Pyroglutamyl Peptides Research Articles

Presence of Modified Peptides with High Bioavailability and Angiotensin-Converting Enzyme Inhibitory Activity in Japanese Fermented Soybean Paste (Miso).

Not only free amino acids and normal short-chain peptides but also modified amino acids, such as N-acetyl- and N-formyl amino acids, monoamines, polyamines, and modified peptides, such as isomerized aspartyl peptides, pyroglutamyl peptides, and diketopiperazines, were identified in Japanese fermented soy paste (miso) prepared using different fungal starters, rice, barley, and soybean-koji. One hour after oral administration of water extract of soybean-koji miso to rats, the modified peptides increased significantly in the lumen upon the ingestion, while the normal peptides did not. In the blood from the portal vein and abdominal vena cava, 17 and 15 diketopiperazines, 16 and 12 isomerized aspartyl peptides, and 2 and 1 pyroglutamyl peptides significantly increased to approximately 10-400 nM, respectively. The modified peptides, which increased in rat blood, showed angiotensin-converting enzyme (ACE) inhibitory activity in a dose-dependent manner, indicating multiple ACE inhibitory peptides with high bioavailability in miso. Among them, l-β-Asp-Pro showed the highest ACE inhibitory activity (IC50 4.8 μM).

Saltiness Enhancement through the Synergism of Pyroglutamyl Peptides and Organic Acids.

Excess sodium intake poses health risks, prompting the exploration of taste modulators to reduce the salt content in low-sodium foods yet maintain salty perception. Previous research found a subthreshold synergistic effect among pyroglutamyl dipeptides on saltiness enhancement. This study investigated the subthreshold synergistic effect of pyroglutamyl peptides and organic acids on saltiness perception. Pyroglutamyl dipeptides (pgluE, pgluV), pyroglutamyl tripeptides (pgluVL and pgluVC), and organic acids (malic acid and succinic acid) were explored in a model system and subsequently in commercial brown onion sauce. The detection thresholds of peptides (pgluE, pgluV, pgluVL, and pgluVC) were determined to be 646, 77, 273, and 221 μmol/L, respectively, and the subthreshold synergistic effect of the pyroglutamyl tripeptides and organic acids was determined using the isobologram method. One of the eight combinations of pyroglutamyl tripeptides with pyroglutamyl dipeptide (pgluV) showed a subthreshold synergistic effect, whereas four combinations of tripeptides with malic acid and one combination with succinic acid exhibited a subthreshold synergistic effect. In commercial brown onion sauce, 25 and 30% salt reductions were achieved using the combinations of the tripeptides with malic acid and succinic acid, respectively. This research lays the foundation for future investigations into the potential combinations of pyroglutamyl peptides and organic acids for saltiness enhancement in low-sodium foods.

Metabolic fate of peptides in a rice protein hydrolysate in rat intestine and blood after oral administration

The objective of this study was to elucidate the metabolic fate of peptides in a rice protein hydrolysate (RPH) in rats. Peptides present in RPH and its in vitro exopeptidase digest were identified using liquid chromatography–tandem mass spectrometry. One hour after an oral administration of RPH (250 mg/kg body weight), the levels of in vitro exopeptidase-resistant pyroglutamyl peptides in the lumen and ileal tissue significantly increased, whereas those of exopeptidase-susceptible peptides remained unchanged. Among them, the concentrations of pyroGlu-Leu, pyroGlu-Val, and pyroGlu-Lys significantly yet slightly increased in portal blood, while those of other hydrophilic ones (pyroGlu-Gln, pyroGlu-Glu, etc.) remained unchanged. However, all of the pyroglutamyl peptides were stable in rat ileal extract and blood plasma. These findings indicate that the pyroglutamyl peptides can enter enterocytes and resist to peptidases, while small amounts are transported to the blood, suggesting that pyroglutamyl peptides in enterocytes might be returned to the lumen.

Identification of short-chain pyroglutamyl peptides in Japanese salted fermented soy paste (miso) and their anti-obesity effect

Miso, a paste of salted fermented soybean, is a seasoning used extensively in traditional Japanese cuisine. Herein, pyroglutamyl peptides present in miso were identified by a liquid chromatography-tandem mass spectrometry (LC-MS/MS), detecting precursor ions, which generated immonium ion of pyroglutamyl residue. By using this method, 13 pyroglutamyl peptides were identified in four types of miso. Administration of the water extract prepared from 0.6 g soybean miso/kg body weight/day significantly suppressed high fat diet-induced weight gain. A similar effect was exerted by the hydrophobic pyroglutamyl peptide fraction, including pyroglutamyl proline (pEP), pEV, pEI, and pEL. Administration of a mixture of synthetic pEP, pEV, pEI, and pEL in a ratio to that in miso or pEL alone also suppressed the weight gain in a dose dependent manner. These results suggest that the short-chain hydrophobic pyroglutamyl peptides present in miso are effective in suppressing high fat diet-induced obesity.

