Flammulina velutipes

Article Cite This: J. Agric. Food Chem. 2017, 65, 10368-10381

pubs.acs.org/JAFC

Proteomic Investigation of Metabolic Changes of Mushroom (Flammulina velutipes) Packaged with Nanocomposite Material during Cold Storage Donglu Fang,† Wenjian Yang,*,‡ Zilong Deng,§ Xinxin An,† Liyan Zhao,† and Qiuhui Hu*,† †

College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, P.R. China College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, Jiangsu 210046, P.R. China § Department of Food Science & Technology, Oregon State University, Corvallis, Oregon 97331-6602, United States ‡

S Supporting Information *

ABSTRACT: Metabolic changes of mushroom (Flammulina velutipes) applied with polyethylene (PE) material (Normal-PM) or nanocomposite reinforced PE packaging material (Nano-PM) were monitored using tandem mass tags (TMT) labeling combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique. A total of 429 proteins were investigated as differentially expressed proteins (DEPs) among treatments after a cold storage period. A total of 232 DEPs were up-regulated and 65 DEPs were down-regulated in Nano-PM packed F. velutipes compared to that of NormalPM. The up-regulated DEPs were mainly involved in amino acid synthesis and metabolism, signal transduction, and response to stress while the down-regulated DEPs were largely located in mitochondrion and participated in carbohydrate metabolic, amino acid synthesis and metabolism, and organic acid metabolic. It was also revealed that Nano-PM could inhibit the carbohydrate and energy metabolism bioprocess, promote amino acids biosynthesis, enhance antioxidant system, and improve its resistance to stress, resulting in a further extended shelf life of F. velutipes. KEYWORDS: nanocomposite packaging, Flammulina velutipes, TMT, proteome, storage

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INTRODUCTION Enoki mushroom (Flammulina velutipes) is low in fat and rich in essential amino acids, dietary fibers, and bioactive polysaccharides as well as its tasty favor, leading to the fourth rank of mushroom consumption in the world. However, fresh F. velutipes is extremely perishable due to its high moisture content and the absence of a cuticle on the surface of mushroom cap or stem.1 The conventional preservation methods of fresh mushroom were dehydration, dipping in chemical preservative agent, modified atmosphere packaging, and bioactive coatings.2 Nevertheless, most of them were difficult to operate and overlooked the cost or safety issues. Nanocomposite materials have attracted increasing interest for food packaging due to their antimicrobial activity, oxygen scavenging ability, barrier properties, and enzyme immobilization.3 In our previous study, a polyethylene based packaging material incorporated with nanoparticles (nano-Ag, nano-TiO2, nano-SiO2) was developed for fresh mushroom preservation.4 Compared to the polyethylene (PE) based packaging materials (Normal-PM), nanocomposite packaging materials (Nano-PM) could retain nutritional components and flavors and inhibit microbial growth, leading to extended mushroom shelf life.4,5 However, study related to the protein expression and metabolic alterations of mushroom packed with Nano-PM remains unclear. Proteomic techniques are typically applied to identify the novel proteins and explore the biological and metabolic dynamics of organism in response to environmental stressors. Compared to gel-based approach such as 2D-PAGE (twodimensional polyacrylamide gel electrophoresis) and 2D-DIGE (two-dimensional differential gel electrophoresis), the gel free © 2017 American Chemical Society

strategy eliminates the major experimental errors and improves testing accuracy.6,7 Liu et al. utilized iTRAQ-coupled twodimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique to investigate the proteome change of the transformation of mycelia into fruit-bodies in F. velutipes.8 It was also reported that iTRAQ-MS/MS was effective to identify the alteration of protein expression that occurred during the postharvest maturation of Agaricus bisporus.9 However, the studies on mushroom proteomic were still limited. Therefore, in order to gain more proteomic data and depict the potential mechanism of Nano-PM on F. velutipes preservation, TMT-labeling combined with LC-MS/MS were applied. It was hypothesized that Nano-PM could decrease the expression of F. velutipes proteins related with energy metabolism and upregulated the protein expression involved in stress resistance resulting in a longer shelf life of F. velutipes. The objectives of this study were (1) to identify the proteins in fresh mushrooms, Nano-PM, and Normal-PM packed mushrooms after 21 days of storage; (2) to distinguish the difference of protein expressions between Nano-PM and Normal-PM packed mushrooms after 21 days of storage; (3) to illuminate the potential mechanism of Nano-PM on F. velutipes preservation. Received: Revised: Accepted: Published: 10368

September 25, 2017 November 1, 2017 November 6, 2017 November 7, 2017 DOI: 10.1021/acs.jafc.7b04393 J. Agric. Food Chem. 2017, 65, 10368−10381

Article

Journal of Agricultural and Food Chemistry

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coupled with an Agilent 1200 high-performance LC system (Agilent Technologies, Santa Clara, CA) at a 0.3 mL/min flow rate. The labeled and dried peptides were reconstituted in buffer A (98% H2O, 2% ACN, pH 10.0), and loaded onto the column. After that, the peptides were eluted using gradient buffer B (20 mM ammonium in 90% acetonitrile, pH 10.0) in the following order: 2−6% in 3 min, 6−25% in 30 min, 25%−38% in 10 min, and 90% holding for 10 min. Elution was measured at 214 nm wavelength, and 10 combined fractions were vacuum-dried until LC-MS/MS analysis. Mass Spectrometry. Peptides were resuspended with nano-RPLC buffer A (0.1% FA, 2% ACN). The online Nano-RPLC was employed on the Easy-nLC 1000 System (Thermo Fisher Scientific Inc., San Jose, CA, USA). The samples were loaded on a trap C18 column (PepMap100, diameter 3 μm, 75 μm × 20 mm, NanoViper, Thermofisher Dionex) and washed by nano-RPLC buffer A at 2 μL/ min for 10 min. An elution gradient of 5−35% acetonitrile (0.1% FA) in 70 min was used on an analytical C18 column (PepMap100, diameter 2 μm, 75 μm × 150 mm, NanoViper, Thermofisher Dionex). Data acquisition was performed with a Q Exactive System (Thermo Fisher Scientific Inc., San Jose, CA) fitted with a Nanospray. The Q Exactive instrument was operated using a data-dependent top-20 method with 70 000 resolution for the full MS scans, 175 000 resolution for highenergy collisional dissociation (HCD) MS/MS scans and a dynamic 30s exclusion. Full MS scans were acquired in the Orbitrap mass analyzer over the range of 300−1800 m/z with a mass resolution of 70 000 at 200 m/z. The 12 most intense peaks with charge state ≥2 were fragmented in the HCD collision cell with 27% normalized collision energy. The tandem mass spectra were acquired in the Orbitrap mass analyzer with a mass resolution of 35 000 at 200 m/z. Protein Identification and Quantification. The MS/MS data were analyzed using Proteome Discoverer 1.3 software with the SEQUEST search engine, constrained with a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.02 Da. Data was searched against a Swiss-Prot complete uniprot-Agaricales database with a 1% false discovery rate (FDR) criteria. Carbamido methylation on cysteine and TMT isobaric labeling of lysine were specified as fixed modifications and oxidation on methionine was specified as a variable modification. A protein ratio was reported as a median value of the ratios for all quantifiable spectra of the peptides pertaining to the corresponding protein. For protein quantitation, we required at least two unique spectra. Differentially expressed proteins were defined by the criteria of a fold change (FC) ≥ 1.2 or ≤ 0.83 (P < 0.05). Bioinformatics Analysis. Hierarchical cluster analysis (HCL), Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using OmicsBean software (http://www.omicsbean.com:88/). RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Total RNAs were extracted from mushroom samples by Trizol reagent (Takara). The Prime Script cDNA Synthesis Kit (Takara) was used to reversely transcribe cDNA. qPCR was performed on an Applied Biosystems 7500 instrument (Foster City, CA) via SYBR Premix EX Taq (Takara). Primer sequences are shown in Table S3. The geometric means of gapdh were applied as internal references to normalize the expression of targets genes.11 Statistical Analysis. Differences among the validated gene expression levels were analyzed using Duncan’s test (SAS system, version 9.0, SAS Institute, Cary, NC) at a significance level of P < 0.05. Data were reported as the mean value and standard deviation of three replications.

