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. 2026 Oct 1;11(40):60808–60817. doi: 10.1021/acsomega.6c03766

Chemical and Proteomic Characterization of Bioactive Protein-Enriched Fraction from Schizophyllum commune with Anticancer Activity in HeLa Cells

Saranya Sedtananun †,‡,*, Lueacha Tabtimmai †,‡, Chanapat Butsukon †, Chanikan Sonklin ‡,§, Kiattawee Choowongkomon ∥
PMCID: PMC13670856  PMID: 42890085

Abstract

Schizophyllum commune, commonly known as the split gill mushroom, is an edible fungus recognized as a source of bioactive compounds with potential applications in the food and biotechnology industries. In this study, protein-enriched fractions were isolated from the fruiting bodies of S. commune using ion-exchange chromatography coupled with a bioassay-guided approach. Cytotoxic activity was evaluated in HeLa cells using MTT and colony formation assays. Among the fractions, the protein-enriched fraction F2 exhibited antiproliferative activity with an IC50 value of 0.37 μg/mL and significantly inhibited colony formation, while showing minimal effects on normal HaCaT cells. Flow cytometry and fluorescence staining further supported the involvement of apoptosis in F2-treated HeLa cells. Chemical and proteomic characterization of the protein-enriched fraction F2 by SDS-PAGE and LC–MS/MS identified 64 protein groups. Gene ontology analysis revealed proteins involved in diverse biological processes, while physicochemical profiling demonstrated a heterogeneous protein composition with isoelectric points ranging from 4 to 7. These findings provide insight into the chemical and proteomic composition of protein-enriched fraction F2. The results support further investigation of S. commune-derived protein-enriched fractions as a source of bioactive proteins, particularly in bioactivity-guided fractionation studies.

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1. Introduction

Edible mushrooms have gained increasing attention as sustainable bioresources rich in diverse bioactive compounds, including polysaccharides, proteins, and peptides, which contribute to their functional and biological properties. , Among them, Schizophyllum commune Fr., commonly known as the split gill mushroom, is a widely distributed wood-decaying basidiomycete species. While extensive studies have focused on mushroom-derived polysaccharides, particularly for their immunomodulatory and antioxidant activities, protein-enriched fractions from edible fungi remain comparatively underexplored despite their potential as bioactive components. , In the case of S. commune, research has predominantly focused polysaccharides, and only a limited number of functional proteins have been reported, including a fibrinolytic enzyme, hydrolysate peptides, and mycoprotein extracts. However, direct evidence linking specific mushroom proteins to anticancer activity remains limited.

Proteomics is a powerful tool for investigating the nutritional profiles of foods, detecting allergens, and mapping essential biological pathways. It plays a pivotal role in understanding the health impacts of food and improving food safety. Recent advancements in biochemical technologies have made proteomics increasingly important for exploring the diverse potential of edible fungi, especially those with medicinal applications. , Proteomics can also be used to identify mushroom proteins with potential health benefits, such as antioxidant, anti-inflammatory, and antimicrobial activities. For example, Lau et al. conducted a proteomic analysis to compare the antihypertensive effects of proteins from edible mushrooms, identifying those with the greatest effects. A study by Domingo et al. reported the proteomic profile of Lentinula edodes (shiitake), revealing proteins involved in metabolism, growth, and replication, including a serine protease inhibitor associated with antioxidant and antibacterial properties. Furthermore, partial purification of mushroom proteins has been explored; for instance, protein-enriched fractions from Phallus indusiatus demonstrated selective cytotoxic activity by effectively inhibiting cancer cell proliferation without affecting normal cells. Investigating mushroom-derived proteins, particularly from under-researched species like S. commune, presents a valuable opportunity for developing novel bioactive components and functional ingredients.