Factors affecting levels of ferulic acid, ethyl ferulate and taste-active pyroglutamyl peptides in sake

Factors affecting ferulic acid, ethyl ferulate and taste-active pyroglutamyl (pGlu) peptides levels in sake were analyzed using small-scale sake brewing tests on eighteen rice samples with differing cultivar variety, cropped year and area, and polishing rate. Ferulic acid concentration in sake was highly positively correlated with its content in rice (r=0.782**, double asterisk indicates 1% significance level), feruloylesterase (FE) activity (r=0.804**) and feruloylated saccharide forming activity (FSFA) (r=0.619**) in the rice koji. The results suggested that ferulic acid in rice induced FE activity and FSFA, and these two enzymes accelerated the formation of ferulic acid in sake mash. The concentration of bitter-tasting peptides in sake was highly positively correlated with crude protein content in rice (r=0.786**), and negatively correlated with acid carboxypeptidase (ACP) activity to (pGlu)LFGPNVNPWH (r=-0.612**), fermentation length (r=-0.820**), and pyroglutamyl leucine ((pGlu)L) concentration in sake (r=-0.502*; 5% significance level). The observation suggested that bitter-tasting peptides are initially formed in sake mash in accordance with protein content in rice, and are then hydrolyzed to smaller peptides, such as (pGlu)L. An ACP specific to the hydrolysis of bitter-tasting peptides was suggested by the observation that ACP activity to (pGlu)LFGPNVNPWH was significantly correlated (-0.612**) to their formation whereas an ACP to Cbz-Glu-Tyr was not (r=0.220). It was suggested that (pGlu) oligo-peptide ethyl esters were formed during the decomposition of bitter-tasting peptides to which the ACP to (pGlu)LFGPNVNPWH might contribute.

P-318 - Japanese rice wine (Sake)-derived pyroglutamyl peptides have anti-inflammatory effect on dextran sulfate sodium (DSS)-induced acute colitis in mice

Inflammatory bowel disease (IBD) is partly caused by dysfunction of immune system and overproduction of inflammatory cytokines. It has been reported that several fermented foods have improving effect of IBD. We previously determined the anti-inflammatory effect of Japanese rice wine (Sake)-derived peptide, pyroglutamyl-leusin (pyroGlu-Leu), pyroglutamyl-tyrosine (pyroGlu-Tyr), and pyroglutamyl-asparaginyl-isoleucine (pyroGlu-Asn-Ile). Anti-colitic activity was evaluated using a dextran sulphate sodium (DSS)-induced colitis mouse model. Orally administrating artificially synthesized pyroGlu-Leu (0.1 mg/kg body weight), pyroGlu-Tyr (1.0 mg/kg body weight) and pyroGlu-Asn-Ile (0.1 and 1.0 mg/kg body weight) exhibited a significant protective effect against colitis in mice. Additionally, administrating pyroGlu-Leu and pyroGlu-Asn-Ile normalized colitis-induced colonic dysbiosis, whereas pyroGlu-Tyr did not. In vitro study, all 3 peptides, pyroGlu-Leu (4 microM), pyroGlu-Tyr (40 microM), and pyroGlu-Asn-Ile (400 microM) suppressed LPS-induced NO production in RAW264.7 cell. These results suggest that the identified pyroglutamyl peptides exhibited an anti-colitic activity via different mechanisms.

Structure, Content, and Bioactivity of Food-Derived Peptides in the Body.

Orally administered peptides are assumed to be degraded into amino acids in the body. However, our recent studies revealed some food-derived prolyl and pyroglutamyl peptides with 2-3 amino acid residues in the blood of humans and animals, while most of the peptides in the endoproteinase digest of food protein are degraded by exopeptidase. Some food-derived dipeptides in the body display in vitro and in vivo biological activities. These facts indicate that the biological activities of food-derived peptides in the body rather than those in food are crucial to understanding the mechanism of the beneficial effects of orally administered peptides.