MATERIALS AND METHODS

Packaging Materials Preparation. Nanoparticles including nanoAg, nano-TiO2, and nano-SiO2 were obtained from a commercial company (Nanjing Haitai Nanotechnology Company, China). NanoPM was prepared using three native nanomaterials (nano-Ag, nanoTiO2, and nano-SiO2) and PE according to the methods from our previous study.4 The prepared films with thickness about 40 μm were used to make 25 × 25 cm2 bags with a high-speed incising and sealing machine (SDD-A500/1200, Zhaoyuan Packaging Machiner Co., Shandong, China). Normal-PM, PE-based materials without any nanopowder, was applied as the control. Mushroom Preparation and Storage Conditions. Fresh F. velutipes (strain F3-46) were obtained from a local mushroom company (Jiangsu Tianfeng Biological Technology Co., Ltd., China) and selected according to their whiteness, shape, closed veil, and developmental stage (10−15 cm stipe length) with no visual defect. Being precooled at 4 °C and 90% relative humidity (RH) for 24 h, mushrooms were randomly packaged into Nano-PM and Normal-PM (250 g for each bag). The packaged mushrooms were stored at 4 °C and 90% RH for 21 days. Then, fresh F. velutipes (F), 21 days storage of Normal-PM packaged F. velutipes (P) and Nano-PM packaged F. velutipes (N) were sampled and stored at −80 °C for further protein analysis. Three independent biological replications were performed. Protein Extraction and Trypsin Digestion. Protein extraction and digestion were conducted based on published protocols with minor modification.10 Mushroom samples (2 g) were ground into powders using liquid nitrogen and extracted with 20 mL of acetone containing 10% w/v trichloroacetic acid. The samples were kept at −20 °C overnight and then centrifuged at 15 000g for 15 min at 4 °C. Discarding the supernatant, the precipitates were washed twice with methanol buffer (90% methanol, 50 mM tris-HCl pH 7.8) and air-dried. The sample powders were then resuspended and homogenized by sonication on ice with 5 mL of lysis buffer (50 mM tris-HCl, pH 7.2, 2% (v/v) βmercaptoethanol, and 1 mM phenyl methanesulfonyl fluoride) for 15 min. An equal volume of ice-chilled tris-saturated phenol (pH 7.8) was added and eddied. After 30 min incubation at 4 °C, the mixture was centrifuged at 5 000g for 30 min. The phenol phase was collected and precipitated for 1 h at −20 °C by adding five volumes of 0.1 M chilled ammonium acetate dissolved in methanol. The protein pellets were collected after being centrifuged at 5 000g for 30 min at 4 °C and washed twice with ice-chilled methanol, twice with ice chilled 80% acetone, and once with 100% acetone. The proteins were air-dried at 4 °C, and the Bradford assay was used to determine protein concentration. In total, 100 μg of proteins from each sample were incorporated into 100 μL of 100 mM tetraethylammonium bromide (TEAB) solution. A volume of 5 μL of tris(2-carboxyethyl) phosphine (TCEP, 200 mM) was added and incubated at 55 °C for 1 h. Samples were then mixed with 5 μL of 375 mM iodacetamide (IAA) and reacted in the darkness for 30 min. Five volumes of chilled acetone were added, and the mixture was stored at −20 °C overnight. After centrifugation at 8 000g for 10 min at 4 °C, the protein precipitate was collected and air-dried for 2−3 min. The protein samples were added with 100 μL of 100 mM TEAB. Trypsin digestion was carried out overnight at 37 °C with a 1:40 (w/w) ratio of trypsin to protein. TMT Labeling. Ten-plex TMT labeling (Thermo Fisher Scientific Inc., San Jose, CA) was performed according to the manufacturer’s instructions. In total, 0.8 mg of TMT powders was reconstituted with 41 μL of anhydrous acetonitrile at room temperature (RT), and the reagents were dissolved by vortexing for 5 min. Each sample was labeled by adding 41 μL of tag, followed by incubation for 1 h at RT. The reaction was quenched with the additional 8 μL of 5% hydroxylamine for 15 min incubation at RT. Samples were then pooled, vacuum-dried, and stored at −80 °C until further analysis. F group was labeled with 126, 127C, 127N; N group was labeled with 128C, 128N, 129C; P group was labeled with 129N, 130C, 130N; and the mixed protein of the above three samples were labeled with 131. High pH Reversed Phase Fractionation. The fractionation of peptides was performed using a Narrow-Bore NX-C18 column (150 mm × 2.1 mm, diameter 5 μm, Agilent Technologies, Santa Clara, CA)

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RESULTS Identification and Comparison of Differentially Abundant Protein. To understand proteomic change in F. velutipes with Nano-PM and Normal-PM packaging during cold storage, we applied TMT for determining the protein profiles. According to our criteria (false discovery rate ≤0.05, unique peptide number ≥2), 2283 proteins were identified from F, N, and P groups. Protein abundances that changed more than 1.2 folds and P < 0.05 were selected as differentially expressed proteins 10369