Cancer remains a major global health challenge, ranking as one of the leading causes of mortality worldwide. Despite advances in screening and treatment, conventional approaches such as surgery, radiotherapy, and chemotherapy are often associated with adverse effects and therapeutic resistance. − These limitations have driven increasing interest in natural products as sources of bioactive compounds with potential biological activities. , Among these, mushrooms have long been utilized in traditional medicine, and recent studies have highlighted their diverse functional properties, including antioxidant and antiproliferative activities. ,, In particular, mushroom-derived compounds have been reported to modulate cellular processes such as apoptosis, which plays a critical role in regulation cell survival and proliferation. ,

Due to their unique bioactivities, mushroom proteins have attracted increasing interest as bioactive components from natural sources. In addition, there is growing interest in using natural extracts as alternatives to pure compounds, driven by their longstanding role in traditional applications and the potential for synergistic effects among multiple active components. This study aims to further explore the potential of partially purified mushroom protein through a bioassay-guided fractionation approach. Protein-enriched fractions were obtained using chromatographic separation and subsequently characterized by SDS–PAGE and LC–MS/MS analyses to determine their protein composition and physicochemical characteristics. The biological activity of the fractions was evaluated using in vitro assays to assess cytotoxic and antiproliferative effects. These findings provide insights into the chemical composition and biological properties of mushroom protein-enriched fractions and support further investigation as bioactive components.

2. Materials and Methods

2.1. Materials and Reagents

The dried fruiting bodies of S. commune were obtained from the Chaiya Mushroom Farm in Surat Thani Province, Thailand. The mushrooms were ground into a fine powder and passed through 60 μm mesh sieve. HeLa (human cervical cancer cell line) and HaCaT (human normal keratinocytes cell lines) were obtained from ATCC (Manassas, VA, USA). All other chemicals and solvents used in this study were of analytical grade.

2.2. Extraction of Crude Proteins from Mushroom Fruiting Bodies

The crude protein solution was extracted using a previously reported method with minor modifications. Briefly, the powder from fruiting bodies of S. commune (25 g) was soaked in 500 mL of chilled 5% acetic acid containing 50 mM 2-mercaptoethanol for 16–18 h at 4 °C under constant stirring. Then, the mixture was filtered using cheesecloth to separate the solid and supernatant portions. The proteins in the supernatant were precipitated by the addition of (NH4)2SO4 to 80% saturation and kept at 4 °C overnight. The protein pellet obtained after centrifugation at 10,000 rpm for 30 min and was subjected to dialysis against 10 mM Tris-HCl buffer (pH 8.0) for 36–48 h at 4 °C. Finally, the dialyzed protein (crude protein) was filtered through a 0.45 μm Millex filter unit and stored at 4 °C.

2.3. Partial Purification of Proteins

The crude protein extract was purified using Fast Protein Liquid Chromatography (FPLC) on an AKTA prime plus system (Amersham Pharmacia), equipped with a HiTrap DEAE FF column (2.0 cm × 5 cm, GE, USA). The column was equilibrated with 10 mM Tris-HCl buffer (pH 8.0), and bound proteins were eluted with stepwise concentrations of NaCl (final concentration 0.05, 0.25, 0.5, and 1 M) in the same buffer. Protein-containing fractions were pooled and concentrated using Amicon Ultra centrifugal filters (3 kDa cutoff, Millipore) at 3500 rpm. The concentrated protein fractions, referred to as protein-enriched fractions, were then assessed for protein content and molecular weight using the Bradford assay and SDS-PAGE, respectively (Figure S1 in the Supporting Information file).

2.4. Cell Cytotoxicity Assay

The cytotoxic potential of protein-enriched fractions was assessed on HaCaT (human normal keratinocytes) and HeLa (cervical carcinoma) cell lines, using the MTT assay. Cells were plated at a density of 5 × 103 cells per well in a 96-well plate and incubated at 37 °C with 5% CO2 for 24 h. Following incubation, the cells were treated with different concentrations of protein-enriched fractions. After 72 h of exposure, MTT solution (5 mg/mL) was added, and the wells were incubated for 2–3 h at 37 °C. The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm with a reference wavelength of 630 nm using a SpectraMax iD3Multi-Mode Microplate Reader (Molecular Devices, USA). Cell viability was calculated as a percentage relative to the untreated control group. Cell survival was calculated as

cellsurvival(%)=[(experimental‐blank)÷(control‐blank)]×100

Cytotoxicity assays were performed in triplicate, with each condition replicated. The half-maximal inhibitory concentration (IC50) was determined using GraphPad Prism 8.0 software.

