Peptidomics of Peptic Digest of Selected Potato Tuber Proteins: Post

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Peptidomics of Peptic Digest of Selected Potato Tuber Proteins: Post-Translational Modifications and Limited Cleavage Specificity Subin R. C. K. Rajendran,† Beth Mason,‡ and Chibuike C. Udenigwe*,† †

Department of Environmental Sciences, Dalhousie University, Truro, Nova Scotia B2N 5E3, Canada Verschuren Centre for Sustainability in Energy and the Environment, Cape Breton University, Sydney, Nova Scotia B1P 6L2, Canada

‡

S Supporting Information *

ABSTRACT: Bioinformatic tools are useful in predicting bioactive peptides from food proteins. This study was focused on using bioinformatics and peptidomics to evaluate the specificity of peptide release and post-translational modifications (PTMs) in a peptic digest of potato protein isolate. Peptides in the protein hydrolysate were identified by LC−MS/MS and subsequently aligned to their parent potato tuber proteins. Five major proteins were selected for further analysis, namely, lipoxygenase, α-1,4glucan phosphorylase, annexin, patatin, and polyubiquitin, based on protein coverage, abundance, confidence levels, and function. Comparison of the in silico peptide profile generated with ExPASy PeptideCutter and experimental peptidomics data revealed several differences. The experimental peptic cleavage sites were found to vary in number and specificity from PeptideCutter predictions. Average peptide chain length was also found to be higher than predicted with hexapeptides as the smallest detected peptides. Moreover, PTMs, particularly Met oxidation and Glu/Asp deamidation, were observed in some peptides, and these were unaccounted for during in silico analysis. PTMs can be formed during aging of potato tubers, or as a result of processing conditions during protein isolation and hydrolysis. The findings provide insights on the limitations of current bioinformatics tools for predicting bioactive peptide release from proteins, and on the existence of structural modifications that can alter the peptide bioactivity and functionality. KEYWORDS: peptidomics, bioactive peptides, bioinformatics, in silico, potato proteins, post-translational modifications

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INTRODUCTION Food protein-derived bioactive peptides are important ingredients for developing functional foods and nutraceuticals for preventative healthcare. Depending on their sequence and structure, peptides have been shown to display several bioactivities that are important in modulating aberrant health conditions in cardiovascular, immune, gastrointestinal, and nervous systems.1,2 Peptides can modulate specific factors responsible for the functioning of the body for general health benefits,3 and can also be used in developing novel functional biomaterials.4,5 A number of plant proteins are currently utilized as sources for the production of bioactive peptides.6,7 The pharmaceutical industry has extensively used bioinformatic tools for drug development during recent decades. Molecular docking and ligand pharmacore prediction are often utilized during drug discovery.8 Similarly, in areas of food bioscience research, there is an increasing trend in the use of in silico models to predict the presence of bioactive peptides in protein precursors.9−11 In silico hydrolysis can be conducted with the BIOPEP “enzyme action” tool,9 or with PeptideCutter, an ExPASy tool that can cleave a given protein or peptide sequence based on the cleavage specificity of proteases.12 Furthermore, the probability of bioactivity of the peptides generated experimentally or in silico can then be predicted based on scores assigned by PeptideRanker13 as recently discussed.11 The bioinformatic tools have the potential to accelerate the prediction of novel bioactive peptides from a large protein database. However, the structure−function relationships of peptides for different bioactivities have not © XXXX American Chemical Society

yet been completely defined, and this can impede the rapid discovery of bioactive peptides from food proteins. Furthermore, bioactive peptides are considered to be generated in their native forms. However, in a complex food matrix or during production (e.g., enzymatic hydrolysis), peptides can undergo chemical modifications and matrix interactions that can alter their functionalities.14 The native precursor proteins are also known to undergo changes physiologically, or during isolation and hydrolysis, that can alter their structural forms, a process known as post-translational modifications (PTMs). Given the high chemical reactivity of peptides and their susceptibility to structural modifications, there is a need to consider PTMs in elucidating and understanding the structural relationships that govern food peptide bioactivity. In the present study, we conducted peptidomic analysis of potato protein hydrolysate generated with pepsin, in an attempt to further understand the structural basis of the hydrolysate’s previously reported bioactivity.15 In silico analysis was also performed on selected proteins identified from the potato protein isolate, based on the relative abundance of constituent peptides in the protein hydrolysate. The aim of the study was to explore the in silico and peptidomic data of the potato protein hydrolysate to gain insights on protein cleavage patterns, PTMs, and peptide structure−PTM relationships. Received: January 26, 2016 Revised: March 3, 2016 Accepted: March 6, 2016