DOI: 10.1021/acs.jafc.7b04393 J. Agric. Food Chem. 2017, 65, 10368−10381

Article

Journal of Agricultural and Food Chemistry (DEPs). Following this criterion, 429 DEPs were identified in total. The volcano plot is a versatile figure illustrating fold-change analysis and t test simultaneously. Figure 1 showed the differential expression pattern of proteins in three groups: F. velutipes packed with Nano-PM versus fresh F. velutipes (N/F), F. velutipes packed with Normal-PM versus fresh F. velutipes (P/F), and F. velutipes packed with Nano-PM versus F. velutipes packed with Normal-PM (N/P). There were more differently upregulated proteins in response to N/F and P/F group. In the Venn diagram (Figure 2A), only 11 proteins out of the 429 DEPs overlapped in N/F, P/F, and N/P groups. A total of 176 proteins were up-regulated and 5 proteins were down-regulated in the comparison of the N/F groups, 117 proteins were up-regulated and 28 proteins were down-regulated in the P/F comparison, and 232 proteins were up-regulated and 65 proteins were downregulated in the N/P group comparison (Figure 2B). Two upregulated DEPs were overlapped in N/F, P/F and N/P groups, while no mutual down-regulated DEPs was observed. As one of the nonmodel organism, little work has been done to support and characterize the identified mushroom proteins. In our study, there were 66, 57, and 122 uncharacterized DEPs in N/F, P/F, and N/P groups, respectively (Figure 2B). The detailed results of the characterized DEPs in N/P group were shown in Table 1. The other two groups (N/F and P/F) were summarized in Tables S1 and S2. Hierarchical cluster analysis was presented as a heat map to distinguish protein abundance among N/F, P/F and N/P. As illustrated in Fig. S1, DEPs in F. velutipes packed with Nano-PM after storage period were found to be distinctly expressed. Thus, different strategies might be varied according to the packaging material under cold storage conditions. Therefore, we focused on the analysis of DEPs in N/P group to illustrate the mechanism of Nano-PM on F. velutipes preservation in comparison with Normal-PM. GO Annotations and KEGG Pathway. On the basis of the 297 identified DEPs data including 175 characterized and 122 uncharacterized proteins in N/P group, we divided 175 characterized DEPs into two groups (up-regulated and downregulated proteins) and gained their functional information via Uniprot (http://www.uniprot.org/). GO annotations were classified into cellular component, molecular function, and biological process. In the cellular component group, the upregulated DEPs were mainly distributed in the cytoplasm (50%), cell part (10%), and nucleus (9%) (Figure 3A), while the mitochondria (35%), cytoplasm (31%), and membrane (18%) were mainly down-regulated DEPs (Figure 4A). The biological processes analysis indicated that protein in Nano-PM packed F. velutipes were up-regulated mainly in amino acid synthesis and metabolic (16%), protein transport (15%), signal transduction (11%), translation (11%), and response to stress (11%) (Figure 3B). In down-regulated DEPs, they were mainly involved in carbohydrate metabolic (32%), amino acid synthesis and metabolic (24%), and organic acid metabolic process (12%), suggesting more nutrient consumption of Normal-PM packed samples during storage (Figure 4B). Regarding to the molecular function, binding (33%) and oxidoreductase activity (15%) were the predominant in up-regulated DEPs (Figure 3C) while downregulated DEPs were mainly exhibited binding, hydrolase activity and transferase activity (Figure 4C). Thus, it was speculated that the alterations of the packaging efficiency among treatments were mainly due to the changes in the metabolic process and chaperone function.

Figure 1. Volcano plots of data sets showing the relationship between the magnitude of differentially expressed proteins in F, N, and P groups. Nano-PM packaged mushroom vs fresh mushroom (N/F), normal-PM packaged mushroom vs fresh mushroom (P/F), and Nano-PM packaged mushroom vs Normal-PM packaged mushroom (N/P). Each point represented a protein; red points were differentially expressed proteins and black points were expressed proteins without a significant (P < 0.05) difference. 10370


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Figure 2. (A) Venn diagrams showing the overlap in differential protein expression between the three groups of F. velutipes (N/F, P/F, and N/P). Shown is the total number of significantly differentially expressed proteins as well as the numbers of up-regulated and down-regulated proteins. (B) The histogram shows the number of up-regulated and down-regulated differentially expressed proteins in each group. The pie chart shows the distribution of the functionally annotated or unchartered proteins in value and percentage.

KEGG pathway analysis was applied to collect the information on protein functions in the metabolic process (Table 2). Eight DEPs in N/P group were associated with 12 specific KEGG

pathways. Three DEPs were involved in carbohydrate and lipid metabolic (path, cci01110); five DEPs were mainly distributed in energy metabolism (path, cci01100); three DEPs were 10371


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Journal of Agricultural and Food Chemistry Table 1. List of Differentially Expressed Proteins in N/P Groupa no.

Uniprot IDb

Up-Regulated Proteins 1 A0A060ILH1 2 A0A067NC76 3 A0A067NU58 4 A0A067PB22 5 A0A067TUE1 6 A0A0C9TA46 7 A0A0C9WWA5 8 A0A0C9YA87 9 A0A0D0C5B5 10 A0A0D0CK06 11 A0A0D2ML80 12 A0A0D2MQA7 13 A0A0D2MQH0 14 A0A0D7A242 15 A0A0D7A8Y9 16 A0A0D7AP59 17 A0A0D7AV20 18 A0A0D7AYV4 19 A0A0D7AYW4 20 A0A0D7AZK2 21 A0A0D7B251 22 A0A0D7B3E9 23 A0A0D7B3W1 24 A0A0D7B4Q6 25 A0A0D7B532 26 A0A0D7B555 27 A0A0D7B5S8 28 A0A0D7B6H0 29 A0A0D7B6K0 30 A0A0D7B773 31 A0A0D7B945 32 A0A0D7B9Z1 33 A0A0D7BA85 34 A0A0D7BB34 35 A0A0D7BB98 36 A0A0D7BCK4 37 A0A0D7BDB9 38 A0A0D7BDF4 39 A0A0D7BDW1 40 A0A0D7BFH8 41 A0A0D7BFU2 42 A0A0D7BG76 43 A0A0D7BH34 44 A0A0D7BI58 45 A0A0D7BIQ6 46 A0A0D7BJ57 47 A0A0D7BJN7 48 A0A0D7BKE3 49 A0A0D7BLF9 50 A0A0D7BM46 51 A0A0D7BNQ8 52 A0A0D7BNY3 53 A0A0D7BQ86 54 A0A0D7BQ93 55 A0A0D7BQQ3 56 A0A0D7BR14 57 A0A0D7BRQ3 58 A0A0D7BSN6 59 A0A0D7BSY4 60 A0A0D7BT08

protein name Heat-shock protein 90 Obg-like ATPase 1 Phospholipid-transporting ATPase Glycoside hydrolase family 31 protein Phosphotransferase Eukaryotic translation initiation factor 3 subunit J Ubiquitinyl hydrolase 1 Phosphatidylserine decarboxylase proenzyme 2 GTP-binding nuclear protein Obg-like ATPase 1 S-(hydroxymethyl)glutathione dehydrogenase Saccharopine dehydrogenase [NAD(+), L-lysine-forming] Glutathione peroxidase GroES-like protein p-loop containing nucleoside triphosphate hydrolase protein Arginyl-tRNAsynthetase TIP120-domain-containing protein Cytoplasmic protein Eukaryotic translation initiation factor 3 subunit J Outer dynein arm light chain 8 PP2C-domain-containing protein HIT-like protein Autophagy-related protein 3 Eukaryotic translation initiation factor 3 subunit A ARM repeat-containing protein Mitogen-activated protein kinase HCP-like protein Methionine sulfoxide reductase B Farnesyl-diphosphate farnesyltransferase NAD(P)-binding protein Glycosyltransferase family 2 protein Myosin regulatory light chain cdc4 Tryptophan synthase beta subunit-like PLP-dependent enzyme Myo-inositol-1-phosphate synthase Polyubiquitin-tagged protein recognition complex, Npl4 component DUF1348-domain-containing protein Aldo/keto reductase Phosphatidylserine decarboxylase proenzyme 2 Urease accessory protein ureG Nonspecific serine/threonine protein kinase Coatomer subunit gamma Lanosterol 14-alpha-demethylase Aldo/keto reductase Vacuolar protein sorting-associated protein 60 Small monomeric GTPase Formate dehydrogenase Nonmuscle myosin heavy chain b Kinesin heavy chain YTH-domain-containing protein Phospho-2-dehydro-3-deoxyheptonate aldolase Aldo/keto reductase Kinase-like protein ARM repeat-containing protein Regulator of G protein signaling superfamily NAD(P)-binding protein Aldehyde dehydrogenase RNA-binding domain-containing protein Tryptophan synthase HCP-like protein Mitogen-activated protein kinase 10372

