Identification of Products from Thermal Degradation of Tryptophan

Jun 21, 2019 - Pentapeptides were heated in a laboratory oven at 220 °C for 10 min. ... calibrated with a 0.1 M sodium formate solution before each c...
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Cite This: J. Agric. Food Chem. 2019, 67, 7448−7454

Identification of Products from Thermal Degradation of Tryptophan Containing Pentapeptides: Oxidation and Decarboxylation Maria Bikaki and Nikolai Kuhnert* Department of Life Sciences & Chemistry, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany

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S Supporting Information *

ABSTRACT: In this contribution, we investigate the thermal decomposition of four pentapeptides containing a tryptophan moiety. Pentapeptides were heated at 220 °C, and the resulting reaction mixtures were investigated by HPLC coupled to highresolution mass spectrometry and tandem mass spectrometry. A total of 95 thermal decomposition products could be observed and resolved by chromatography. In detail, we report on the structure assignment of two types of reaction products common to investigated peptides and introduce two decomposition mechanisms. Pentapeptides react with oxygen to produce hydroxyltryptophan derivatives. In addition, we observe the C-terminal decarboxylation of two peptides to form N-acyl tryptamine derivatives. KEYWORDS: pentapeptides, thermal treatment, degradation products, chemical mechanisms, tryptophan modifications

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INTRODUCTION During food processing, the chemical composition of food changes dramatically with up to 70% of the original chemical constituents in raw material being converted to processing compounds.1 Thermal treatment including cooking, steaming, baking, frying, or roasting are most commonly employed inducing such chemical transformations. Food processing products contribute significantly to the sensory properties of a given food and are frequently associated with either beneficial or adverse health effects of the food.2 Additionally thermal treatment of food is believed to be a main contributor to human evolution leading to a vast array of Darwinian selection advantages.3 The most intensely studied reaction occurring during food processing is the Maillard reaction, summarizing all processes occurring if sugars react with amino acids giving a myriad of different products. For certain foods like coffee or cocoa significant contributions have been made to unravel the chemistry behind thermal treatment.4,4b Similarly, selected primary and secondary metabolites have been investigated with respect to their thermal reactivity, including sugars, amino acids, polyphenols, and others. The fate of proteins and their related shorter peptides has been investigated on some occasions,5,6 without a clear picture emerging on their reactivity and possible reaction products. Hidalgo et al.7 has reported on the protein backbone change cleavage induced by cysteine thiol side chains and aspartic and glutamic acid side chains. The decomposition of asparagine residues to form acrylamide8 has been reported as well as dehydration reactions of serine and threonine side chains.9 This lack of knowledge is presumably related to the nature of the reaction products forming a complex mixture, hence posing a tremendous analytical challenge. Our research group has previously introduced a series of analytical strategies based on high-resolution and tandem mass spectrometry to address this analytical challenge, for example, in tea and cocoa fermentation and coffee roasting, which we apply here.1,10−13 © 2019 American Chemical Society

In this contribution, we have investigated the thermal decomposition of a series of pentapeptides with amino acid sequences commonly observed in food. The study of shorter peptides has the following advantages. First, short peptides with judiciously chosen amino acid sequences can serve as a suitable model system for more complex proteins giving insight into basic mechanistic pathways. Second, a study of thermal peptide decomposition in the absence of further reaction partners allows furthermore a simplification of a real food system facilitating the exploration of chemical reactivity under harsh conditions. Finally, short peptides are commonly encountered in many food products as secondary metabolites in animals and plants with hormonal function and in particular those processed by fermentation yielding proteolytic peptide degradation products such as in many dairy products (cheese, yoghurt), soy products, or cocoa. On frequent occasions, such short peptides have been suggested as key aroma precursors.14 Recently, such short peptides have received plenty of attention due to their biological effects, affecting human health. They have been termed bioactive peptides. From a chemical point of view, understanding how foodderived bioactive peptides behave under thermal conditions similar to which food industry applies would be a beneficial tool for peptide engineering and peptide synthesis. The role of individual and adjacent amino acids of the peptide is considered crucial, bringing many questions at the foreground related to the role which each amino acid’s side chain or stereochemistry might play on the thermal-degraded products formation. Working on custom pentapeptides simplifies the examined system, and subsequent chemical mechanisms observed lead to a better understanding in this field. For this purpose, two pairs of Received: Revised: Accepted: Published: 7448