2.5. Clonogenic Assay

The clonogenic assay was used to assess the long-term colony-forming inhibition of HeLa cells, following the modified method of Samatiwat et al. HeLa cells were plated at a density of 600 cells per well in 6-well plates. After 24 h of incubation, the medium was replaced with a complete medium containing protein-enriched fraction F2 and F3 at IC50 concentrations. After an additional 48 h of incubation, the cells were maintained in fresh medium for 8 more days, with the medium changed every fourth day. The colonies were then fixed with absolute methanol and stained with a 0.5% crystal violet solution. The number of colonies in each well was counted under a microscope, and the percentage of protein-enriched fraction F2 and F3-treated colonies relative to the DMSO-treated control colonies was calculated.

2.6. Morphological Examination using Fluorescence Microscopy

Apoptosis in HeLa cells was examined using triple fluorescent staining with AnnexinV-FITC, 7-AAD, and Hoechst 33342 for cell apoptosis. Briefly, HeLa cells were plated at a density of 5 × 103 cells per well in a 6-well plate. After 24 h of incubation, the cells were treated with medium containing protein-enriched fraction F2. After 48 h of treatment, cells were washed twice with 0.01 M PBS and suspended in 200 μL of binding buffer. Cells were then incubated with the fluorescent dyes for 30 min at 4 °C in the dark room. The cellular nuclear morphology was immediately observed under an inverted microscope (Nikon ECLIPSE Ts2, Nikon, USA). Fluorescent images were analyzed using Pixit Pro software (Lanoptik Technologies Ltd., China).

2.7. Annexin V Apoptosis Assay

Quantitative analysis of live, early/late apoptotic and dead cells was performed using the Muse Annexin V/Dead Cell Assay Kit (Millipore, Darmstadt, Germany) with the Muse Cell Analyzer (Millipore) according to the manufacturer’s instructions. Briefly, the assay identifies phosphatidylserine (PS) exposed on the outer membrane of apoptotic cells using a premixed reagent that includes fluorescently labeled Annexin V, and 7-Aminoactinomycin D (7-AAD) as a marker for dead cells. HeLa cells were treated with protein-enriched fraction F2 for 24 h at 37 °C, while a control group of cells was incubated in growth medium under the same conditions. The cells were then analyzed according to the manufacturer’s instructions. During all cytofluorometric analyses, cell debris and clumps were excluded by appropriate gating.

2.8. Proteomic Profiling Analysis

The protein-enriched fraction F2 sample was cleaned using Biorad P6̅ gel spin column and reduced with 10 mM DL-Dithiothreitol (DTT) at 65 °C for 30 min in the dark. Then, sulfhydryl groups were alkylated with 25 mM iodoacetamide (IAA) for 20 min at room temperature in the dark. Protein sample was subsequently digested with a trypsin solution for 16 h. The digestion was terminated by adding 1% formic acid, followed by centrifugation at 14,000 rpm for 10 min. Proteomic analysis was performed using a LC–MS 6545XT QTOF (Agilent Technologies, Santa Clara, CA, USA). The mobile phases consisted of (A) 0.1% formic acid in water and (B) 95% acetonitrile containing 0.1% formic acid. The samples were directly loaded onto Agilent Peptide Map (2.1 × 150 mm, 2.7 μm) maintained at 60 °C and separated over 85 min run with a flow rate of 0.4 mL/min. Data-dependent acquisition (DDA) of MS and MS/MS spectra was performed over m/z ranges of 40–1700 and 25–1000, respectively. The source was operated under the following conditions: capillary voltage, 4000 V; nozzle voltage, 500 V; nebulizer (N2), 35 psi; and drying gas flow rate, 13 L/min. Agilent.d files were converted to mzXML format using MSConvert and OpenMS. The raw data were searched against Schizophyllaceae genome database using MaxQuant software (version 2.6.3). A total of 28,741 protein sequences from the Schizophyllum genome database were included in the analysis. Finally, 64 protein groups were identified (Table S1 in the Supporting Information file).