gene name

fold change

HSP90 PLEOSDRAFT_1046650 PLEOSDRAFT_1056164 PLEOSDRAFT_1073828 GALMADRAFT_235701 HCR1 K443DRAFT_684818 PSD2 GYMLUDRAFT_262859 GYMLUDRAFT_242240 HYPSUDRAFT_135998 HYPSUDRAFT_64362 HYPSUDRAFT_52546 FISHEDRAFT_76950 FISHEDRAFT_46516 FISHEDRAFT_35012 CYLTODRAFT_494795 CYLTODRAFT_433313 HCR1 CYLTODRAFT_381731 CYLTODRAFT_432544 CYLTODRAFT_432386 CYLTODRAFT_401315 TIF32 CYLTODRAFT_379714 CYLTODRAFT_424444 CYLTODRAFT_445296 CYLTODRAFT_357360 CYLTODRAFT_400821 CYLTODRAFT_492352 CYLTODRAFT_437313 CYLTODRAFT_398934 CYLTODRAFT_490588 CYLTODRAFT_422491 CYLTODRAFT_490653 CYLTODRAFT_422025 CYLTODRAFT_421559 PSD2 CYLTODRAFT_421360 CYLTODRAFT_436222 CYLTODRAFT_420788 CYLTODRAFT_421031 CYLTODRAFT_372018 CYLTODRAFT_420435 CYLTODRAFT_419810 CYLTODRAFT_420103 CYLTODRAFT_488242 CYLTODRAFT_419339 CYLTODRAFT_487560 CYLTODRAFT_345810 CYLTODRAFT_98287 CYLTODRAFT_440678 CYLTODRAFT_434746 CYLTODRAFT_486880 CYLTODRAFT_449491 CYLTODRAFT_417814 CYLTODRAFT_343194 CYLTODRAFT_434369 CYLTODRAFT_342383 CYLTODRAFT_417069

1.35 1.21 1.21 1.20 1.32 1.21 1.78 1.28 1.20 1.36 1.20 1.26 1.24 1.32 1.20 1.25 1.26 1.40 1.47 1.32 1.34 1.22 1.26 1.29 1.21 1.33 1.24 1.22 1.28 1.40 1.21 1.75 1.22 1.43 1.22 1.20 1.23 1.21 1.27 1.27 1.29 1.24 1.24 1.46 1.27 1.75 1.25 1.35 1.20 1.23 1.26 1.22 1.22 1.34 1.28 1.25 1.50 1.23 1.23 1.29


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Journal of Agricultural and Food Chemistry Table 1. continued no.

Uniprot IDb

Up-Regulated Proteins 61 A0A0D7BU18 62 A0A0D7BU47 63 A0A0D7BUG5 64 A0A0D7BUY1 65 A0A0D7BV79 66 A0A0D7BVK4 67 A0A0D7BVM9 68 A0A0D7BW84 69 A0A0L6W765 70 A0A0L6WF67 71 A0A0L6WG41 72 A0A0L6WN22 73 A0A0L6WNK9 74 A0A0L6WP77 75 A0A0L6WRY1 76 A0A0L6WS22 77 A0A0L6WUV2 78 A0A0L6WX24 79 A0A0L6WX93 80 A0A0L6WYS8 81 A0A0L6X344 82 A0A0W0EUD7 83 A0A0W0EX60 84 A0A0W0F1F6 85 A0A0W0G4C3 86 A0A137Q6R5 87 A0A137QV71 88 A0A137QXR2 89 A0A146H5F4 90 A0A146HQY8 91 A0A146HRF0 92 A0A146I7T7 93 A0A151 V8F3 94 A0A151VAC5 95 A0A151VBD0 96 A0A151VDA9 97 A0A151VDU1 98 A0A151VGT0 99 A0A151VGV4 100 A0A151VTR3 101 A0A151VVB9 102 A0A151VVT7 103 A0A151VZR2 104 A0A151W3U2 105 A0A151W664 106 A0PFJ4 107 A8NGS2 108 A8NLE1 109 A8NQ29 110 A8NY17 111 A8NZV3 112 A8P5F5 113 B0CPH9 114 B0CYU2 115 B0CZU7 116 B0D6H6 117 B0DWN9 118 B5ABT0 119 D2JY93 120 D6RLQ2

protein name

gene name

fold change

Mob1/phocein DIS3-like exonuclease 2 VID27 cytoplasmic protein Tubulin alpha chain Sec1-like protein UV excision repair protein Rad23 1-aminocyclopropane-1-carboxylate deaminase Formate dehydrogenase Aldehyde dehydrogenase Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform Geranylgeranyl pyrophosphate synthase Putative chorismate mutase Eukaryotic translation initiation factor 4E type 2 Charged multivesicular body protein 1 Nascent polypeptide-associated complex subunit beta Putative aryl-alcohol dehydrogenase S-(hydroxymethyl)glutathione dehydrogenase Acetyl-coenzyme A synthetase Elongation factor 3 CAP-Gly domain-containing linker protein 2 Glutathione peroxidase Tryptophan synthase Putative histidine-tRNA ligase Putative UDP-N-acetylglucosaminediphosphorylase Putative Crg1 Calmodulin Vigilin 1 Elongation factor 3 26S proteasome regulatory subunit Protein kinase C Eukaryotic translation initiation factor 5 Autophagy-related protein 3 Nucleolar protein 58 Aldehyde dehydrogenase Heat shock protein sti1 Serine/threonine-protein kinase sid2 RasGEF domain-containing serine/threonine-protein kinase X CCR4-NOT transcription complex subunit 7 Versicolorin reductase Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase Mitochondrial Rho GTPase 1 Elongation factor 3 Eukaryotic translation initiation factor 3 subunit C Protein transport protein sec71 Putative asparagine synthetase [glutamine-hydrolyzing] NADPH–cytochrome P450 reductase CMGC/GSK protein kinase Ubiquitinyl hydrolase 1 Peptidyl-prolyl cis−trans isomerase 3-isopropylmalate dehydratase Superoxide dismutase Glutamate dehydrogenase Predicted protein Predicted protein Predicted protein CipC1 protein, concanamycin induced protein C Synaptobrevin-like protein Putative kinesin-1 6,7-dimethyl-8-ribityllumazine synthase CMGC/DYRK/YAK protein kinase

CYLTODRAFT_416744 CYLTODRAFT_416332 CYLTODRAFT_364376 CYLTODRAFT_468249 CYLTODRAFT_386664 CYLTODRAFT_363929 CYLTODRAFT_385789 CYLTODRAFT_416290 J132_10835 J132_07524 J132_07175 J132_08831 J132_07417 J132_06153 J132_01090 J132_03211 J132_11192 J132_07120 J132_08415 J132_05588 J132_10117 WG66_19726 WG66_18822 WG66_17200 WG66_4028 AN958_02030 AN958_03124 AN958_11047 MCHLO_01663 MCHLO_07849 MCHLO_07952 MCHLO_12712 NOP58 aldA_0 sti1 sid2 gefX CNOT7 stcU dhdh GEM1 tef3 NIP1 sec71 asn1 cyp450 CC1G_03802 CC1G_05800 CC1G_05429 CC1G_00469 CC1G_06963 CC1G_05483 LACBIDRAFT_301830 LACBIDRAFT_292975 LACBIDRAFT_292707 LbCipC1 LACBIDRAFT_255034 N/A N/A CC1G_14245