February 14, 2019 April 26, 2019 June 21, 2019 June 21, 2019 DOI: 10.1021/acs.jafc.9b01056 J. Agric. Food Chem. 2019, 67, 7448−7454

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Journal of Agricultural and Food Chemistry pentapeptides with different sequence were chosen and investigated.

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Table 2. High-Resolution Mass (UHPLC-ESI-Q-TOF-MS) Data of Thermal-Degraded Products of FAKAW and Their Parent Ions (M + H)

MATERIALS AND METHODS

Chemicals and Reagents. Custom pentapeptides used in this study were synthesized from Synpeptide Co., Ltd., Shanghai, China, and they were used without further purification. All reagents used in this study were HPLC grade, and they were purchased from Sigma-Aldrich (Germany). Milli-Q water (18.2MΩ cm at 25 °C) was used throughout all experiments. Sample Preparation. Pentapeptides were heated in a laboratory oven at 220 °C for 10 min. The solid peptide standards (50 μg each) were placed in an open glass vial, heated, and then cooled down at room temperature. The nonvolatile thermal-degraded products were collected by extraction inside from the glass tube with milli-Q water (50 μL). 1:10 Dilutions of the stock samples were directly used for HPLC-MS measurements. Direct infusions on an ion trap mass spectrometer were performed when targeted fragmentation was required. UHPLC-ESI-Q-TOF-MS. HPLC experiments were performed on an Agilent 1260 HPLC system using a Peptide 2.7 μm C18 column (2.1 × 250 mm, 2.7 μm particle size) along with the recommended guard column. The sample injection volume was 3 μL. The binary solvent system used consisted of Milli-Q water (Solvent A) and acetonitrile (Solvent B), both containing 0.1% formic acid. The solvents flow rate was kept constant at 0.250 mL/min, and the column temperature was set at 25 °C. The gradient profile used was starting with 5% B, increasing to 100% B in 80 min, followed by washing with 100% B until 90 min, and decreasing to 5% B until 95 min. The column was reequilibrated for the next measurement at 5% B for 20 more minutes. The effluent HPLC system was connected to an Impact HD ultrahigh resolution ESI-Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to an electrospray ionization source (nebulizer pressure of 2 bar, dry gas flow rate of 8 L/min, and dry gas temperature of 200 °C). All data were acquired in both negative- and positive-ion mode. In this work, only results in the positive ion mode are presented. Both full scan spectra and MS/MS data sets were recorded. The TOF analyzer was calibrated with a 0.1 M sodium formate solution before each chromatographic run. Mass calibration and charge deconvolution of HPLC-TOF-MS data was performed using DataAnalysis 4.2 (Bruker, Germany). All results presented in this study were analyzed manually using both tandem MS and high-resolution mass data. When required, further manual targeted fragmentation was performed with an ion-trap mass spectrometer fitted with an ESI source (HCT-Ultra, 215 Bruker Daltonics, Bremen, Germany) in positive ion mode.