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DOI: 10.1021/acs.jafc.6b00418 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Properties, Functions, and Peptidomic Data of the Five Selected Proteins in the Peptic Potato Protein Hydrolysatea protein

function

lipoxygenase lipid metabolism α-1,4 glucan phosphorylase L-1 carbohydrate metabolism isozyme annexin calcium and phospholipid binding patatin-2-Kuras 3 lipid metabolism and storage protein polyubiquitin protein degradation a

no. of AA residues

−10 log P

coverage (%)

total no. of peptides identified

unique peptides

area under the tR curve

861 966

395.73 384.81

88 83

277 250

4 243

1.18 × 109 1.02 × 109

314

358.36

87

89

89

3.01 × 108

386

361.04

85

202

7

1.50 × 109

77

151.85

93

12

12

2.15 × 107

AA, amino acids; P, probability; tR, retention time.

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quantitated based on the area under the tR curve for each peptide. This was then used to calculate the percentages of the modified to unmodified peptides in a locus with a PTM as follows:

MATERIALS AND METHODS

Materials. Russet potatoes were purchased from a local store in Truro, NS, Canada. Pepsin from porcine gastric mucosa was purchased from Sigma-Aldrich (Oakville, ON, Canada). All the other reagents used for the experiments were purchased from Fisher Scientific Co. (Ottawa, ON, Canada). Preparation of the Potato Protein Hydrolysate. Protein isolation from potato tuber extract was conducted as earlier reported based on isoelectric protein precipitation at pH 5.15 The isolated protein fraction was collected, lyophilized, and stored at −20 °C prior to hydrolysis. The protein isolate was suspended in water to form aqueous slurry (5% w/v) and subsequently hydrolyzed with pepsin (from porcine gastric mucosa) at an enzyme/substrate ratio of 1:100 (w/w) at 37 °C and pH 2 for 1 h to mimic the gastric digestion phase.15 Thereafter, hydrolysis was terminated by heating the mixture at 90 °C for 15 min and the sample was then freeze-dried. LC−MS/MS Analysis. Q-Exactive Orbitrap analyzer outfitted with a nanospray source and EASY-nLC nano-LC system (Thermo Fisher, San Jose, CA, USA) was used to perform the LC−MS/MS analysis at the Mass Spectrometry Facility at SPARC BioCentre, The Hospital for Sick Children (Toronto, ON, Canada), as previously reported.15 Peptidomics. PEAKS software (Bioinformatic Solutions Inc., Waterloo, ON, Canada) was used to analyze the LC−MS/MS data.16 The data analysis of the spectra was performed based on the approach reported by Broeckx et al.17 The database (DB) workflow module, a combination of DB (UniProt, Solanum tuberosum) search module and de novo sequencing, was performed with the following parameters: a precursor mass tolerance of 10 ppm using monoisotopic mass, and a fragment mass tolerance of 0.02 Da. The PEAKS Q module was then used for quantitation of the peptides based on the total area under the LC retention time (tR) curve. The potato proteins were identified based on their constituent peptide coverage, and their abundance in the hydrolysate was determined based on the total area under the LC tR curve of their constituent peptides. Five potato proteins were selected for further analysis based on their high coverage, abundance, confidence level, and physiological functions. Peptide PTM Analysis. The SPIDER feature of PEAKS was used to identify the peptides with PTMs based on mass difference. A peptide identification filter, false discovery rate (FDR) of 0.5%, was set for each sample, and protein identification was based on the presence in each protein of at least two unique peptides, which are not shared by multiple proteins. In addition, the protein identification reliability score (−10 log P, where P is the probability of identification) was set at a threshold of 17.5, corresponding to confident identifications. PTM Frequency. PTMs were accepted at a locus based on a threshold Ascore (a probability based score indicating the likelihood of modification occurring at a locus18) of 20. Frequency of occurrence of PTMs in a protein, based on the peptides aligned to it, was calculated manually using the formula Xm/∑X × 100; where Xm is the number of amino acid residues with PTMs in a given protein, and ∑X is the total number of particular modifiable amino acid residues (Met, Asn, Gln, or all three) in the protein. PTM Profiling. A minimal ion intensity of 5% was kept as the threshold to identify the loci with PTMs in the major proteins. The relative abundance and amount of peptides in proteins can be