1.23 1.26 1.41 1.33 1.22 1.26 1.29 1.24 1.53 1.62 1.27 1.40 1.29 1.25 1.28 1.23 1.25 1.29 1.41 1.53 1.24 1.21 1.22 1.34 1.51 1.44 1.21 1.32 1.22 1.27 1.43 1.35 1.39 1.34 1.21 1.23 1.28 1.25 1.34 1.21 1.21 1.27 1.42 1.20 1.21 1.25 1.28 1.21 1.34 1.21 1.33 1.22 1.29 1.44 1.67 1.55 1.40 1.32 1.24 1.23

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Journal of Agricultural and Food Chemistry Table 1. continued no.

a

Uniprot IDb

protein name

gene name

fold change

Up-Regulated Proteins 121 G8A540 122 Q33DK7 123 Q5EGJ1 124 Q7Z9L9 125 R4HKU2 126 U3LL74 127 V2WPV3 128 V2X493 129 V2X759 130 V2XBH5 131 V2XCQ8 132 V2XCT1 133 V2XH17

Putative flavonol reductase/cinnamoyl-CoA reductase Putative serine/threonine protein kinase Translation elongation factor EF1-alpha Tubulin alpha chain Myosin regulatory light chain Translation elongation factor 1-alpha Heat shock protein hsp98 Bar adaptor protein hob3 Protein phosphatase 2c ptc3 Formate dehydrogenase C-1-tetrahydrofolate synthase Flavonol synthase DNA helicase

N/A stk-4 tef1 N/A cdc4 tef1 Moror_13235 Moror_3105 Moror_14013 Moror_6768 Moror_6556 Moror_13784 Moror_258

1.24 1.31 1.23 1.40 1.70 1.26 1.64 1.27 1.26 1.25 1.22 1.26 1.28

Down-Regulated Proteins 1 A0A067PDE9 2 A0A067P039 3 A0A0C9T9Z0 4 A0A0C9XYD0 5 A0A0D0BYL2 6 A0A0D7ALN1 7 A0A0D7AN89 8 A0A0D7AVE1 9 A0A0D7AWT8 10 A0A0D7B205 11 A0A0D7B7K2 12 A0A0D7B9N8 13 A0A0D7BDY3 14 A0A0D7BFN5 15 A0A0D7BG48 16 A0A0D7BHH5 17 A0A0D7BKM4 18 A0A0D7BL46 19 A0A0D7BN64 20 A0A0D7BVN2 21 A0A0D7BW47 22 A0A0G2SY46 23 A0A0L6WDP8 24 A0A0L6WDQ8 25 A0A137Q717 26 A0A137QKG3 27 A0A137QKY8 28 A0A137QSD1 29 A0A146HG47 30 A0A151VVF5 31 A0A151W487 32 A8NHB2 33 A8NHX8 34 A8NXG9 35 B0CR39 36 B0CUW3 37 B0D316 38 B0D620 39 D2JY92 40 G8A543 41 G8A549 42 V2WTU3

Succinyl-CoA:3-ketoacid-coenzyme A transferase Beta-glucosidase Trehalase Protein disulfide-isomerase Histone H4 Alpha-mannosidase Arginine biosynthesis bifunctional protein ArgJ, mitochondrial V-type ATPase DnaJ-domain-containing protein Succinyl-CoA:3-ketoacid-coenzyme A transferase Methylmalonate-semialdehyde dehydrogenase Mitochondrial half-size ABC transporter Thiolase Beta-hexosaminidase Bifunctionalacetylglutamate kinase/N-acetyl-gamma-glutamyl-phosphate reductase Citrate synthase Flavocytochrome c Glutathione S-transferase Isovaleryl-CoA dehydrogenase Eukaryotic translation initiation factor 3 subunit C Sulfate adenylyltransferase Trehalase Putative enoyl-CoA hydratase 2 Actin-1 Extracellular metalloproteinase Beta-glucosidase Acetylornithine aminotransferase, mitochondrial Putative fumarate reductase Aldo-keto reductase Isocitratelyase Putative ATPase YjoB Glucuronyl hydrolase Alpha-mannosidase Alcohol dehydrogenase Methylmalonate-semialdehyde dehydrogenase Acetylornithine aminotransferase Arginine biosynthesis bifunctional protein ArgJ, mitochondrial Predicted protein FDS protein Putative exo-1,3-beta-glucanase Putative exobeta-1,3-glucanase Plasma membrane ATPase

PLEOSDRAFT_1052683 PLEOSDRAFT_21598 PLICRDRAFT_45215 K443DRAFT_673954 GYMLUDRAFT_176462 FISHEDRAFT_35735 FISHEDRAFT_63484 CYLTODRAFT_495350 CYLTODRAFT_426899 CYLTODRAFT_457115 CYLTODRAFT_423662 CYLTODRAFT_355389 CYLTODRAFT_421627 CYLTODRAFT_488998 CYLTODRAFT_489283 CYLTODRAFT_420207 CYLTODRAFT_419120 CYLTODRAFT_451353 CYLTODRAFT_429664 NIP1 MET3 NTH J132_10447 J132_10451 AN958_01924 AN958_08211 AN958_07918 AN958_04613 MCHLO_04945 ACU-7 yjoB CC1G_11511 CC1G_01530 CC1G_00301 LACBIDRAFT_301007 LACBIDRAFT_305721 LACBIDRAFT_324786 LACBIDRAFT_233707 N/A N/A N/A Moror_7712

0.81 0.82 0.78 0.82 0.81 0.74 0.60 0.61 0.81 0.81 0.76 0.79 0.81 0.49 0.72 0.76 0.79 0.81 0.78 0.64 0.77 0.82 0.80 0.15 0.68 0.81 0.51 0.80 0.78 0.62 0.81 0.58 0.77 0.79 0.71 0.70 0.61 0.45 0.65 0.80 0.42 0.76

N/F: F. velutipespacked with Nano-PM vs F. velutipespacked with Normal-PM. bUniprot: http://www.uniprot.org/.

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Figure 3. Bioinformatics analysis of up-regulated and characterized proteins in N/P through Gene Ontology (GO) in three domains: cellular component, biological process, and molecular function.

participated in arginine and proline metabolism (path, cci00330). Other DEPs had relationship with valine leucine

and isoleucine biosynthesis (path, cci00290), peroxisome biogenesis (path, cci04146), alanine aspartate and glutamate 10375


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Figure 4. Bioinformatics analysis of down-regulated and characterized proteins in N/P through Gene Ontology (GO) in three domains: cellular component, biological process, and molecular function.

leucine and isoleucine degradation (path, lbc00280), inositol phosphate metabolism (path, lbc00562), and propanoate metabolism (path, lbc00640). On the basis of the cellular

metabolism (path, cci00250), nitrogen metabolism (path, cci00910), snare interactions in vesicular transport (path, lbc04130), other glycan degradation (path, cci00511), valine 10376


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organism), qRT-PCR was performed to confirm the TMT data and investigate the dynamic transcriptional expression patterns of five representative DEPs. It was demonstrated that the validated genes were generally consistent with the TMT data (Figure 5), indicating the results of TMT labeling combined with 2D LC-MS/MS technique were reliable. The Nano-PM groups had significantly lower mRNA expression levels on two genes encoding β-glucosidase and trehalase; whereas, the genes involved in redox homeostasis responded to stress exhibited higher patterns (P < 0.05).