RESULTS AND DISCUSSION In order to understand the chemical behavior during thermal processing of food peptides, four selected custom pentapeptides Table 1. List of Pentapeptides Investigated and Their High Resolution Monoisotopic Neutral Mass MW (Da)

FAKAW WAKAF FAQAW FAEAW

621.3274 621.3274 621.2910 622.2751

molecular formula

theor m/z (M + H)

exp m/z (M + H)

error (ppm)

RT (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

C9H18N3O2 C14H27N6O5 C12H26N5O3 C12H25N4O4 C21H35N6O4 C12H17N2O3 C12H16N3O C20H30N5O4 C12H16N3O C22H35N6O5 C14H18N3O3 C32H44N7O7 C32H45N7O7 C18H27N4O3 C18H23N4O2 C24H35N6O6 C25H35N6O8 C22H31N6O5 C25H35N6O7 C33H44N7O7 C35H47N6O8 C36H44N7O10 C33H40N7O7 C36H44N7O9

200.1394 359.2037 288.2030 289.1870 435.2714 237.1234 218.1288 404.2292 218.1288 463.2663 276.1343 638.3297 319.6685 347.2078 327.1816 503.2613 547.2511 459.2350 531.2562 650.3297 679.3450 734.3144 646.2984 718.3195

200.1394 359.2033 288.2037 289.1868 435.2714 237.1235 218.1283 404.2293 218.1294 463.2668 276.1345 638.3298 319.6689 347.2067 327.1819 503.2605 547.2511 459.2363 531.2554 650.3313 679.3462 734.3149 646.2982 718.3191

0.0 1.3 −2.5 0.8 0.2 −0.6 2.2 −0.2 −2.9 −1.0 −1.0 −0.2 −1.2 3.1 −1.0 1.5 0.0 −2.7 1.5 −2.5 −1.7 −0.6 0.2 0.6

2.7 3.2 3.9 7.0 9.9 10.2 12.0 15.0 17.5 19.1 20.9 21.0 21.5 22.4 23.8 24.2 24.5 28.9 29.5 34.0 35.8 36.7 39.4 40.8

Table 3. High-Resolution Mass (UHPLC-ESI-Q-TOF-MS) Data of Thermal-Degraded Products of FAQAW and Their Parent Ions (M + H)

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peptide sequence

no.

with different sequences, reported previously in food sources, were subjected to thermal treatment and subsequent LC−MS (Table 1). Table S1 shows identity of sequences of all natural dietary peptides obtained from BioPepDB along with their putative functions. Pentapeptides selected carried a C-terminal tryptophan moiety and an N-terminal phenylalanine moiety or an N-

no.

molecular formula

theor m/z (M + H)

exp m/z (M + H)

error (ppm)

RT (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

C12H19N4O5 C11H22N5O4 C20H31N6O5 C20H30N5O6 C22H25N2O3 C20H31N6O5 C31H40N7O9 C14H18N3O3 C19H26N5O5 C22H31N6O6 C24H34N7O7 C31H40N7O8 C31H40N7O9 C30H40N7O8 C37H35N5O7 C30H39N6O7 C30H40N7O5 C32H40N7O8 C45H47N6O5 C35H40N7O10

299.1350 288.1666 435.2350 436.2191 365.1860 435.2350 654.2882 276.1343 404.1928 475.2300 532.2514 638.2933 654.2882 626.2933 330.6263 595.2875 578.3085 650.2933 751.3602 718.2831

299.1362 288.1678 435.2358 436.2203 365.1870 435.2346 654.2872 276.1348 404.1930 475.2299 532.2519 638.2932 654.2888 626.2970 330.6266 595.2872 578.3084 650.2922 751.3593 718.2822

−4.0 −3.9 −1.7 −2.8 −2.7 1.1 1.5 −1.9 −0.3 0.2 −0.8 0.1 −1.0 −0.6 −1.1 0.5 0.2 1.6 1.3 1.3

10.0 10.0 10.1 14.3 16.0 17.2 19.8 21.3 23.0 23.7 24.0 24.9 25.9 27.1 29.6 31.6 32.4 37.6 40.1 45.7

terminal tryptophan moiety and a C-terminal phenylalanine to assist in compound identification by LC-UV analysis due to characteristic chromophores indicating the formation of C- or N-terminal breakdown products. Additionally, natural proteolytic enzymes with exopeptidase activity have a tendency to cease degradation at aromatic amino acid residues,15,16 hence 7449