2.9. Database Search and Functional Annotation

FASTA files containing all protein sequences were retrieved from UniProtKB for further analysis. The GRAVY score was calculated using an online tool (https://www.gravy-calculator.de/), while the molecular weight and isoelectric point were determined using the ProtParam tool on the Expasy server. The identified proteins were then annotated with gene ontology (GO) terms for molecular function, biological process, and cellular component using Blast2GO.

2.10. Statistical Analysis

All experiments were performed in triplicate. Data are presented as the mean ± standard deviation (SD). Statistical significance was evaluated using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparisons test, with GraphPad Prism software (version 8.0). Differences were considered statistically significant at p < 0.05 and highly significant at p < 0.01.

3. Results and Discussion

3.1. Extraction and Partial Purification of Protein-Enriched Fractions from S. commune

Mushrooms produce a variety of proteins that exhibit important biological and pharmacological properties. , In this study, crude proteins were extracted from S. commune fruiting bodies by 80% (NH4)2SO4 precipitation. The crude proteins extract was partial purified by anion-exchange chromatography on a HiTrap DEAE FF column, resulting in four peaks, with fractions F2 and F3 showing the highest absorbance at 280 nm (Figure A). SDS-PAGE analysis revealed proteins with molecular weights ranging from approximately 10 to 175 kDa (Figure B). Partial purification provides a balance between protein yield and purity while preserving protein bioactivity and reducing processing time and costs. These protein bands may correspond to various proteins from S. commune, consistent with previous studies.

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Partial purified proteins from the fruiting bodies S. commune. (A) Crude protein was fractionated on a HiTrap DEAE FF column with a stepwise increasing concentration of NaCl (0.05 M NaCl, 0.25 M NaCl, 0.5 M NaCl, 1 M NaCl); (B) SDS PAGE results of different chromatographic fractions (Marker, F1, F2, F4, F3).

3.2. Cytotoxicity and Antiproliferative Activity of the Protein-Enriched Fraction in HeLa Cells

The cytotoxic effects of protein-enriched fractions F1, F2, F3, and F4 on HeLa cell lines were evaluated using an MTT assay across different concentrations. As shown in Figure A, protein-enriched fractions F2 and F3 exhibited the greatest inhibitory effects at 2.9 μg/mL. Cell viability decreased in a concentration-dependent manner. Interestingly, treatment of HeLa cells with protein-enriched fractions F2 and F3 at concentrations up to 0.73 μg/mL resulted in a measurable reduction in cell viability. In comparison, normal human keratinocytes (HaCaT) cells exhibited relatively higher viability, maintaining approximately 75–80% survival at the lowest concentrations tested (Figure B). HaCaT cells were used as a nontumorigenic human keratinocyte model to preliminarily assess the cytotoxic selectivity of the protein-enriched fractions. These findings suggest a degree of differential sensitivity between the two cell lines under the tested conditions. As indicated in Figure C,D, a comparison of the growth inhibitory effects between protein-enriched fraction F2 and F3 revealed that fraction F2 (IC50 = 0.37 μg/mL) exhibited a stronger growth inhibitory effect than fraction F3 (IC50 = 2.52 μg/mL). Protein-enriched fractions F2 and F3 indicated preliminary cytotoxic selectivity by inhibiting the proliferation of HeLa cells while exhibiting low cytotoxicity toward HaCaT cells. The selective cytotoxicity of protein-enriched fractions F2 and F3 toward cancer cells, while sparing normal cells, highlights their potential as a promising candidate for therapeutic development. Therefore, protein-enriched fractions F2 and F3 at concentrations of 0.37 and 2.52 μg/mL were used in the subsequent assays.

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Effect of the protein-enriched fraction from the fruiting bodies of S. commune on cell survival of HeLa and HaCAT cells. (A) Protein fraction of F1–F4 on survival rate (%) of HeLa cells; (B) Protein-enriched fraction of F2–F3 on survival rate (%) of HaCAT cells; (C, D) IC50 of protein-enriched fraction F2 and F3 on survival rate (%) of HeLa cells. Un, untreated cells; F2, protein-enriched fraction 2; F3, protein-enriched fraction 3. Data values are means of the independent triplicate experiments ± SD.