% modified peptides =

∑ A m /∑ A × 100

where ∑Am is the total of area of peptides with the modified locus, and ∑A is the total of area of peptides with the locus. Protein Modeling for PTM Visualization. To visualize PTMs in the native structure of proteins, SWISS-MODEL tool19 was used to build the 3D structures for the major potato proteins. The structures were predicted based on templates from other plant homologues available in Protein Data Bank. PyMOL Molecular Graphics System (Version 1.7.4 Schrödinger, LLC) was used to observe the structures and to label amino acid residues with PTMs. In Silico Analysis. In silico analysis was conducted as previously reported.11 Sequences of the five selected potato proteins were obtained from UniProtKB and subjected to in silico hydrolysis using ExPASy PeptideCutter (Swiss Institute of Bioinformatics) with pepsin at pH ≥ 2. The peptides resulting from in silico hydrolysis were then compared to those identified by peptidomics with consideration for parameters such as number of cleavage sites, cleavage specificity, and size of the peptides.

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RESULTS AND DISCUSSION Peptidomic Data Analysis. In the PEAKS DB workflow, the database search module enabled peptide identification based on homology search in protein databases whereas de novo sequencing was used for identification of novel peptides. In total, 3667 peptides were characterized based on their molecular weights derived from the LC−MS/MS spectra.15 Based on parameters such as −10 log P, protein coverage, area under the tR curve (relative abundance), and varying physiological roles, five proteins were selected for further analysis, namely, lipoxygenase (accession no. Q9SC16), α-1,4 glucan phosphorylase L-1 isozyme (P04045), annexin (Q9M3H3), patatin-2-Kuras 3 (Q42502), and polyubiquitin (E2I6L5). The different features of these proteins, their physiological function, and peptidomic data are shown in Table 1. Lipoxygenase had the highest confidence level based on the −10 log P value and a high coverage (88%), which indicates the percentage of the protein sequence covered by the identified peptides. However, based on area under the tR curve (indicating relative abundance), it was observed that patatin is the most abundant protein in the fraction. Patatin homologues are the major storage proteins in potato,20 and their high abundance is supported by the electrophoretic profile of the potato protein isolate.15 PTM Analysis. PTM assessment of the hydrolysate resulted in the identification of peptides, in the selected proteins, that contain oxidized Met and deamidated Glu/Asn residues (Figure 1). We present two hypotheses for explaining the PTMs observed in the protein hydrolysates. The first one takes B

DOI: 10.1021/acs.jafc.6b00418 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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(solubilization and precipitation) on the protein native structure should also be taken into consideration when evaluating their susceptibility to PTMs. PTM Profiling and Visualization. PTM profiling in the lipoxygenase and patatin proteins at different loci containing modifications is depicted in Figure 2. It is clear that amino acid

Figure 1. Frequency of PTMs occurring in the selected proteins present in the potato protein hydrolysate fraction.