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Figure 1. Thermal oxidation of FAKAW heated at 220 °C for 10 min. (a) Zoomed base peak chromatogram (BPC) of FAKAW heated at 220 °C for 10 min in positive ion mode using an UHPLC-ESI-Q-TOF-MS. Identification of numbered peaks is presented in Table 2. Peak nos. 12 and 13 show the oxidized mother ion. (b) Mass spectrum, which reveals oxidation of FAKAW [M + H + O]+, m/z 638.3, 12 obtained with an UHPLC-ESI-Q-TOF mass spectrometer (positive ion mode), and (c) MS2 spectrum of the oxidation product at m/z 638.3.

Figure 2. MS2, MS3, and MS4 spectra of oxidized FAKAW obtained from an ion trap mass spectrometer in positive ion mode, proving that the oxidation took place on the pyrrole ring of tryptophan.

accumulating such peptides in typical fermented foods such as cocoa, dairy, or soy adding particular relevance to these sequences from a dietary composition perspective.

With application of similar conditions to those in real food systems, samples were heated at 180 °C for 10 min in solid form in a laboratory oven and at 220 °C for 10 min both under 7450

DOI: 10.1021/acs.jafc.9b01056 J. Agric. Food Chem. 2019, 67, 7448−7454

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Figure 3. Proposed oxidation mechanism of the FAKAW.

at the C-terminus of the pentapeptides examined: oxidation and decarboxylation of tryptophan. Chemical mechanisms of these reactions are proposed. Oxidation. Oxidative reactions and the modifications that oxygen causes on proteomics are well investigated over the last few decades17 mainly in medicine or health-related fields. In terms of foods, few things are known regarding food protein oxidation including in many cases controversial results and conclusions. The complicated nature of the food system is one reason for this limitation. However, protein oxidation is considered to be responsible for the undesirable color (myoglobin in meat) and flavor (methionine oxidative products in milk) changes. Our results confirm the challenges that food scientists face in terms of food protein oxidation. The two pentapeptides examined, FAKAW and WAKAF, were thermally treated under atmospheric conditions. For this reason, the first expected occurring reaction is oxidation. With Phe and Trp having the highest rate constants of reactivity among all 20 proteinogenic amino acids,18 we expected to obtain oxidative product of the mother pentapeptide in both cases. However, oxidation of the intact (precursor) peptide was observed only in the case of FAKAW at m/z 638.3 [M + 15.99] ([C32H44N7O7]+) 12 (Figure 1a), having higher intensity as a doubly charged ion at m/z 319.6 13 (Table 2). The extracted ion chromatogram (Figure S1) at m/z 319.6 [M + 15.99] ([C32H44N7O7]2+) 13 reveals an oxidation of the mother pentapeptide with one O added. The MS2 spectrum (Figure 1c) shows a peak at m/z 165.1 B, which is the c1 ion of FAKAW, indicating that the N-terminus part of the pentapeptide was not oxidized. At the same time, the presence of both peaks m/z 221.09 A and 292.1 C, oxidized y1 and y2 ions, respectively, prove that the oxidation took place at the C-terminus of the pentapeptide and more specifically on the tryptophan. In order to clarify on which position the oxygen molecule was attached, targeted MS3 fragmentation was performed on the oxidized y1 ion m/z 221.09 A. Direct infusions were performed on an ion trap mass spectrometer in positive ion mode. Fragmentation of 220.7 A gave a base peak ion of m/z

