the catalyst. The catalyzed reaction exhibited a first order dependence on both [PtIV] and [AgI] whereas the order with respect to tryptophan concentration was found to be less than unity. Increasing ionic strength and dielectric constant was found to decrease the oxidation rate. The suggested oxidation mechanism involves formation of a silver(I)-tryptophan intermediate complex in a pre-equilibrium step, which confirmed by both spectral and kinetic evidences. The complex reacts with the oxidant by an inner-sphere mechanism leading to decomposition of the complex in the rate-determining step. The final oxidation products of tryptophan were identified as the corresponding aldehyde (indole-3-acetaldehyde), ammonium ion and carbon dioxide.

Kinetics and mechanistic approach to the chromic acid oxidation of l-tryptophan with a spectral detection of chromium(III) product

The kinetics of chromic acid oxidation of l-tryptophan in H2SO4 medium at a constant ionic strength of 0.6moldm−3and at 25°C have been investigated spectrophotometrically. The reaction exhibits a 3:2 stoichiometry (tryptophan:chromic acid). The reaction shows a first order dependence on [chromic acid], a fractional-first order dependence on [tryptophan] and fractional-second order dependence on [acid]. Increasing ionic strength and dielectric constant had no significant effect on the oxidation rate. The final oxidation products of tryptophan were identified as the corresponding aldehyde (indole-3-acetaldehyde), ammonium ion and carbon dioxide. A spectral evidence for the formation of chromium(III) product was obtained. The experimental results suggest formation of an intermediate complex between the protonated tryptophan and chromic acid which decomposes in the rate-determining step to yield the products. The mechanism of such reaction has been proposed. The thermodynamic parameters with respect to the first step of the suggested mechanism were evaluated and discussed.

Characterization of the Degradation Products of a Color-Changed Monoclonal Antibody: Tryptophan-Derived Chromophores

We describe the characterization of degradation products responsible for color change in near UV-visible light-irradiated and heat-stressed monoclonal antibody (mAb) drug product in liquid formulation. The treated samples were characterized using reversed-phase HPLC and size-exclusion HPLC with absorption spectroscopy. Both methods showed color change was due to chromophores formed on the mAb but not associated with the formulation excipients in both light-irradiated and heat-stressed mAb samples. These chromophores were further located by a new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy. Mass spectrometry identified the major tryptophan oxidation products as kynurenine (Kyn), N-formylkynurenine (NFK), and hydroxytryptophan (OH-Trp). The absorption spectra showed that each of the tryptophan oxidation products exhibited a distinct absorption band above 280 nm shifted to the longer wavelengths in the order of OH-Trp < NFK < Kyn. The Kyn-containing peptide was detected by absorption at 420 nm. No new absorption bands were observed for either methionine or histidine oxidation products. This confirmed that tryptophan oxidation products, but not methionine and histidine oxidation products, were responsible for the color change. It is worth noting that a new oxidation product with the loss of hydrogen (2 Da mass decrease) for Trp-107 of the heavy chain was identified in the heat-stressed mAb sample. This oxidized tryptophan residue exhibited a distinct absorption band at the maximum absorbance wavelength 335 nm, which is responsible for the color change to yellow. This study showed that the new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy is useful to identify tryptophan oxidation products as chromophores responsible for color change in stressed mAb drug product.

UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products: IMPLICATIONS FOR HUMAN LENS AGING AND CATARACT FORMATION

Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320-400 nm, 100 milliwatts/cm(2), 45 min to 2 h), young human lenses (20-36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.

Structural Characterization of New Microcystins Containing Tryptophan and Oxidized Tryptophan Residues

Microcystins are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Eight of the (more than) 90 microcystin variants presently characterized, contain the amino acid tryptophan. The well-researched oxidation products of tryptophan; kynurenine, oxindolylalanine, and N-formylkynurenine, have been previously identified in intact polypeptides but microcystin congeners containing oxidized tryptophan moieties have not been reported. Liquid chromatography-tandem mass spectrometric analysis of an extract of Microcystis CAWBG11 led to the tentative identification of two new tryptophan-containing microcystins (MC‑WAba and MC-WL), as well as eight other microcystin analogs containing kynurenine, oxindolylalanine and N‑formylkynurenine (Nfk). Investigation of one of these congeners (MC‑NfkA) by nuclear magnetic resonance spectroscopy was used to verify the presence of Nfk in the microcystin. Liquid chromatography-mass spectrometry analysis of a tryptophan oxidation experiment demonstrated that tryptophan-containing microcystins could be converted into oxidized tryptophan analogs and that low levels of oxidized tryptophan congeners were present intracellularly in CAWBG11. MC-NfkR and MC-LNfk were detected in standards of MC-WR and MC-LW, indicating that care during storage of tryptophan-containing microcystins is required.

Comprehensive Analysis of Nonenzymatic Post-Translational β-Lactoglobulin Modifications in Processed Milk by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

Nonenzymatic post-translational protein modifications (nePTMs) result in changes of the protein structure that may severely influence physiological and technological protein functions. In the present study, ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) was applied for the systematic identification and site-specific analysis of nePTMs of β-lactoglobulin in processed milk. For this purpose, β-lactoglobulin, which had been heated with lactose under conditions to force nePTM formation (7 d/60 °C), was screened for predicted modifications by using full scans and enhanced resolution scan experiments combined with enhanced product ion scans. Thus, the main glycation, glycoxidation, oxidation, and deamidation products of lysine, arginine, methionine, cysteine, tryptophan, and asparagine, as well as the N-terminus, were identified. Using these MS data, a very sensitive scheduled multiple reaction monitoring method suitable for the analysis of milk products was developed. Consequently, 14 different PTM structures on 25 binding sites of β-lactoglobulin were detected in different milk products.

