Automated high-throughput proteomic analysis of stored blood cells from a large cohort of non-domestic felids

Significance of the topic

Blood transfusion is a vital intervention to treat severe anemia in animals, yet matching donor and recipient blood types in non-domestic felids remains challenging due to limited knowledge of their blood group diversity. In zoological settings, the ability to store and crossmatch blood for rare or endangered cat species can be lifesaving and help manage emergencies when compatible donors are unavailable. High-throughput proteomic profiling of stored red blood cells offers insights into protein stability over time and potential biomarkers for blood compatibility.

Objectives and study overview

This study aimed to develop and validate an automated, high-throughput bottom-up proteomic workflow for analyzing stored red blood cells from a large cohort of non-domestic cat species. Key goals included evaluating sample stability across storage durations, characterizing the proteome diversity among 18 felid species, and assessing whether proteomic signatures could reflect phylogenetic relationships and inform transfusion compatibility.

Methodology and instrumentation

An automated sample preparation platform (Thermo Scientific AccelerOme) processed pelleted red blood cells from 136 individual cats across 18 species, stored for 0, 7, 14, or 28 days. The workflow involved lysis, DNA removal, reduction, alkylation, trypsin/LysC digestion, peptide cleanup, and pooling without manual intervention. Peptides (200 ng) were separated on a Thermo Scientific Vanquish Neo UHPLC with an EASY-Spray C18 column (150 µm × 15 cm) at 2–2.5 µL/min and 50 °C using a 13-minute gradient.

Data-independent acquisition was performed on a Thermo Scientific Orbitrap Astral mass spectrometer with 199 windows (380–980 m/z). MS1 scans at 240 K resolution and DIA MS2 scans captured a dynamic range spanning seven orders of magnitude. Acquired data were written directly to the Thermo Scientific Ardia Advanced Tower server and processed in real time by Proteome Discoverer 3.1 SP1 with the CHIMERYS 2.0 search algorithm, enabling parallel searches and automated multiconsensus analysis.

Main results and discussion

Across 1,072 injections, the workflow identified over 53,000 peptides mapping to more than 7,000 protein entries. On average, each sample yielded over 10,000 peptides corresponding to approximately 1,500 unique proteins after redundancy correction. No significant loss of sensitivity was detected over a 16-day acquisition period. Principal component analysis of peptide abundances demonstrated clear clustering by species that mirrored known phylogenetic relationships among the felid suborder. Protein signal intensities covered a seven-order magnitude dynamic range, highlighting the platform’s capability for deep proteome coverage.

Benefits and practical applications

• The fully automated AccelerOme workflow reduces hands-on time and improves reproducibility for large-scale proteomic studies.
• The vanquish Neo–Orbitrap Astral combination delivers high throughput (100+ samples/day) with deep coverage and wide dynamic range.
• Real-time data acquisition and processing via the Ardia platform streamline data management and accelerate analysis turnaround.
• Proteomic profiling of stored blood cells can inform compatibility assessments and optimize transfusion strategies in non-domestic felids.

Future trends and potential applications

Advancements in automation and instrument sensitivity will enable routine proteomic monitoring of stored blood products in zoological medicine. Integration with machine learning could predict transfusion compatibility based on proteomic signatures. Expanding the workflow to other wildlife species may support broader conservation and veterinary initiatives. Moreover, scaling database resources and reducing redundancy will improve protein identification accuracy and facilitate biomarker discovery.

Conclusion

This study demonstrates a robust high-throughput proteomic workflow for archived red blood cells from diverse non-domestic felids. Automation at every stage ensures reproducibility, while the Orbitrap Astral system achieves deep coverage and maintains sensitivity over large sample sets. The approach offers valuable insights into proteome stability during storage and species-specific protein patterns that reflect evolutionary relationships, with direct implications for improving blood transfusion safety in exotic animal care.

References

No external literature references were provided in the original document.