Comparative phosphoproteome analysis to identify candidate phosphoproteins involved in blue light-induced brown film formation in Lentinula edodes

Materials treatment and protein extraction

The L. edodes strain L901 which is a new hybrid strain was obtained from the Zhejiang Academy of Agricultural Sciences. Fungal mycelia were grown at 22 °C under red- and blue-light conditions (LED light sources) for 22 d. The light intensity approximately 100 lux and the incubator illuminated all day. Fungal mycelia were grown were grown in the potato dextrose agar media. Samplings were taken after mycelial changed colour under blue light conditions. The determination of total polysaccharides was performed according to Zhang’s description (Zhang et al., 2018). For protein extraction, a proper amount of sample was ground in liquid nitrogen into a cellular powder and then transferred to a 5-mL centrifuge tube. The samples were treated with four volumes of lysis buffer (10 mM dithiothreotol, 1% protease inhibitor, and 1% phosphatase inhibitor) and then sonicated three times. The supernatant was centrifuged for 10 min at 4 °C and 5,500 g with an equal volume of Tris equilibrium phenol. The supernatant was taken and precipitated overnight with a fivefold volume of 0.1 M ammonium acetate/methanol. The protein precipitation was washed sequentially with methanol and acetone. The protein was redissolved in 8 M urea, and the protein concentration was determined using a bicinchoninic acid assay kit (P0012, Beyotime, Shanghai, China) according to the manufacturer’s instructions.

Trypsin digestion, TMT labeling, and HPLC fractionation

For digestion, the final concentration of dithiothreotol in the protein solution was 5 mM and was reduced at 56 °C for 30 min. The 11-m final concentration of iodoacetamide was incubated at room temperature for 15 min. Finally, the urea concentration of the sample was diluted to less than 2 M. Trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion.The trypsinase-hydrolyzed peptide segments were desalted using a Strata X C18 (Phenomenex) and then freeze-dried in a vacuum. The peptide segment was dissolved in 0.5 M Triethylammonium bicarbonate and labeled according to the instructions of the TMT kit (90066,Thermo-Scientific, Rockford, IL, USA). The simple operation was as follows: the labeled reagent was dissolved in acetonitrile after thawing, incubated at room temperature for 2 h after mixing with the peptide segment, desalinated after mixing with the labeled peptide segment, and freeze-dried in a vacuum.

The tryptic peptides were fractionated using high pH reverse-phase HPLC on an Agilent 300Extend C18 column (5-µm particles, 4.6-mm ID, 250-mm length). Briefly, peptides were first separated using a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into six fractions and dried by vacuum centrifugation.

Affinity enrichment

Peptide mixtures were first incubated with an immobilized metal ion affinity chromatography (IMAC) microsphere suspension and vibrated in loading buffer (50% acetonitrile and 6% trifluoroacetic acid). IMAC microspheres were used Ti. The IMAC microspheres enriched with phosphopeptides were collected by centrifugation, and the supernatant was removed. To remove nonspecifically adsorbed peptides, the IMAC microspheres were washed with loading buffer and 30% acetonitrile plus 0.1% trifluoroacetic acid, sequentially. To elute the enriched phosphopeptides from the IMAC microspheres, elution buffer containing 10% NH₄OH was added, and the enriched phosphopeptides were eluted with vibration. The resulting peptides were desalted with C18 ZipTips (Millipore) and lyophilized for the LC-MS/MS analysis.

LC-MS/MS analysis

The tryptic peptides were dissolved in 0.1% formic acid and directly loaded onto a home-made reversed-phase analytical column (15-cm length and 75-µm i.d.). The gradient increased from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min and to 80% in 3 min. It was then held at 80% for the last 3 min, at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system.

The peptides were subjected to an NSI source followed by MS/MS in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1,800 for a full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using normalized collision energy (NCE) set as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. The data-dependent procedure alternated between one MS scan and 20 MS/MS scans with a 15.0-s dynamic exclusion. The automatic gain control was set at 5E4. The fixed first mass was set as 100 m/z.

Database search

The MS data were retrieved using Maxquant (v1.5.2.8) using the following search parameter settings: the database was Lentinula_edodes_uniprot (https://www.uniprot.org/proteomes/?query=organism:5353&sort=score); an anti-database was added to calculate the false positive rate (FDR) caused by random matching and a common contamination library was added to eliminate the contamination proteins from the results. Trypsin/P was specified as the cleavage enzyme, allowing up to four missing cleavage events. The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification, and acetylation and oxidation of Met were specified as variable modifications. The FDR was adjusted to <1%, and the minimum score for modified peptides was set >40.