Table 2. KEGG Pathway Analysis of Differentially Expressed Proteins in N/P Groupa Uniprot IDb

protein name

Up-Regulated Proteins A8NY17 3-isopropylmalate dehydratase

KEGG pathway ID

metabolism in KEGG

cci00290 cci01100 cci01110

A8NZV3 A8P5F5

Superoxide dismutase Glutamate dehydrogenase

cci04146 cci00250 cci00330

B0DWN9

Synaptobrevin-like protein

Down-Regulated Proteins B0D316 Arginine biosynthesis bifunctional protein ArgJ

cci00910 cci01100 lbc04130

lbc00330 lbc01100 lbc01110

A8NHX8 B0CR39

B0CUW3

Alpha-mannosidase Methylmalonatesemialdehyde dehydrogenase

Acetylornithine aminotransferase

cci00511 lbc00280 lbc00562 lbc00640 cci01100 lbc00330 lbc01100 lbc01110

a KEGG: http://www.omicsbean.com:88/. uniprot.org/.

b

valine leucine and isoleucine biosynthesis energy metabolism carbohydrate and lipidmetabolism peroxisome biogenesis alanine, aspartate and glutamate metabolism arginine and proline metabolism nitrogen metabolism energy metabolism snare interactions in vesicular transport

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DISCUSSION F. velutipes is a typical low-temperature commercial mushroom in the world due to its high nutritional value, unique flavor, and taste.12 However, harvested fresh mushrooms deteriorate quickly because of rapid respiration, metabolism and physical damage, microbial attack, and water loss.13 In our previous studies, we have proved that Nano-PM combined with cold storage was effective on F. velutipes preservation.4 Nevertheless, more studies were required to understand the different mechanisms of molecular and physiological alterations between conventional and nanopackaging under cold storage. This study was to investigate how different packaging material could alter protein expression in F. velutipes during cold storage using a TMT-coupled 2D LC-MS/MS technique. In total, 2283 proteins were identified and quantified from Nano-PM, NormalPM and fresh F. velutipes groups, demonstrating that TMT was applicable for multiplexed proteomic profiling beyond the conventional two-way comparative proteomic studies. It was also revealed that some proteins corresponding to some central metabolic pathways in N/P group were up-regulated or downregulated and they might play an important role in F. velutipes response to different packaging. The following four functional categories including carbohydrate and energy metabolism, amino acids biosynthesis and metabolism, signaling pathways, stress response and defense were selected to evaluate the data sets. The biological relevance of these DEPs in N/P group was discussed below. Proteins Associated with Carbohydrate and Energy Metabolism. From GO analysis of N/P group (Figure 4), we found that 35% of down-regulated DEPs with annotation were distributed in mitochondria and 32% was involved in carbohydrate metabolism process, indicating relatively lower energy metabolism of Nano-PM treatment under cold storage compared to that of Normal-PM. It showed that some DEPs participated in energy metabolism pathways including tricarboxylic acid cycle (TCA), glyoxylate cycle, starch and sucrose metabolism, and glycolysis. Citrate synthase (CS) is a key enzyme of multiple important metabolic pathways in cells. CS catalyzes the product from the condensation of oxaloacetate and acetyl-coenzyme A, forming citrate through the TCA process and glyoxylate cycle.14 Isocitratelyase (ICL), as a unique enzyme from the glyoxylate cycle, provided an anaplerotic pathway of TCA cycle, allowing the growth of C2 compounds by bypassing the CO2-generating steps of the TCA cycle.15 Both CS and ICL had lower expression level in F. velutipes from Nano-PM treatment compared to that of Normal-PM, which demonstrated that Nano-PM could inhibit cellular energy metabolism of mushroom fruiting body during postharvest cold storage. As a result, developed Nano-PM slowed down the loss of nutrients including carbohydrate, amino acids, and organic acid in F. velutipes. The results were consistent with our previous study that Nano-PM was able to absorb and oxidize

arginine and proline metabolism energy metabolism carbohydrate and lipidmetabolism other glycan degradation valine leucine and isoleucine degradation inositol phosphate metabolism propanoate metabolism energy metabolism arginine and proline metabolism energy metabolism carbohydrate and lipidmetabolism

Uniprot: http://www.

localization of central DEPs and their biological function, a putative model was applied to illustrate the metabolic pathways and possible roles in up- or down-regulated identified proteins in F. velutipes packed with Nano-PM (Figure 6). qPCR Validation. Because of the limitation of available specific antibodies for the edible mushroom species (nonmodel

Figure 5. QPCR validation of five proteins of different abundance at the mRNA level. All measurements were expressed as the mean ± SD (n = 3). *Indicates a significant difference at P < 0.05 for each protein comparison. 10377


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Figure 6. Overview of the main metabolic pathway and possible roles in up- or down-regulated identified differentially expressed proteins in F. velutipes packed with Nano-PM compared to that with Normal-PM (N/P). EMP, Embden−Meyerhof−Parnas; TCA, tricarboxylic acid cycle; ETC, electron transport chain; CS, citrate synthase; ICL, isocitratelyase; FDH, formate dehydrogenase; ALDH, aldehyde dehydrogenases; β-Glu, β-glucosidase; SDH, saccharopine dehydrogenase; TRPS, tryptophan synthase; GDH, glutamate dehydrogenase; CM, chorismate mutase; MAPK, mitogen-activated protein kinase; MIPS, myo-inositol-1-phosphate synthase; RGS, regulator of G protein signaling; PKC, protein kinase C; ROS, reactive oxygen species; GPX, glutathione peroxidase; SOD, superoxide dismutase; MsrB, methionine sulfoxide reductase B; MetO, methionine sulfoxide.

group suggested that Nano-PM could postpone the cell wall degradation during storage to maintain the mushroom cell integrity. Trehalose, as one of the important carbohydrate storages in fungi, could be hydrolyzed by trehalase into glucose as energy supplement.21 Moreover, trehalose is involved in stress response and could prevent biological cells from stress during abiotic stresses.22 Liu et al. found that trehalose hydrolysis by trehalase is an important physiological process for fungal spore germination and the resumption of mycelium growth.7 In our study, the protein trehalase expression in Nano-PM packed F. velutipes was down-regulated, suggesting the inhibited growth of fruiting body and enhanced resistant capability. Proteins Associated with Amino Acids Biosynthesis and Metabolism. Mushroom senescence and response to abiotic stress involve many dynamic changes in proteins. However, little information was known for proteomics change of postharvest F. velutipes.23 In this study, several proteins related with protein synthesis, degradation, and folding were identified and quantified. According to the GO analysis of the N/P group (Figure 3B), 16% up-regulated DEPs with annotation were involved in amino acids biosynthesis and metabolism. Saccharopine dehydrogenase (SDH) is a key enzyme of Llysine biosynthesis via the amino adipic acid (AAA) pathway. SDH could produce L-lysine from L-α-aminoadipate.24 According to Less et al., the ectopic overexpression of the SDH gene would occur when plants were subjected to salt and/or osmotic