To further confirm the antiproliferative effects of protein-enriched fractions F2 and F3 against HeLa cells, a clonogenic assay was conducted to assess the colony-forming ability of HeLa cells. The clonogenic assay identifies viable cells capable of proliferating after the protein-enriched fractions are removed from the culture medium. Moreover, clonogenicity serves as an indirect measure of the neoplastic potential of cancer cells. HeLa cells were treated with the IC50 concentration of protein-enriched fractions F2 and F3, and the number of colonies formed was counted. As illustrated in Figure A, the number of colonies was reduced following treatment with protein-enriched fractions F2 and F3 compared with the untreated control (Un). The survival fraction was calculated and showed a significant reduction in HeLa cell survival, with the greatest and statistically significant inhibitory effect observed for protein-enriched fraction F2 (Figure B). The reduction in colony formation following treatment with protein-enriched fractions F2 and F3 indicates decreased colony-forming ability of HeLa cells, consistent with their antiproliferative activity. The lower surviving fraction observed for protein-enriched fraction F2 is consistent with the MTT assay results showing inhibition of HeLa cell growth. These findings further support the antiproliferative effect of protein-enriched fraction F2.

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Effect of protein-enriched fractions of F2 and F3 from the fruiting bodies of S. commune on colony formation by HeLa cells. (A) HeLa cells were treated with IC50 of F2 and F3, the colony formation was monitored; (B) the number of colonies counted, and the survival fraction calculated. Un, untreated cells; F2, protein-enriched fraction 2; F3, protein-enriched fraction 3. Data values are means of the independent triplicate experiments ± SD.

3.3. Protein-Enriched Fraction F2 Induces Apoptosis-Associated Morphological Changes in HeLa Cells

Morphological changes in HeLa cells following exposure to protein-enriched fraction F2 were observed under a phase-contrast microscope. In the untreated group, HeLa cells cultured as monolayers overnight in DMEM culture medium showed normal morphology (Figure ). In contrast, treatment of HeLa cells with protein-enriched fraction F2 at 1/2IC50 and IC50 concentrations resulted in cell shrinkage, rounding, loss of contact with neighboring cells, and detachment from the culture surface. Previous studies have reported that mushroom-derived proteins induced noticeable morphological alterations in cancer cells. In the present study, treatment with the protein-enriched fraction F2 significantly altered the morphology of HeLa cells. These morphological alterations are consistent with regulated cell death, although they are not sufficient on their own to establish the mode of cell death.

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Visualization of apoptotic cell population after the treatment under a fluorescence microscope, the treated HeLa cells were visualized using Annexin-FITC/7AAD staining. IC50 and 1/2IC50 of each protein-enriched fraction from the fruiting bodies of S. commune were used for evaluation. Bright field, Annexin-FITC, 7AAD, and merged were photographed. The arrow indicated apoptotic cell populations. Un, untreated cells; F2, protein-enriched fraction 2.

The loss of plasma membrane integrity allows the dye to penetrate dead cells. To determine whether the observed cell death was associated with apoptosis, nuclear morphology and cell membrane integrity in HeLa cells treated with protein-enriched fraction F2 were assessed using triple fluorescence staining with Annexin V-FITC, 7-AAD, and Hoechst 33342 under fluorescence microscope (Figure ). The observed fluorescence patterns suggest apoptosis in HeLa cells following treatment with protein-enriched fraction F2. The absence of red fluorescence in untreated cells is consistent with cell viability, whereas the strong green fluorescence in treated cells is consistent with apoptotic cell death. , The increase in green fluorescence is likely associated with phosphatidylserine externalization during early apoptosis, as detected by Annexin V-FITC staining. This is consistent with previous studies reporting phosphatidylserine exposure as a hallmark of apoptosis that enables the discrimination of viable and apoptotic cells. Furthermore, the nuclear alterations observed with Hoechst 33342 staining are consistent with apoptotic cell death. Chromatin condensation, nuclear fragmentation, and apoptotic body formation are well-established morphological features of apoptosis. Hoechst 33342 staining of untreated HeLa cells showed intact nuclei with a slightly oval shape, smooth edges, and uniformly distributed chromatin, whereas cells treated with protein-enriched fraction F2 revealed nuclear changes, including chromatin condensation, fragmentation, and apoptotic body formation. These findings suggest that protein-enriched fraction F2 is associated with nuclear alterations consistent with apoptotic cell death rather than necrosis.