Identification of pyroglutamyl peptides with anti-colitic activity in Japanese rice wine, sake, by oral administration in a mouse model

Two pyroglutamyl peptides with anti-colitic activity were identified in Japanese rice wine, sake, by oral administration of a small dose (0.1–1.0 mg/kg body weight) and in vivo activity-guided fractionation. Compounds in sake were fractionated by preparative isoelectric focusing followed by preparative reversed phase-liquid chromatography. Anti-colitic activity was evaluated using a dextran sulphate sodium (DSS)-induced colitis mouse model. The final active fraction contained three pyroglutamyl peptides: pyroglutamyl-tyrosine (pyroGlu-Tyr), pyroglutamyl-asparaginyl-isoleucine (pyroGlu-Asn-Ile) and pyroglutamyl-asparaginyl-isoleucyl-asparagyl-asparaginyl-proline (pyroGlu-Asn-Ile-Asp-Asn-Pro). Orally administrating artificially synthesized pyroGlu-Tyr (1.0 mg/kg body weight) and pyroGlu-Asn-Ile (0.1 and 1.0 mg/kg body weight) exhibited a significant protective effect against colitis in mice, whereas pyroGlu-Asn-Ile-Asp-Pro showed no significant effect. Additionally, administrating pyroGlu-Asn-Ile normalized colitis-induced colonic dysbiosis, whereas pyroGlu-Tyr did not. These results suggest that identified pyroglutamyl peptides exhibited an anti-colitic activity via different mechanisms.

Antidepressant-like effect of food-derived pyroglutamyl peptides in mice

The N-terminal glutamine residue, exposed by enzymatic cleavage of precursor proteins, is known to be modified to a pyroglutamyl residue with a cyclic structure in not only endogenous but also food-derived peptides. We investigated the effects of wheat-derived pyroglutamyl peptides on emotional behaviors. Pyroglutamyl leucine (pyroGlu-Leu, pEL) and pyroglutamyl glutaminyl leucine (pyroGlu-Gln-Leu, pEQL) exhibited antidepressant-like activity in the tail suspension and forced swim tests in mice. pEQL exhibited more potent antidepressant-like activity than pEL after i.p. and i.c.v. administration. pEQL exhibited antidepressant-like activity at a lower dose than Gln-Gln-Leu, suggesting that pyroglutamyl peptide had more potent activity. To examine whether pyroglutamyl peptides increased hippocampus neurogenesis, associated with the effects of antidepressants, we measured 5-bromo-2′-deoxyuridine (BrdU) incorporation. pEL and pEQL increased BrdU-positive cells in the dentate gyrus of the hippocampus. Intriguingly, pEL did not increase hippocampal mRNA and protein expression of brain-derived neurotrophic factor (BDNF), which is a factor associated with both neuropoietic and antidepressive effects. Thus, pyroglutamyl peptides may enhance hippocampal neurogenesis via a pathway independent of BDNF. We also confirmed that pEL and pEQL were produced in the subtilisin digest of major wheat proteins, glutenin and gliadin, after heat treatment. pEL and pEQL are the first peptides derived from wheat proteins to be shown to exhibit an antidepressant-like activity.

Anti-inflammatory effect of pyroglutamyl-leucine on lipopolysaccharide-stimulated RAW 264.7 macrophages

AimsFood-derived peptides have been reported to yield a variety of health promoting activities. Pyroglutamyl peptides are contained in the wheat gluten hydrolysate. In the present study, we investigated the effect of pyroglutamyl dipeptides on the lipopolysaccharide (LPS)-induced inflammation in macrophages. Main methodsRAW 264.7 macrophages were treated with LPS and various concentrations of pyroglutamyl-leucine (pyroGlu-Leu), -valine (pyroGlu-Val), -methionine (pyroGlu-Met), and -phenylalanine (pyroGlu-Phe). Cell viability/proliferation and various inflammatory parameters were measured by the established methods including ELISA and western blotting. The binding of fluorescein isothiocyanate-labeled LPS to RAW 264.7 cells was also measured fluorescently. Key findingsAll the tested dipeptides significantly inhibited the secretion of nitric oxide, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 from LPS-stimulated RAW 264.7 macrophages. Above all, pyroGlu-Leu inhibited the secretion of all these inflammatory mediators even at the lowest dose (200μg/ml). PyroGlu-Leu dose-dependently suppressed IκBα degradation and MAPK (JNK, ERK, and p38) phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, it did not affect the binding of LPS to the cell surface. SignificanceOur results indicated that pyroGlu-Leu inhibits LPS-induced inflammatory response via the blocking of NF-κB and MAPK pathways in RAW 264.7 macrophages.

