Proteomic Profiling Reveals Differential Protein Patterns in Galdieriasulphurariaextracts replacing fetal bovine serum in muscle cell culture

Proteomic profiling of Galdieria sulphuraria extracts as fetal bovine serum (FBS) replacements in muscle cell culture

Significance of the topic

The development of cultivated meat and other cell culture–based food products requires ethically acceptable, sustainable, and cost-effective serum alternatives to fetal bovine serum (FBS). Protein-rich algal extracts are promising candidates, but their complex composition can include both cytotoxic and growth-promoting components. Understanding which proteins drive those effects is essential to design safe, reproducible, and scalable serum-free supplements for mammalian cell culture.

Aims and study overview

This study evaluated crude (CE) and heat-treated (HE) protein extracts from the extremophilic red microalga Galdieria sulphuraria as partial replacements for FBS in cultures of C2C12 murine myoblasts. The goals were to (1) compare effects of CE and HE on cell viability, proliferation, morphology and differentiation; (2) characterize protein composition differences by label-free proteomics; and (3) identify protein groups depleted or enriched by heat treatment that correlate with cytotoxicity or growth support.

Methodology and sample workflow

- Biomaterial: Crude and heat-treated aqueous protein extracts prepared from Galdieria sulphuraria.
- Cell model: C2C12 myoblasts cultured under reduced-serum conditions with CE or HE supplementation; cell viability, metabolic activity, morphology, proliferation and capacity for terminal myogenic differentiation were assessed by microscopy and functional assays (qualitative in the report).
- Proteomic sample preparation: Proteins subjected to tryptic digestion using the PreOmics iST sample preparation kit; peptide clean-up; samples normalized to 500 ng/µL and 500 ng injected per analysis.
- LC–MS acquisition: nanoElute 2 nano-LC with a 22-minute elution gradient coupled to a timsTOF HT mass spectrometer operated in dia-PASEF mode.
- Data analysis: DirectDIA label-free quantitation (LFQ) processed in Spectronaut (Biognosys); downstream multivariate analyses included principal component analysis (PCA), hierarchical clustering and heat-map visualization.

Used instrumentation

• PreOmics iST sample preparation kit for streamlined tryptic digestion and peptide clean-up.
• nanoElute 2 nano-LC system (Bruker Daltonics) with a short 22 min gradient.
• timsTOF HT mass spectrometer (Bruker Daltonics) operated in dia-PASEF mode for high-sensitivity data-independent acquisition.
• Spectronaut (Biognosys) for directDIA LFQ processing and quantitative proteomic analysis.

Main results and discussion

- Biological outcomes: Crude algal extracts (CE) induced marked cytotoxicity in C2C12 cells, manifested as reduced viability, decreased metabolic activity, altered morphology and impaired proliferation. In contrast, heat-treated extracts (HE) supported cell viability and metabolic function under serum-reduced conditions and allowed progressive replacement of up to 80% of FBS during extended culture while preserving the ability of myoblasts to undergo terminal differentiation.

- Proteomic outcomes: Label-free proteomic profiling revealed a clear separation between CE and HE samples by PCA, indicating reproducible qualitative and quantitative compositional differences. Hierarchical clustering and heat-map visualization showed that heat treatment led to loss (likely precipitation or denaturation) of distinct protein groups present in CE that correlate with cytotoxic effects. Simultaneously, certain proteins were relatively enriched or more detectable in HE, consistent with selective retention of growth-promoting components or exposure of functional peptides following heat treatment.

- Interpretation: Heat inactivation appears to remove or neutralize algal proteins that are deleterious to mammalian cells while preserving or enriching factors that support proliferation and differentiation. The observed changes suggest that targeted processing of algal extracts can convert a cytotoxic crude preparation into a functional supplement suitable for long-term mammalian cell culture.

Practical benefits and applications

• Provides a proof-of-concept for using heat-treated Galdieria sulphuraria extracts as partial FBS replacements in muscle cell culture, addressing ethical and sustainability drivers in cultivated meat production.
• Demonstrates a streamlined proteomics workflow (iST kit + timsTOF HT + Spectronaut) that enables rapid characterization of complex algal extracts for QC and optimization.
• Identifies an experimental strategy—remove cytotoxic fractions by heat treatment and analyze residual proteome—to guide formulation of serum-free or serum-reduced media based on algal biomass.

Future trends and potential uses

• Protein-level target identification: Follow-up studies should identify specific cytotoxic proteins and growth-promoting factors (by MS/MS-driven identification, peptide mapping, or orthogonal fractionation) to enable selective depletion or enrichment.
• Bioprocess optimization: Scale-up of extraction and controlled heat-treatment parameters can be optimized to maximize yield of beneficial components while minimizing loss of labile growth factors.
• Component replacement and synergy: Identified algal factors could be recombinantly produced, combined with defined supplements, or used in hybrid formulations to create fully defined serum-free media.
• Safety, reproducibility and regulatory pathways: Comprehensive biochemical and toxicological profiling, lot-to-lot QC by targeted proteomics, and demonstration of consistent performance in relevant cell lines will be necessary for regulatory acceptance in food applications.
• Integration with multi-omics: Combining proteomics with metabolomics and glycomics can clarify non-protein contributors to cytotoxicity and growth support and enable rational media design.

Conclusion

Heat-treated protein extracts of Galdieria sulphuraria represent a promising approach to replace a substantial fraction of FBS in C2C12 myoblast culture. The combination of a rapid proteomics workflow and functional cell assays allowed identification of compositional changes caused by heat treatment that correlate with a switch from cytotoxic to growth-permissive behavior. Systematic identification and process control of extract components can accelerate development of algal-based, serum-free supplements for cultivated meat and other cell culture applications.

References

The original report describes analytical methods and results obtained using PreOmics iST sample preparation, nanoElute 2 LC, timsTOF HT operated in dia-PASEF mode, and Spectronaut for directDIA LFQ processing. Additional bibliographic citations were not provided in the source document.