Identification of Oxidation Sites and Covalent Cross-Links in Metal Catalyzed Oxidized Interferon Beta-1a: Potential Implications for Protein Aggregation and Immunogenicity

Oxidation via Cu(2+)/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu(2+)/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl)benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated, and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe), tryptophan, and Tyr residues were identified throughout the primary sequence. Covalent cross-links via 1,4- or 1,6-type addition between primary amines and DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl)propanoic acid, an oxidation product of Phe and Tyr) were detected. There was no evidence of disulfide bridge, Schiff base, or dityrosine formation. The chemical cross-links identified in this work are most likely responsible for the formation of covalent aggregates of IFNβ1a induced by oxidation, which have previously been shown to be highly immunogenic.

Qualitative and quantitative evaluation of derivatization reagents for different types of protein-bound carbonyl groups

Mass spectrometry (MS) of 'carbonylated proteins' often involves derivatization of reactive carbonyl groups to facilitate their enrichment, identification and quantification. Among the many reported reagents, 2,4-dinitrophenylhydrazine (DNPH), biotin hydrazide (BHZ) and O-(biotinylcarbazoylmethyl) hydroxylamine (ARP) are the most frequently used. Despite their common use in carbonylation research, their reactivity towards protein-bound carbonyls has not been quantitatively evaluated in detail, to the best of our knowledge. Thus we studied the reactivity and specificity of these reagents towards different classes of reactive carbonyl groups (e.g. aldehydes, ketones and lactams), each being represented by a synthetic peptide carrying an accordingly modified residue. All three tagging reagents were selective for aliphatic aldehydes and ketones. Lactams and carbonyl-containing tryptophan oxidation products, however, were labelled only at low levels or not at all. Whereas DNPH derivatization was efficient under the published standard conditions, the derivatization conditions for BHZ and ARP had to be altered. Acidic conditions provided quantitative labelling yields for ARP. Peptides derivatized with DNPH, BHZ and ARP fragmented efficiently in tandem mass spectrometry, when the experimental conditions were chosen carefully for each reagent. Importantly, the tested carbonylated peptides did not cross-react with amino groups in other proteins present during sample preparations or enzymatic digestion. Thus, it appears favourable to digest proteins first and then derivatise the reactive carbonyl groups more efficiently at the peptide level under acidic conditions. The carbonylated model peptides used in this study might be valid internal standards for carbonylation proteomics.

A novel approach for the selective determination of tryptophan in blood serum in the presence of tyrosine based on the electrochemical reduction of oxidation product of tryptophan formed in situ on graphite electrode

In this study, a novel method was proposed for the selective determination of tryptophan (TRP) in blood serum in the presence of tyrosine. This method is based on the electrochemical reduction of 2-amino-3-(5-oxo-3,5-dihydro-2H-indol-3-yl)-propionic acid (5-O-3,5DH-TRP) formed by the oxidation of TRP on the electrochemically treated pencil graphite (ETPG) electrode surface at a suitable potential value. The parameters affecting the TRP determination were deeply investigated. The optimal pH value was determined as 3. The highest reduction current intensity was obtained at the accumulation potential and time values of +0.95V and 120s, respectively. The reduction peak current values of 5-O-3,5DH-TRP versus TRP concentration at the ETPG electrode showed linearity in the range from 0.5μM to 50.0μM (R2=0.9962) with a detection limit of 0.05μM (S/N=3). The reduction peak intensity of 5-O-3,5DH-TRP on the ETPG electrode showed no significant change in the presence of different interfering substances. The analytical application of the proposed novel method was successfully tested by using human blood serum samples.

Metabolomics Profiling of Cell Culture Media Leading to the Identification of Riboflavin Photosensitized Degradation of Tryptophan Causing Slow Growth in Cell Culture

As more protein biopharmaceuticals are produced using mammalian cell culture techniques, it becomes increasingly important for the biopharmaceutical industry to have tools to characterize the cell culture media and evaluate its impact on the cell culture performance. Exposure of the cell culture media to light, temperature stress, or adventitious introduction of low-level organisms during preparation can lead to the generation of chemical degradants or metabolites of the media components, which are potentially detrimental to the cell culture process. In this work, we applied a liquid chromatography-mass spectrometry based metabolomics methodology for the investigation of a media lot used for a mammalian cell culture process that had resulted in low growth rate and failure to meet required viable cell density (VCD). The study led to the observation of increased levels of tryptophan oxidation products and a riboflavin degradant, lumichrome, in the malfunctioning media lot, relative to working media lots. A compound found 7-fold higher in the working media lots appeared to be tetrahydropentoxyline, a condensation product of glucose and tryptophan. A second compound found at an over 50-fold higher level in the malfunctioning media lot with a proposed molecular formula of C(21)H(17)N(3)O(3) from high-resolution mass spectrometry (HRMS) analysis remains unknown, although it is confirmed to be a degradant of tryptophan in the media. A study of the cell culture media performed under stress conditions using fluorescent light and heat showed that the media powder was highly resistant to light-induced degradation, while solution media could be easily degraded after brief light exposure. It is therefore suspected that inadvertent exposure of the media to light during preparation and storage has resulted in the poor performance of the media causing the low growth and VCD in the cell culture process.