ethylene resulting in a relatively lower respiration rate in mushroom.4 Moreover, it was found formate dehydrogenase (FDH) was up-regulated in N/P group. FDH is a set of enzymes that catalyzes the oxidation of formate to carbon dioxide and supplies mushroom cell with additional ATP.16 According to Bykova et al., FDH activity would be increased due to the accumulation of formic acid under low oxygen concentration.17 Meanwhile, low oxygen concentration was able to cause anaerobic damage to the mushroom cell.18 Aldehyde dehydrogenases (ALDH), widely spread among white-rot basidiomycete fungi, was effective on mitigation of anaerobic damage via detoxification of aromatic aldehydes to their corresponding carboxylic acids.19 ALDH was up-regulated in Nano-PM compared to that of the Normal-PM treatment, resulting in less anaerobic damage during storage. Soluble sugars and their metabolites are major contributors to mushroom quality. As a heterogeneous group of exotype glycosyl hydrolases, β-glucosidase catalyzes the hydrolysis of β-glucosidic linkages in β-D-glucosides and oligosaccharides, producing glucose monomers by breaking down the β-1,4 glucosidic bonds of cellobiose.20 β-glucosidase exists in all kingdoms of life and plays important roles in fundamental biological processes. According to the Uniprot database (www.uniprot.org/uniprot/ A0A067P039), β-glucosidase is involved in cellulose degradation pathway as a part of glycan metabolism. Cellulose is major part of cell wall and the down-regulated DEP of β-glucosidase in N/P 10378


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Journal of Agricultural and Food Chemistry stress.25 In our results, SDH was up-regulated in the N/P group, which was a spontaneous strategy of the mushroom fruiting body responding to stress. Tryptophan synthase (TRPS) participates in the tryptophan (Trp) biosynthesis from indole glycerol phosphate and L-serine. The Trp biosynthetic pathway leads to the production of many secondary metabolites with diverse functions.26 Zhao, Williams, and Last found that induction of the synthesis of Trp enzymes and camalexin was triggered by oxidative stress.27 This could explained the up-regulation of TRPS protein expression observed in our study. Similarly, 3isopropylmalate dehydratase involved in L-leucine biosynthesis pathway was also up-regulated in the N/P group. Glutamate dehydrogenase (GDH) is one of the key roles in maintaining the balance of carbon and nitrogen. GDH returns the carbon in amino acids back into reactions of carbon metabolism and the TCA cycle.28 The up-regulation of GDH expression in Nano-PM packed F. velutipes could enhance its resistance to cold stress or low oxygen conditions. In addition, two elongation factors (gene name of tef1 and tef3) showed significantly higher expression levels in the N/P group. Elongation factors are an essential component of eukaryotic protein synthesis, catalyzing the translocation of peptidyl t-RNA from the aminoacyl to the peptidyl site on the ribosome in a GTP-dependent reaction.29 The family of protein disulfide isomerases (PDI) was proven to play a vital role in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum.30 The differential expression profiles of PDI in F. velutipes with different packaging hints at abnormalities of growth due to misfolded proteins during storage. These findings could give some important implications altered response of different mushroom packaging formulations at the molecular level under cold storage. Proteins Associated with Signaling Pathways. Cell signaling, crucial to achieving both a hypometabolic state and reorganizing multiple metabolic pathways, could optimize longterm viability during cold storage. The expression of mitogenactivated protein kinase (MAPK), myo-inositol-1-phosphate synthase (MIPS), regulator of G protein signaling (RGS), protein kinase C (PKC), and bar adaptor protein hob3 (hob3) increased in the N/P group. The cellular control switches are regulated via an extensive network of interactive and intracellular signal transduction pathways, such as the mitogen-activated protein kinase family (MAPKs). They participate and regulate cell functions including gene expression, proliferation, differentiation, mitosis, apoptosis, and cell survival.31 Good et al. found that MAPK pathways were in charge of cell cycle arrest and mating in response to pheromone stimulation in yeast.32 MIPS is the key rate-limiting enzymein phospholipid biosynthetic process, catalyzing the conversion from D-glucose 6-phosphate to 1L-myo-inositol 1phosphate. The phospholipid precursor (myo-inositol) was then derived from 1L-myoinositol 1-phosphateby dephosphorylation and participated in the synthesis of phosphatidyl inositol (PtdIns), which involved in signal transduction pathways from eukaryotes cell.33 RGS proteins is associated with intracellular signal transduction and responsible for the rapid turnoff of G protein-coupled receptor signaling pathways.34 The overexpression of RGS might be a stress response to extracellular stimuli, but its exact function and regulating mechanism remain unclear. PKC is a family of protein kinase enzymes and activated by signals such as increased concentration of diacyl glycerol or calcium ions.35 Hence, PKC enzymes play important roles in

several signal transduction, regulating cell activation, differentiation, proliferation, death, and effector functions.36 Hob3 is one of the two BAR (BIN/Amphiphysin/Rvs) adapter family proteins from the fission yeast amphiphysin family proteins Rvs161p and Rvs167p.37 Hob3 protein could locate to the division area forming a ring or some patches at the growing poles and integrate cell polarity signals generated by actin and vesicle dynamics with central regulators of cell cycle. Cell division control protein 42 (cdc42) was recruited by hob3 to the membrane area contacting the contractile ring, where cdc42 activated by gef1 gene would in turn regulate and activate other target molecules required for cytokinesis.38 According to Zhou et al., the deletion of hob3 would cause cell elongation and multiseptation in Saccharomyces cerevisiae.39 In the study, higher activity of the DEPs suggests that these enzymes including MAPK, MIPS, RGS, PKC, and hob3 might play an important role in the process of F. velutipes preservation using Nano-PM. Proteins Associated with Stress Response and Defense. The oxidative stress hypothesis is considered to be related with fruit ripening and senescence because the free radicals can damage all cell components including lipids, proteins, and DNA.40 Redox homeostasis in cell is important for organism and regulated by the interaction between reactive oxygen species (ROS) and the cellular antioxidant system.41 In this study, higher level of glutathione peroxidase (GPX), superoxide dismutase (SOD), methionine sulfoxide reductase B (MsrB) and heat shock proteins (HSPs) of Nano-PM packed F. velutipes were accumulated during cold storage compared to that of Normal-PM treatment. GPX can reduce lipid hydroperoxides to their corresponding alcohols and scavenge free radicals of hydrogen peroxide. Furthermore, glutathione (GSH) can be catalyzed by GPX into oxidized glutathione (GSSH), which then participates in the glutathione-ascorbate cycle pathway.42 SOD is considered as primary defenses against the potent toxicity of ROS, alternately catalyzing the dismutation of the superoxide (O2−) radical into either ordinary molecular oxygen (O2) or hydrogen peroxide (H2O2).43 The expression of SOD protein was up-regulated by 1.33-fold in the N/P group. This result was consistent with our previous study, indicating that Nano-PM treatment could significantly inhibit the accumulation of ROS in F. velutipes.44 MsrB is one of antioxidant repair enzymes performing the enzymatic reduction of methionine sulfoxide (MetO), the oxidized form of the amino acid methionine (Met), back to methionine. Oxidation of methionine residues in tissue proteins can cause them misfolded or otherwise render them dysfunctional.45 Therefore, the up-regulation of MsrB protein expression in N/P group in turn reduced the damage of proteins in F. velutipes caused by ROS. HSPs at different molecular weights are widely known to be involved in cell response to oxidative stress. Without the presence of stress, HSPs are associated with the stabilization of ancient and newly synthesized proteins in cell.46 By reestablishing protein conformation and maintaining cellular homeostasis, HSPs are able to protect cells from external stimulus, such as temperature and metal stress.47 In our study, HSP 90 and HSP 98 were identified and up-regulated in F. velutipes packed with Nano-PM, compared to that of Normal-PM treatment. Vassilev, Plesofsky-Vig and Brambl suggested that HSP 98 played a role in the functioning of ribosomes in translation during the stress response, stabilizing the translational complexes in cells.48 HSP 90 is a chaperone protein contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The fraction of heat 10379