Identification of Pyroglutamyl Peptides in Japanese Rice Wine (Sake): Presence of Hepatoprotective PyroGlu-Leu

Japanese rice wine, sake, is made from steamed rice, water, and lactic acid by "multiple parallel fermentation" with mold (Aspergillus oryzae) and yeast (Saccharomyces cerevisiae). Nineteen pyroglutamyl peptides were identified in commercially available sake. Among them, pyroGlu-Leu and pyroGlu-Gln were the major constituents. PyroGlu-Leu has been demonstrated to attenuate hepatitis and colitis in animal models. Commercial products (n = 5) contained pyroGlu-Leu at concentrations ranging from 40 to 60 μM (10-15 mg/L). The pyroGlu-Leu content in sake mash increased during the fermentation processes. However, no pyroGlu-Leu was produced by yeast inoculated into preheated mash. Furthermore, addition of (13)C-Leu to the mash did not increase the ratio of pyroGlu-(13)C-Leu to pyroGlu-(12)C-Leu. On the other hand, digestion of steamed rice with A. oryzae proteases increased the pyroGlu-Leu content. These results indicate that pyroGlu-Leu in sake is produced from rice proteins by digestion with A. oryzae proteases.

Identification of a Hepatoprotective Peptide in Wheat Gluten Hydrolysate against d-Galactosamine-Induced Acute Hepatitis in Rats

A hepatoprotective peptide, pyroglutamyl leucine (pyroGlu-Leu), was identified in wheat gluten hydrolysate through an in vivo activity-guided fractionation approach based on D-galactosamine-induced acute hepatitis in rats and fractionation of peptides with large-scale preparative ampholine-free isoelectric focusing. The active acidic fraction predominantly consisted of pyroglutamyl peptides and free pyroglutamic acid. Pyroglutamyl peptides were derivatized with phenyl isothiocyanate after removal of a pyroglutamyl residue by pyroglutamate aminopeptidase. The derivatives were purified by reversed-phase HPLC and subjected to sequence analysis. The active fraction contained pyroGlu-Ile, pyroGlu-Leu, pyroGlu-Gln, pyroGlu-Gln-Gln, and free pyroGlu. Ingestion of pyroGlu-Leu at 20 mg/kg body weight significantly decreased serum aspartate and alanine aminotransferases to approximately 30% and 20% of those values of the vehicle group, respectively, which were near the normal levels. Thirty minutes after ingestion of pyroGlu-Leu at 20 mg/kg, the concentration of pyroGlu-Leu in portal blood plasma increased to approximately 2 μM.

Profiling of N-Acetylated Protein Termini Provides In-depth Insights into the N-terminal Nature of the Proteome

N-terminal processing of proteins is a process affecting a large part of the eukaryotic proteome. Although N-terminal processing is an essential process, not many large inventories are available, in particular not for human proteins. Here we show that by using dedicated mass spectrometry-based proteomics techniques it is possible to unravel N-terminal processing in a semicomprehensive way. Our multiprotease approach led to the identification of 1391 acetylated human protein N termini in HEK293 cells and revealed that the role of the penultimate position on the cleavage efficiency by the methionine aminopeptidases is essentially conserved from Escherichia coli to human. Sequence analysis and comparisons of amino acid frequencies in the data sets of experimentally derived N-acetylated peptides from Drosophila melanogaster, Saccharomyces cerevisiae, and Halobacterium salinarum showed an exceptionally higher frequency of alanine residues at the penultimate position of human proteins, whereas the penultimate position in S. cerevisiae and H. salinarum is predominantly a serine. Genome-wide comparisons revealed that this effect is not related to protein N-terminal processing but can be traced back to characteristics of the genome.

S.24.02 The cell biology of Abeta generation

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Aβ(N3E-42) and Aβ(N3Q-42) was investigated. These amyloid-β peptides were expressed as fusion proteins with either the pre–pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Aβ(N3Q-42) after β- and γ-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Aβ-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.