into account the conditions that the proteins were subjected to during processing, i.e., protein isolation and enzymatic hydrolysis. The second hypothesis considers possible modification of the intact protein during the physiological processes in the potato tuber. Both hypotheses have been supported by previous studies with other proteins. Methionine oxidation has been shown to be a physiologically relevant process that occurs as proteins age or as proteins interact with reactive oxygen species.21 Methionine can undergo either a two-electron oxidation to methionine sulfoxide or a one-electron oxidation to methionine radical cations.22 Both mechanisms require the catalytic support and assistance from neighboring groups, which stabilize electron-deficient reaction centers.21 In our study, PTMs were observed to occur mostly in loci in close proximity to Ala, Glu, Asp, and Thr residues. In contrast, Asn and Gln deamidation can occur as a result of the action of proteases.23 Therefore, it is likely that the PTMs observed in the potato peptides originated as a result of processing, or from natural physiological processes in the potato tuber. Of the five selected proteins, annexin and polyubiquitin did not show any PTMs at the threshold of 5% ion intensity. The findings clearly demonstrated that Met oxidation is the predominant PTM in the peptides, and lipoxygenase and patatin were observed to have the highest number of PTMs (Figure 1). PTM Frequency. PTMs occurring in the five major proteins (at a threshold of Ascore ≥20) were assessed using PEAKS. The residues with PTMs in the proteins were compared to the total number of residues that are capable of being modified to give the PTM frequency. PTM frequency gave an indication of the susceptibility of a protein to PTMs. Based on the hypothesis that PTMs can be induced by physiological processes, lipoxygenase was predicted to have higher modifications, especially Met oxidation, due to its key biological role in lipid oxidation. Similarly, patatin is a storage protein that possesses lipase activity, and was also predicted to harbor PTMs. Our findings indicate that peptides derived from both lipid associated proteins are more susceptible to modifications than others. Figure 1 shows that patatin has the highest total PTM frequency, and lipooxygenase has the highest frequency of Met oxidation. However, based on the first hypothesis, all the proteins within the fraction have equal probability for PTMs as a result of exposure to processing conditions, which was not the case observed in this study. It can also be argued that the probability to harbor more PTMs is higher in lipooxygenase and patatin since they are the most abundant proteins in the fraction based on their constituent peptides (Table 1). Furthermore, the possible effect of the isolation process

Figure 2. Percentage of the modified to unmodified peptides in the peptide loci containing PTMs (with ion intensity >5%) for (A) patatin and (B) lipoxygenase.

residues in certain loci are more susceptible to modification than the others. Considering the amino acid residues located in close proximity to the PTM-containing loci (see Supporting Information), it was observed that Ala is present at the Nterminal of a number of the PTM sites, although a clear trend was not observed in this regard. To elucidate any protein structural characteristics underlying the occurrence of the observed PTMs, we modeled the 3D structures of lipoxygenase, α-1,4 glucan phosphorylase L-1 isozyme, and patatin by homology modeling using SWISSMODEL. Template homologue proteins with 66%, 61%, and 88% identity were used for each of these proteins, respectively. The structures were differentially labeled and visualized in PyMOL Molecular Graphics System (Version 1.7.4 Schrödinger LLC) as shown in Figure 3A−C. Interestingly, all the modified loci of patatin were present on α-helices (Figure 3A). M85 and M331 have the highest profiles of modification compared to other oxidized Met residues. Similarly, lipoxygenase was observed to have four loci with Met oxidation (Figure 2) including M25, which is not present on an α-helix (Figure 3C). Moreover, the latter has a very low profile of modified peptides (Figure 2) compared to the other loci. M690, the only PTM on the α-1,4 glucan phosphorylase L-1 isozyme, is also present on an α-helix (Figure 3B). Based on the findings, the presence of PTMs appears to be dependent on the secondary structure, predominantly occurring in α-helices, of the locus. Differential interaction of solvent with the secondary structures can lead to differences in the susceptibility of their C

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Figure 3. 3D protein structures of (A) patatin, (B) α-1,4 glucan phosphorylase L-1 isozyme, and (C) lipoxygenase depicting the PTMs.