atmospheric conditions. The LC−MS signals of thermal degradation products were largely identical in both cases; however, at lower temperature only 10% reaction yield was observed, whereas at the higher temperature the reaction yield was estimated at 40−80%, allowing acquisition of tandem MS data for all major degradation products. Hence, all further work was continued at the higher temperature regime. To investigate the formation of thermal-degraded products favored for each individual pentapeptide and to explain the chemical reactions that occur, data were analyzed manually, taking into account both high-resolution mass data and fragmentation patterns. Tables 2 and 3 present the highresolution masses of all thermal-degraded products obtained from FAKAW and FAQAW, respectively. Base peak chromatograms (Figures 1a and S1) are also presented. In total, 95 thermal-degraded products were identified as heat derivatives of the four examined pentapeptides. FAQAW produced the least number of thermal-degraded products (19 products were identified taking into account peaks with intensity higher than 104), whereas FAEAW gave the highest number of thermaldegraded products among the four pentapeptides examined (28 products were identified). In this work, an effort is made to explain the chemical mechanism hidden behind the thermal decomposition products, which are formed on the C terminus of tryptophan derivative. In order to estimate the amount of the precursor pentapeptide, which was degraded during the heating process, the ratio of the peak areas of the heated pentapeptide versus the unheated one was calculated and expressed in percentage. Only 19.2% of the FAQAW was degraded being the most thermostable pentapeptide among the four pentapeptides examined. FAEAW and FAKAW showed similar thermostability with 25.4% and 26.4% of the precursor being degraded, respectively. WAKAF was the least thermostable pentapeptide. 85.8% of the precursor WAKAF was degraded, proving that the peptides’ sequence plays a significant role on their thermostability. Investigation of Thermal Degraded Products. Our interest is focused on two chemical reactions, which took place 7451

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Figure 4. Thermal decarboxylation of FAQAW heated at 220 °C for 10 min. (a) Mass spectrum, which reveals decarboxylation of FAQAW [M + H − COO−]+, m/z 578.3 17 obtained with an UHPLC-ESI-Q-TOF mass spectrometer (positive ion mode), (b) MS2 spectrum of the decarboxylated product ion at m/z 578.3 17, (c) zoomed spectrum of (b) showing the fragment ion at m/z 232.1 J.

174.7 D followed by lower intensity ions of m/z 203.7 F and m/z 157.7 E/G. Further fragmentation of both MS3 peaks gave a base peak ion of m/z 156 G, fragment of F and m/z 156 E, fragment of D (Figure 2). Figure S2 presents a proposed fragmentation scheme of the fragment ions obtained from the oxidized FAKAW. An oxidation chemical mechanism is also suggested (Figure 3). Although the origin of the hydroxyl derivative is considered tentative, based on additional fragmentation data on FAQAW (Figure S6 and S7), which is oxidized as well, we believe that the regiochemistry is clearly defined at the 2-OH position on the indole ring based on a characteristic fragment ion at m/z 147 (see Figure S7). At this point, a further attempt to explain the origin of the hydroxyl radical in our samples was made. We have identified minor peroxo species (Figure S8), leading to the proposed mechanism that involves initial formation of a benzylic radical, which is quenched by dioxygen at the 2-OH position due to resonance. The intermediate peroxo radical propagates the radical chain reaction reminiscent of lipid peroxidation. The peroxo species is further reduced to yield the

oxidized tryptophane moiety. The initial radical might be formed after homolytic bond cleavages (Figure 3). Oxidation of tryptophan is extensively examined in the literature.19 However, most of the studies focus on individual tryptophan oxidation behavior as a single amino acid,20 or they approach the topic from a biological point of view focusing on large protein modifications.18 This is the first time that oxidation of tryptophan is examined on model pentapeptide systems. Due to the electron rich nature of the indole group, tryptophan is easily oxidized. The obtained fragments prove that the oxidation took place on the pyrrole ring of tryptophan. We assume that the hydroxyl radical attacked the C2 carbon of the indole group instead of C3 carbon. Reactions of pyrroles with electrophiles on the C2 and C5 are favored because of the resonance-stabilized intermediate products formation. Nakawaga et al.19a present in detail a mechanistic model of tryptophan oxidation to formylkynurenine. However, none of the oxidative tryptophan products found in the literature come in agreement with the oxidation mechanism proposed in this work. To the best of our 7452