Improved recovery of proteome‐informative, protein N‐terminal peptides by combined fractional diagonal chromatography (COFRADIC)

We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

Mass spectrometric identification of pyroglutamic acid in peptides following selective hydrolysis

The formation of a pyroglutamyl residue by enzymatic cyclization of an amino terminus glutamine residue is a common posttranslational modification [1–3]. Several biologically active peptides, most notably the thyrotropinreleasing hormone $(TRH)^1$ and gonadotropin-releasing hormone (GnRH), were among the first examples where the N-terminal modification was characterized. The widespread occurrence of glutaminyl cyclase in both plants and animals has ensured that the number of naturally occurring pyroglutamyl peptides is rapidly growing. A recent report established formation of pyroglutamic acid (Z) from N-terminal glutamic acid in recombinant monoclonal antibodies [4]. An N-terminus pyroglutamic acid residue impedes Edman sequencing, necessitating the use of a specific enzyme, pyroglutamate aminopeptidase, to liberate a peptide with a free amino terminus, which is then amenable to automated sequence analysis [5]. During the course of studies aimed at characterizing the peptide components of Conus venom, we encountered several examples of pyroglutamyl peptides. We describe here the serendipitous finding of mild hydrolysis conditions, which permit opening of the pyroglutamyl ring, without significant cleavage of primary and secondary amide bonds.

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Aβ(N3E-42) and Aβ(N3Q-42) was investigated. These amyloid-β peptides were expressed as fusion proteins with either the pre–pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Aβ(N3Q-42) after β- and γ-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Aβ-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.

Occurrence of the Free and Peptide Forms of Pyroglutamic Acid in Plasma from the Portal Blood of Rats That Had Ingested a Wheat Gluten Hydrolysate Containing Pyroglutamyl Peptides

In order to determine pyroglutamic acid levels in plasma, we developed a method based on precolumn derivatization of the carboxyl group of pyroglutamic acid with 2-nitrophenylhydrazine. Eight-week-old male SD strain rats were administered 200 mg of an acidic peptide fraction obtained from a commercial wheat gluten hydrolysate containing 0.63 mmol/g pyroglutamyl peptide. After administration, significant amounts of free pyroglutamic acid were observed in the ethanol-soluble fraction of the plasma from the portal vein. In addition, pyroglutamate aminopeptidase digestion of the ethanol-soluble fraction liberated significant amounts of pyroglutamic acid, which indicated the presence of the pyroglutamyl peptide. The presence of the pyroglutamyl peptide in the plasma was further confirmed by size exclusion chromatography. The levels of free and peptide forms of pyroglutamic acid increased significantly and reached a maximum (approximately 40 nmol/mL) at 15 and 30 min after administration, respectively.

Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions

N-terminal pyroglutamate (pGlu) formation from glutaminyl precursors is a posttranslational event in the processing of bioactive neuropeptides such as thyrotropin-releasing hormone and neurotensin during their maturation in the secretory pathway. The reaction is facilitated by glutaminyl cyclase (QC), an enzyme highly abundant in mammalian brain. Here, we describe for the first time that human and papaya QC also catalyze N-terminal glutamate cyclization. Surprisingly, the enzymatic Glu 1 conversion is favored at pH 6.0 while Gln 1 conversion occurs with an optimum at pH 8.0. This unexpected finding might be of importance for deciphering the events leading to deposition of highly toxic pyroglutamyl peptides in amyloidotic diseases.

Isolation and identification of indigestible pyroglutamyl peptides in an enzymatic hydrolysate of wheat gluten prepared on an industrial scale.

An enzymatic hydrolysate of wheat gluten was further digested in vitro with porcine pepsin and pancreatin to obtain an indigestible peptide. Indigestible pyroglutamyl peptide was isolated from the digest by strong cation-exchange, size-exclusion, and reversed-phase chromatographies. The pyroglutamyl peptide was digested with pyroglutamate aminopeptidase, and the digest was reacted with phenyl isothiocyanate. The resultant phenylthiocarbamyl (PTC) peptides were purified by reversed-phase HPLC by using binary gradient elution with ammonium acetate buffer, pH 6.0, and acetonitrile. The PTC peptides were analyzed with an automatic peptide sequencer on the basis of the Edman degradation method with a modified program. Some pyroglutamyl peptides were also analyzed by fast-atom bombardment ionization mass spectrometry without the pyroglutamate amino peptidase digestion. Consequently, pyroGlu-Asn-Pro-Gln, pyroGlu-Gln-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gly-Gln-Gly-Gln, pyroGlu-Gln, pyroGlu-Gln-Pro, pyroGlu-Ile-Pro-Gln, pyroGlu-Ile-Pro, pyroGlu-Gln-Pro-Leu, pyroGlu-Gln-Phe-Pro-Gln, pyroGlu-Ser-Phe-Pro-Gln, pyroGlu-Phe-Pro-Gln, and pyroGlu-Gln-Pro-Pro-Phe-Ser were identified.