Annotation methods and functional enrichment

The GO annotation on the proteomics level was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). First, the system converted the protein ID to UniProt ID, matched the GO ID with the UniProt ID, and then extracted the corresponding information from the UniProt-GOA database based on the GO ID. If there was no protein information queried in the UniProt-GOA database, then algorithm software based on the protein sequence, InterProScan, was used to predict the GO function of the protein.

The KEGG database was used to annotate protein pathways. First, the KEGG online service tool KAAS was used to annotate the submitted proteins, and then KEGG mapper was used to place the annotated proteins into the corresponding pathways in the database. WoLF PSORT, a software for predicting subcellular localization, was used to annotate the submitted proteins for subcellular localization. Fisher’s exact test was used to detect differentially modified proteins against the background of identified proteins. A P-value of less than 0.05 was considered significant. The softwares motif-x and MoMo were used to analyze the models of sequences that contained the amino acids in specific positions of modified 13-mers (six amino acids upstream and downstream of the site) in all the protein sequences.

Characteristics of quantitative phosphoproteomic data in L. edodes mycelia

Using affinity enrichment followed by LC-MS/MS, the phosphoproteomic changes in L. edodes mycelia grown in red or blue light were investigated. A flow chart of our experiment is exhibited in Fig. 1A. Pearson’s correlation coefficient between the two groups showed sufficient reproducibility (Fig. 1B). In this study, 160,949 secondary spectra were obtained by MS analyses. After searching the theoretical protein data, the effective number of spectra was 22,857 and the utilization rate of the spectra was 14.2%. In total, 8,830 peptides and 7,777 phosphorylated peptides were identified. There were 11,224 phosphorylation modification sites on 2,786 proteins, of which 9,243 sites on 2,579 proteins provided quantitative information (Fig. 1C). The first-order mass errors of most spectra are less than 10 ppm, which is in accordance with the high accuracy of the MS (Fig. 1D). Most of the peptides were distributed in 7–20 amino acids, which was in accordance with the general rules of trypsin-based enzymatic hydrolysis and high energy collision dissociation (HCD) fragmentation, indicating that the sample preparation and the quality accuracy of the mass spectrometer reached the standard required (Fig. 1E). The detailed information regarding the identified peptides are listed in Table S1.

Analysis of phosphorylation sites

In L. edodes mycelia, 977 (35.07%) phosphoproteins were modified at a single site, 519 (18.63%) at two sites, and 1,290 (46.3%) at three or more phosphosites (Fig. 2A). Interestingly, some proteins contained a large number of phosphosites. For example, there are 34 phosphosites in a non-specific serine/threonine protein kinase (A0A1Q3E061), 45 phosphosites in a regulatory transcript from a polymerase II promoter-related protein (A0A1Q3ERS8) and 53 phosphosites in a SRC Homology 3 (Sh3) domain-containing protein (A0A1Q3ENM7) (Table S1).

To analyze the density levels of the phosphorylation sites in each protein, the phosphorylated proteome of L. edodes was compared with those of other species. The average number of phosphorylation sites per protein in L. edodes is 3.22, which is similar to the numbers in Bombyx mori (3.07), Nicotiana tabacum (3.05), and Physcomitrella patens (3.44) (Fig. 2B) (Fang et al., 2016; Lu et al., 2019; Shobahah et al., 2017).

Characteristics of the identified phosphoproteins in L. edodes

To predict the possible functions of the identified phosphoproteins, a GO classification analysis was performed. Most of the proteins were classified into three GO categories (Fig. 3A). Specifically, 594 proteins were annotated as ‘metabolic process’, 519 proteins were annotated as ‘cellular process’, and 361 proteins were annotated as ‘single-organism process’. In the cellular component category, the largest terms were ‘cell’ (289 proteins), ‘organelle’ (186 proteins), and ‘macromolecular complex’ (154 proteins). In the molecular function category, ‘binding’ (846 proteins), ‘catalytic activity’ (627 proteins), and ‘transporter activity’ (51 proteins) were the three top dominant terms. The euKaryotic Ortholog Groups annotation clustered all the phosphoproteins into four major categories. The ‘cellular processes and signaling’ category contained the largest number of proteins (Fig. 3B). Most identified phosphoproteins were grouped into 13 subcellular component categories predicted by WoLF PSORT software, including 783 nuclear, 380 cytoplasmic, and 275 mitochondrial proteins (Fig. 3C). The detailed annotation information for all the identified phosphoproteins are listed in Table S2.