The combination of triple fluorescence staining provides a comprehensive assessment of apoptotic progression by simultaneously evaluating plasma membrane integrity and nuclear morphology. Taken together, these findings support the involvement of apoptosis in the cytotoxic effects of protein-enriched fraction F2 on HeLa cells following treatment at 1/2IC50 and IC50 concentrations for 48 h. A standard apoptosis-inducing agent was not included as a positive control in the present study. Future studies incorporating a well-established apoptosis inducer would strengthen the interpretation of the apoptosis-related findings. Additional investigation of apoptosis-related molecular markers and signaling pathways is needed to elucidate the underlying mechanisms. Evaluation of protein-enriched fraction F2 in other cancer cell lines may provide further insight into its anticancer activity.

3.4. Protein-Enriched Fraction F2 Induces Apoptosis in HeLa Cells Assessed by Flow Cytometry

Apoptosis is one of the major mechanisms underlying cytotoxicity. To determine whether protein-enriched fraction F2-induced cell death involved apoptosis, the Muse Annexin V & Dead Cell assay was performed. In this assay, Annexin V was used to detect the externalization of phosphatidylserine on the cell surface, an early hallmark of apoptosis. The dead cell marker 7-AAD was included in the kit to assess cell membrane integrity. Thus, cells negative for both markers (lower left quadrant) were considered viable, cells positive for Annexin V only (lower right quadrant) were in early apoptosis, and cells positive for both Annexin V and 7-AAD were classified as late apoptotic/dead cells (upper right quadrant). Cells positive for 7-AAD only were indicative of necrosis (upper left quadrant).

As illustrated in Figure A, treatment with protein-enriched fraction F2 at 1/2IC50 and IC50 concentrations increased the proportion of early apoptotic cells to approximately 5.65–6.27%. At the IC50 concentration, fewer than 4.59% of cells remained viable, while the majority (89%) progressed to late apoptosis (upper right quadrant). Notably, less than 0.15% of cells were positive for 7-AAD alone (upper left quadrant), indicating minimal necrotic cell death, indicating minimal necrotic cell death under the conditions tested. These findings are consistent with apoptosis as a major mode of cell death induced by protein-enriched fraction F2 and agree with the fluorescence staining results observed in HeLa cells (Figure B).

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Flow cytometric analysis of protein-enriched fraction F2 from the fruiting bodies of S. commune induced apoptosis in HeLa cells using annexin V-FITC and PI double staining. (A) The cancer cell lines were treated with 1/2IC50 and IC50 of protein-enriched fraction F2 and after 24 h cells were stained, and flow cytometric analysis was performed; (B) Quantitative presentation of apoptosis induced by 1/2IC50 and IC50 of protein-enriched fraction F2 in HeLa cells. Un, untreated cells; F2, protein-enriched fraction 2;.

3.5. Protein Identification of Protein-Enriched Fraction F2 using LC–MS/MS Analysis

Protein identification was conducted using MaxQuant software (version 2.6.3), with the UniProt-Schizophyllaceae database containing 28,741 proteins sequences as the reference. Proteomic analysis of protein-enriched fraction F2 revealed a diverse protein composition, reflecting its biochemical complexity. A total of 64 protein groups were identified, covering a wide range of functional categories, including enzymes, structural proteins, and putative bioactive proteins. Selected proteins from the protein-enriched fraction F2 were subjected to protein identification based on the MS data, as shown in Table .