PTMs into consideration. Therefore, it is imperative to validate whether PTMs in food proteins can affect their enzymatic hydrolysis by different food-grade proteases used for producing bioactive peptides. A recent study has applied the in silico approach in predicting the release of peptides from patatin and their associated bioactivity.29 Our peptidomics results indicate that peptic hydrolysate of patatin contains larger peptides than the theoretically generated peptides assessed for bioactivity by Fu et al.29 The smallest peptides detected in the LC−MS/MS analysis were hexapeptides, which can be attributed to the limited MS detection range, but can also be due to limited proteolytic cleavage, or low abundance of low molecular size peptides in the hydrolysates. The number of cleavage sites identified with both PeptideCutter (in silico) and peptidomics (experimental) data for the potato proteins is presented in Table 2. The cleavage specificity of pepsin in silico is clearly

constituent amino acid residues to oxidation during protein isolation and hydrolysis. Further studies are necessary to elucidate the mechanism of PTM occurrence and its relationship to secondary structure of proteins and peptides. The deamidation profile did not yield any distinct trend. Deamidation can occur during protein hydrolysis and was found to vary in the potato peptides depending on the adjacent amino acid residues. PTMs in Protein Digestibility and Bioactivity. PTMs of amino acid residues can induce changes that alter the physicochemical features of peptides and proteins. However, a previous study demonstrated that Met oxidation of casein did not affect in vitro digestibility, and only slightly reduced the nutritional availability of the protein.24 Similarly, we observed that the oxidized Met residues did not visibly influence the cleavage pattern of pepsin in the potato protein hydrolysate, as the peptides had similar cleavage patterns irrespective of the presence of PTMs. Although the value of proteins as food ingredients may appear largely unaffected, there are notable physiological consequences of Met oxidation. The catalytic activities of some enzymes in Arabidopsis have been shown to be reduced as a result of Met oxidation.25 Interestingly, PTMs have also been introduced into peptides to increase their bioactivity, and structure and function relationships have been elucidated for many PTMs.26 The PTMs observed in our study have revealed the need for routine assessment of their occurrence during bioactive peptide production from food proteins, and whether their presence would increase or decrease peptide bioavailability and bioactivity. In Silico Analysis. The primary sequence of the five proteins obtained from UniProtKB were subjected to in silico hydrolysis with pepsin at pH ≥ 2 using ExPASy PeptideCutter. This approach has been used to study the cleavage patterns of proteases in the release of bioactive peptides from food proteins.27,28 However, the in silico approach does not take

Table 2. Number of Cleavage Sites Identified by Peptidomics and in Silico Approaches protein lipoxygenase α-1,4 glucan phosphorylase L-1 isozyme annexin patatin-2-Kuras 3 polyubiquitin

in silico cleavage sites

exptl cleavage sites

234 241

196 200

81 102 17

68 128 11

different from the peptidomics data, and this also presents a limitation in using exclusively in silico tools for a reliable prediction of the value of precursor proteins for bioactive peptide production. The structural and functional basis of the discrepancies between in silico and experimental data needs to be identified and characterized to make this approach more reliable. D

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Journal of Agricultural and Food Chemistry In conclusion, findings from this study indicate that the in silico approach considered as a theoretical basis for prediction of bioactive peptides from precursor proteins needs further refining for more accuracy in cleavage specificity. Indeed, this factor can be greatly affected by the native protein structure, which would be challenging to predict considering the various conditions the proteins are often subjected to prior to enzymatic hydrolysis. Moreover, PTMs occurring in amino acid residues of peptides cannot be predicted using the bioinformatics approach, and this needs to be taken into account for assessing the sequence, solution structure, and functional aspects of proteins and peptides. PTMs have been hypothesized to occur in proteins and peptides as a result of processing conditions, and they can also form in native proteins in their physiological states. From the 3D modeling of lipoxygenase and patatin, the PTMs were predominantly associated with the α-helix secondary structure of the native proteins. Moving forward, it is imperative to understand the basis of the PTM formation, and their implications on peptide bioactivity, bioavailability, and safety for the purpose of human health promotion.

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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.6b00418.

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Amino acid sequence of peptides identified near PTMs (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1 (902) 893-6625. Fax: +1 (902) 893-1404. Funding

Financial support provided by the Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundation for Innovation, Nova Scotia Research and Innovation Trust, and MITACS Canada is gratefully acknowledged. S.R.C.K.R. is a recipient of a Nova Scotia Research and Innovation Graduate Scholarship. Notes

The authors declare no competing financial interest.

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