Protein motifs associated with phosphorylation

Among the identified phosphosites in L. edodes, 8,645 sites occurred at serine residues, 2239 sites at threonine residues, and 340 sites at tyrosine residues (Fig. 4A). To understand the upstream pathway of the identified phosphorylated proteins, a motif analysis was carried out using MOMO and Motif-X software. A number of conserved phosphorylation motifs were enriched in the phosphorylated proteins of L. edodes (Table S3). A total of 7,741 distinct sequences containing 13 residues were obtained, with 6 upstream and 6 downstream residues around each phosphosite (Table S4). The five S-based motifs containing the largest numbers of sequences were ‘sP’, ‘RxxsP’,‘PxsP’‘Gs’, and ‘RRxS’, and the five top T-based motifs were ‘tP’,‘tPP’,‘RxxtP’, RxtP’, and ‘Rxxt’. A Y-based motif, ‘Rxxxxxy’, was identified. Two position-specific heat maps of upstream and downstream amino acids at all the identified phosphorylated serine or threonine sites. For the S-based motifs, strong preferences for glutamic acid, lysine, and arginine upstream, and aspartic acid, glutamic acid, and proline downstream, of the phosphorylation sites were observed. For the T-based motifs, preferences for lysine, proline, and arginine upstream, and aspartic acid and proline downstream, of the phosphorylation sites were observed (Fig. 4C).

Differentially phosphorylated proteins (DPPs) in response to a blue-light treatment

To compare the DPPs between red- and blue-light treated samples, expression profiles of the proteins generated by MeV software are shown in a heatmap (Fig. 5A). The screening of DPPs followed the following criteria: change threshold ≥1.5 times and t-test p-value <0.05. Among these DPPs, 475 sites in 317 phosphorylated proteins were up-regulated and 349 sites in 243 phosphorylated proteins were down-regulated (Fig. 5B and Table S5). Based on the subcellular localizations predicted by WoLF PSORT software, all the DPPs were classified into 10 subcellular components. There were 204 nuclear localized DPPs, 82 cytoplasmic localized DPPs, and 51 plasma membrane localized DPPs (Fig. 5C).

Functional enrichment analysis of the DPPs

To understand the biological functions of these phosphorylated proteins, GO, KEGG and protein domain enrichment analyses of DPPs were carried out. For biological process, cellular component, and molecular function categories, the DPPs were mostly enriched in ‘DNA conformation change’ (Fig. 6A); ‘nucleosome’ (Fig. 6B), and ‘transporter activity’ (Fig. 6C), respectively.

To reveal the metabolic pathways involved in the formation of brown films induced by blue light, the DPPs were further analyzed using the KEGG database. For the up-regulated DPPs, two KEGG pathways, ‘Ribosome biogenesis in eukaryotes’, and ‘ABC transporters’, were significantly enriched’ (Fig. 7A). For the down-regulated DPPs, four enriched KEGG pathways were identified, ‘Valine, leucine and isoleucine degradation’, ‘Phenylalanine metabolism’, ‘Galactose metabolism’, and ‘Fructose and mannose metabolism’ (Fig. 7B). We also found that the total polysaccharides of blue light treatment was significantly lower than that of red light treatment (Fig. S1). A protein domain enrichment analysis revealed that the up-regulated DPPs were enriched in 19 protein domains, with ‘ABC transporter-like’, ‘P-type ATPase’, and ‘HAD-like domain’ being the most highly enriched(Fig. 7C). The down-regulated DPPs were most strongly associated with ‘Glutathione S-transferase, C-terminal-like’, ‘YTH domain’ ‘VPS9 domain’, ‘Domain of unknown function DUF1708’, and ‘High mobility group box domain’(Fig. 7D).

Identification of DPPs related to signal transduction mechanisms and carbohydrate-active enzymes (CAZymes)

To better understand the DPPs related to blue light-induced mycelial brown film formation, a functional classification of DPPs was conducted using euKaryotic Ortholog Groups. A total of 319 DDPs were grouped into 23 subcategories (Fig. S2). For the ‘signal transduction mechanisms’ subcategory, 50 phosphosites in 29 phosphorylated proteins were identified (Table 1). Among these, 30 phosphosites were up-regulated and 20 were down-regulated.

CAZymes, including auxiliary activity (AA), carbohydrate-binding modules (CBM), carbohydrate esterase (CE), glycoside hydrolase (GH), glycosyl transferase (GT), and polysaccharide lyase (PL), were involved in the hydrolysis of plant cell wall polysaccharides and play an important role in the degradation of substrates(Davies & Williams, 2016). In the present study, 13 DPPs were identified as CAZymes, including 11 phosphosites in three CBMs, two phosphosites in two CEs, four phosphosites in three GHs, and six phosphosites in five GTs (Table 2). Interestingly, the GHs were up-regulated, while the CBMs were down-regulated.