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ase chain reaction; DEPs, differentially expressed proteins; TCA, tricarboxylic acid cycle; CS, citrate synthase; ICL, isocitratelyase; FDH, formate dehydrogenase; ATP, adenosine triphosphate; GTP, guanosine triphosphate; ALDH, aldehyde dehydrogenases; SDH, saccharopine dehydrogenase; AAA, aminoadipic acid; TRPS, tryptophan synthase; Trp, tryptophan; GDH, glutamate dehydrogenase; PDI, protein disulfide isomerases; MAPK, mitogen-activated protein kinase; MIPS, myo-inositol-1phosphate synthase; RGS, regulator of G protein signaling; PKC, protein kinase C; hob3, bar adaptor protein hob3; cdc42, cell division control protein 42; ROS, reactive oxygen species; GPX, glutathione peroxidase; GSH, glutathione; GSSH, oxidized glutathione; SOD, superoxide dismutase; MsrB, methionine sulfoxide reductase B; MetO, methionine sulfoxide; HSPs, heat shock proteins

shock proteins will increase in cell from 2% to 6% of cellular proteins when against the stress.49 HSP 90 is essential for viability in eukaryotes and involved in cell cycle control and signal transduction.50 In summary, TMT-coupled 2D LC-MS/MS proteomic profiling technique was applied to investigate differences in protein expression patterns of fresh, Nano-PM, and Normal-PM packed F. velutipes after 21-day cold storage. A total of 2283 proteins were identified from three treatments, in which 429 differentially expressed proteins (DEPs) were selected, demonstrating the feasibility of the TMT approach to multiplexed proteomic profiling. Compared to Normal-PM packed F. velutipes, the up-regulated DEPs in Nano-PM group were mainly distributed in cytoplasm and involved in amino acid synthesis and metabolism, signal transduction, and response to stress, while the down-regulated DEPs were largely located in mitochondrion and participated in carbohydrate metabolic, amino acid synthesis and metabolism, and organic acid metabolic. Generally, this study offers valuable evidence at the molecular level that Nano-PM applied on fresh F. velutipes could preserve the mushroom by reduction of energy metabolism and enhancement of stress resistance.

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(1) Lagnika, C.; Zhang, M.; Nsor-Atindana, J.; Tounkara, F. Extension of mushroom shelf-life by ultrasound treatment combined with high pressure argon. Int. Agrophys. 2014, 28, 39−47. (2) Xue, Z.; Hao, J.; Yu, W.; Kou, X. Effects of processing and storage preservation technologies on nutritional quality and biological activities of edible fungi: a review. J. Food Process Eng. 2017, 40, e12437. (3) De Azeredo, H. M. C. Nanocomposites for food packaging applications. Food Res. Int. 2009, 42, 1240−1253. (4) Fang, D.; Yang, W.; Kimatu, B. M.; Mariga, A. M.; Zhao, L.; An, X.; Hu, Q. Effect of nanocomposite-based packaging on storage stability of mushrooms (Flammulina velutipes). Innovative Food Sci. Emerging Technol. 2016, 33, 489−497. (5) Fang, D.; Yang, W.; Kimatu, B. M.; Zhao, L.; An, X.; Hu, Q. Comparison of flavour qualities of mushrooms (Flammulina velutipes) packed with different packaging materials. Food Chem. 2017, 232, 1−9. (6) Zargar, S. M.; Gupta, N.; Mir, R. A.; Rai, V. Shift from gel based to gel free proteomics to unlock unknown regulatory network in plants: a comprehensive review. J. Adv. Res. Biotech 2016, 1, 19. (7) Chen, L.; Zhang, B.; Cheung; Peter, C. K. Comparative proteomic analysis of mushroom cell wall proteins among the different developmental stages of Pleurotus tuberregium. J. Agric. Food Chem. 2012, 60, 6173−6182. (8) Liu, J.; Chang, M.; Meng, J.; Feng, C.; Zhao, H.; Zhang, M. Comparative proteome reveals metabolic changes during the fruiting process in Flammulina velutipes. J. Agric. Food Chem. 2017, 65, 5091− 5100. (9) Chen, M.; Liao, J.; Li, H.; Cai, Z.; Guo, Z.; Wach, M. P.; Wang, Z. iTRAQ-MS/MS proteomic analysis reveals differentially expressed proteins during post-harvest maturation of the white button mushroom Agaricusbisporus. Curr. Microbiol. 2017, 74, 641−649. (10) Isaacson, T.; Damasceno, C. M. B.; Saravanan, R. S.; He, Y.; Catalá, C.; Saladiá, M.; Rose, J. KC. Sample extraction techniques for enhanced proteomic analysis of plant tissues. Nat. Protoc. 2006, 1, 769− 774. (11) Chai, Y.; Wang, G.; Fan, L.; Zhao, M. A proteomic analysis of mushroom polysaccharide-treated HepG2 cells. Sci. Rep. 2016, 6, 23565. (12) Kalač, P. A review of chemical composition and nutritional value of wild-growing and cultivated mushrooms. J. Sci. Food Agric. 2013, 93, 209−218. (13) Aguirre, L.; Frias, J. M.; Barry-Ryan, C.; Grogan, H. Assessing the effect of product variability on the management of the quality of mushrooms (Agaricus bisporus). Postharvest Biol. Technol. 2008, 49, 247−254. (14) Wienkoop, S.; Saalbach, G. Proteome analysis. Novel proteins identified at the peribacteroid membrane from Lotus japonicus root nodules. Plant Physiol. 2003, 131, 1080−1090. (15) Dunn, M. F.; Ramirez-Trujillo, J. A.; Hernández-Lucas, I. Major roles of isocitratelyase and malate synthase in bacterial and fungal pathogenesis. Microbiology 2009, 155, 3166−3175.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04393. Figure S1, hierarchical clustering of changes in abundance of the differentially expressed proteins of three groups (N/ F, P/F, and N/P), with the green color representing lower than average standardized volume, whereas the red color represents greater than average standardized volume; Tables S1 and S2, lists of differentially expressed proteins in the N/F or P/F groups; and Table S3, summarized primer data of five representative differentially expressed proteins in the N/P group (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-25-85870269. *Phone: +86-25-84399086. ORCID

Wenjian Yang: 0000-0002-7527-8491 Funding

The authors acknowledge financial support from the Natural Science Foundation of Jiangsu Province (Grant No. BK20141009) and the National Natural Science Foundation of China (Grant No. 31401552). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED TMT, tandem mass tags; 2D LC-MS/MS, two-dimensional liquid chromatography-tandem mass spectrometry; PE, polyethylene; Nano-PM, nanocomposite packaging materials; Normal-PM, normal packaging materials; N, F. velutipes packed with nanocomposite packaging materials; P, F. velutipes packed with normal packaging materials; F, fresh F. velutipes; FDR, false discovery rate; FC, fold change; HCL, hierarchical cluster analysis; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes; qRT-PCR, quantitative real-time polymer10380


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Flammulina velutipes

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