DOI: 10.1021/acs.jafc.9b01056 J. Agric. Food Chem. 2019, 67, 7448−7454

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fragment ion ([C13H18N3O]+) (Figure 4c), revealing that the decarboxylation occurred at the C-terminus of tryptophan. Detailed fragmentation scheme is presented in Figure S3. Similar arguments also apply to FAEAW yielding N-acyl tryptamine derivatives following thermal decarboxylation. Rand et al.23 proposed a mechanism for the ketonic decarboxylation of adipic acid (20gr) to form cyclopentanone. The first two steps of this mechanism include deprotonation of the carboxylic acid under basic conditions and decarboxylation at 250−280 °C. Although in this work FAQAW was heated up at 220 °C, we propose a similar chemical mechanism (Figure 5), considering a smaller amount of sample was heated up (50 μg). In this work an effort has been made to explore the chemical mechanisms that are hidden behind thermal degradation of food pentapeptides. Applied heating conditions were similar to these used at a household level. High-resolution and tandem mass spectrometry data proved to be a sufficient analytical tool in identifying selected thermal degradation products obtained after modifications at the C-terminus of the examined pentapeptides. Oxidation and decarboxylation of tryptophan were the main chemical reactions that occurred.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01056. Food-derived bioactive peptides, high-resolution mass data, extracted ion chromatogram, proposed fragmentation schemes, zoomed base peak chromatogram, EIC, Tandem MS, MS3 mass spectrum, and BPCs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 49 421 200 3120. Fax: 49 421 200 3229.

Figure 5. Proposed decarboxylation mechanism of FAQAW.

knowledge, this is the first time that oxidation on the C2 of indole group of tryptophan on pentapeptides is noted. Decarboxylation. Decarboxylation was the second chemical reaction occurring at the C-terminus of both FAQAW and FAEAW. Tryptamine, a tryptophan derivative monoamine, is produced by gut bacteria in human gastrointestinal tract. Recently, Bhattarai et al. showed how tryptamine accelerates the whole-gut transit21 contributing to human health by inhibiting diseases such as the irritable bowel syndrome. In food products, decarboxylation of amino acids mainly occurs due to enzymatic activity, which leads to biogenic amines formation.22 However, thermally treated food products may also include decarboxylation as an essential step that leads to further thermal-degraded products formation. Decarboxylation of asparagine, for instance, is responsible for acrylamide formation in foods.8 Although the thermal decarboxylation mechanism has been reported for free amino acids and in Amadori products yielding Strecker aldehydes to our knowledge, this is the first report in dietary pentapeptides. Figure 4a presents the decarboxylated precursor ion of FAQAW in positive ion mode at m/z 578.3 [M − 43.99] ([C30H40N7O5]+) 17. The MS/MS fragmentation acquired using the auto-MSn mode (Figure 4b) generated a base peak at m/z 347.1 H and two less intense peaks at m/z 191.1 I and 219.1 K, b3, a2, and b2 fragment ions, respectively, which show that CO2 loss took place at the C-terminus of the FAQAW. The fragment ion at m/z 232.1 J corresponds to a decarboxylated y2

ORCID

Nikolai Kuhnert: 0000-0003-1681-8424 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from Jacobs University Bremen is gratefully acknowledged. Furthermore, excellent technical support by Ms. Anja Müller is acknowledged.

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ABBREVIATIONS USED HPLC, high performance liquid chromatography; UHPLC-ESIQ-TOF-MS, ultra-high performance liquid chromatographyelectrospray ionization quadrupole time of flight mass spectrometer; LC−MS, liquid chromatography−mass spectrometry; LC-UV, liquid chromatography ultraviolet; BioPepDB, bioactive peptide database; FAKAW, phenylalanylalanyllysinylalanyltryptophan; FAQAW, phenylalanylalanylglutaminylalanyltryptophan; FAEAW, phenylalanylalanylglutamylalanyltryptophan; WAKAF, tryptophanylalanyllysinylalanylphenylalanine

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DOI: 10.1021/acs.jafc.9b01056 J. Agric. Food Chem. 2019, 67, 7448−7454