1. Selected Protein of Protein-Enriched Fraction F2 from the MS-Data for Protein Identification.

protein ID protein name razor + unique peptides sequence coverage intensity M w (kDa)
D8PSZ1 Cytolysin 10 29.9 14,863,000.0 31.860
D8Q7S3 Carbohydrate-binding module family 13 protein (Fragment) 10 35.4 11,347,000.0 33.007
D8PU51 β-glucosidase 9 12.1 353,580.0 97.870
D8Q9M3 Glucoamylase 8 26.7 844,480.0 60.940
D8PQ92 α-galactosidase 6 14.3 284,810.0 54.862
D8PQS8 Phosphatidylglycerol/phosphatidylinositol transfer protein (Fragment) 6 28.1 347,770.0 15.358
D8Q1P7 Uncharacterized protein 6 77.5 916,230.0 14.843
D8PPJ5 Cerato-platanin (Fragment) 5 49.3 262,090.0 14.126
D8QGA5 Ubiquitin-like domain-containing protein 4 26.6 371,660.0 14.539
D8PNF6 Carbohydrate-binding module family 13 protein 4 60.2 1,303,600.0 16.841
D8PNM2 Chitinase 4 13.0 295,670.0 39.120
D8PRI9 Tripeptidyl-peptidase II 4 12.3 748,070.0 66.754
D8Q1J2 Expressed protein 4 25.1 2,033,500.0 25.443
D8QHR7 SWIRM-domain-containing protein 4 5.3 155,340.0 120.92
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Among the identified proteins, D8PSZ1 (cytolysin-like protein) exhibited the highest intensity (14,863,000.0) with a molecular weight of 31.86 kDa, indicating its high abundance in the protein-enriched fraction F2. This protein showed moderate sequence coverage (29.9%), suggesting reliable identification. Cytolysin-like proteins are generally associated with membrane-interacting activities in various organisms. Literature reports describe cytolysins in mushrooms, such as flammutoxin from Flammulina velutipes and ostreolysin from Pleurotus ostreatus, as well as cytotoxic activities of related proteins in other species. However, in the context of this study, these reports are provided for biological context and do not constitute direct evidence of their involvement in the observed anticancer activity.

Another notable protein, D8Q7S3 (carbohydrate-binding module family 13 protein, fragment), exhibited 35.4% sequence coverage with 10 razor and unique peptides and a molecular weight of approximately 33 kDa. Proteins containing CBM13 domains are typically involved in carbohydrate recognition and binding. Structurally, these domains share similarities with lectins, some of which have been reported to exhibit biological activities, including cytotoxic effects. , Nevertheless, the presence of the CBM13-containing proteins (fragment) is interpreted as part of the compositional complexity of the protein-enriched fraction F2 rather than indicative of a specific biological function.

The uncharacterized protein D8Q1P7 demonstrated the highest sequence coverage (77.5%) with a molecular weight of 14.84 kDa, despite its lower intensity. This suggests that it was well represented in the MS data set, likely due to factors such as protein size and peptide detectability. Therefore, this protein may be a promising candidate for future structural and functional characterization.

The inclusion of molecular weight (Mw) values allows an approximate comparison with SDS-PAGE profiles, where major bands observed in the approximately 30–35 kDa and ∼14–16 kDa regions are consistent with the identified proteins. However, due to the limited resolution of SDS-PAGE and the potential comigration of proteins, direct assignment of individual proteins to specific bands cannot be confirmed.

In summary, MS analysis identified cytolysin-like proteins and carbohydrate-binding module family 13 proteins among the major constituents of the protein-enriched fraction F2. An uncharacterized protein with substantial sequence coverage was also detected, suggesting it is a candidate for further investigation. These findings reflect the protein composition of the bioactive fraction obtained through bioassay-guided fractionation and should not be interpreted as direct evidence of functional causality. It should be noted that this study focuses on the detailed proteomic characterization of the most bioactive protein-enriched fraction (F2), which was selected based on its consistently higher anticancer activity. Accordingly, a comprehensive comparative proteomic analysis across all protein-enriched fractions (F1, F3, and F4) was beyond the scope of the present study. Therefore, the observed anticancer activity is interpreted as being associated with the overall protein composition of the protein-enriched fraction F2, rather than attributed to individual proteins or to direct comparisons among fractions. A limitation of this study is that the active protein(s) were not purified or identified, and functional validation of the candidate proteins was not performed. Future studies should focus on protein purification, protein identification, functional characterization, and comparative proteomic profiling across all fractions to clarify the molecular basis of the observed anticancer activity.

3.6. GO Functional Classification of Proteins Identified in Protein-Enriched Fraction F2

The 64 identified proteins were subjected to GO enrichment analysis using their protein IDs to better understand their biological roles in organisms, tissues, or cells. The enriched GO terms were then organized into three primary ontological categories: biological processes, molecular functions, and cellular components, as illustrated in Figure . Among the annotated molecular functions (Figure A), hydrolase activity (28%), catalytic activity (9%), oxidoreductase activity (6%), transferase activity (6%), structural activity (3%), and other molecular functions (21%) were identified. The cellular component ontology (Figure B) refers to the areas within the cell where a gene product exerts its activity. Proteins were primarily associated with the extracellular region (6%) and other cellular components (10%). The biological process annotation (Figure C) revealed that the proteins were involved in a range of processes, including carbohydrate metabolic processes (20%), biological processes (11%), carbohydrate derivative metabolic processes (8%), and amino acid metabolic processes (5%).

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GO classification of protein-enriched fraction F2 identified by mass spectrometry. (A) Molecular functions; (B) Cellular components; (C) Biological functions.

3.7. Physicochemical Properties of Protein-Enriched Fraction F2

The identified components in protein-enriched fraction F2 were characterized based on their physicochemical properties, including hydrophobicity and isoelectric point (pI). Hydrophobicity is a fundamental property of peptides and proteins that influences their structure and biological function. To evaluate this characteristic, GRAVY (Grand Average of Hydropathy) values, as described by Kyte and Doolittle, were calculated using the amino acid sequences of peptides identified by LC–MS/MS analysis. The GRAVY index represents the average hydropathy of a given peptide or protein, where negative values indicate hydrophilic characteristics and positive values suggest hydrophobicity. Proteins with GRAVY values below 0 are generally associated with globular and water-soluble structures, whereas those with positive values tend to exhibit more hydrophobic and membrane-associated properties. The results showed that the majority of identified peptides in protein-enriched fraction F2 exhibited negative GRAVY values, indicating a predominantly hydrophilic nature (Figure A). This observation is consistent with the presence of proteins associated with extracellular regions, many of which are globular and water-soluble. In addition, the predicted isoelectric points (pI) of the identified peptides were mainly distributed within the range of 4–7 (Figure B), suggesting that most components in protein-enriched fraction F2 are weakly acidic to neutral under physiological conditions.

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Comparison of physicochemical properties of protein-enriched fraction F2. (A) GRAVY score; (B) PI.

4. Conclusions

In summary, the protein-enriched fraction F2 from S. commune, identified through bioassay-guided fractionation, exhibited cytotoxic effects against HeLa cells while showing minimal effects on normal HaCaT cells. LC-MS/MS analysis identified candidate proteins in the bioactive fraction; however, further purification and functional validation are required to determine their direct contribution to the observed anticancer activity. The possible contribution of other coextracted mushroom constituents, including polysaccharides and small metabolites, cannot be excluded. The present study focused on the most bioactive fraction identified through bioassay-guided screening and did not include comparative proteomic analysis across all fractions. Further studies incorporating comprehensive proteomic profiling and functional characterization are needed to clarify the bioactive components and their structure–function relationships.

Supplementary Material

ao6c03766_si_001.xlsx (32.2KB, xlsx)
ao6c03766_si_002.pdf (133.3KB, pdf)

Acknowledgments

This research was funded by King Mongkut’s University of Technology North Bangkok, Contract No. KMUTNB-68-BASIC-36.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c03766.

  • Proteomic identification of proteins in the bioactive protein-enriched fraction from S. commune (Table S1) (XLSX)

  • SDS PAGE gel of different chromatographic fractions from S. commune (Figure S1) (PDF)

S.S.: Methodology, formal analysis, investigation, writingoriginal draft, writingreview and editing, funding acquisition, conceptualization. L.T.: Methodology, formal analysis. C.B.: Methodology, formal analysis. C.S. and L.K.C.: Investigation.

The authors declare no competing financial interest.

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