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

Importance of the topic

Effective blood transfusion in non-domestic felids is critical for veterinary care in zoos and conservation programs. Due to unknown and diverse blood types among wild felid species, transfusion carries high risk of adverse reactions. Additionally, long-term storage of blood products is essential for emergency preparedness in institutions with limited numbers of each species. Understanding proteome stability in stored red blood cells helps assess sample integrity and may inform improved storage and transfusion protocols.

Objectives and study overview

This study aimed to evaluate proteomic changes in stored red blood cells from 18 non-domestic felid species over a 28-day refrigeration period. By leveraging high-throughput, automated workflows, the authors sought to determine whether protein degradation or shifts in proteome profiles occur during storage. The work further intended to establish a robust analytical pipeline suitable for large cohort studies in wildlife medicine.

Methodology and instrumentation

Blood samples from 136 individual felids across 18 species were collected during routine exams at AZA institutions. Fresh whole blood was refrigerated for 0, 7, 14, or 28 days, then pelleted and stored at −80°C. Pelleted red cells underwent bottom-up proteomic sample preparation using a Thermo Scientific AccelerOme automated platform. Key steps included lysis, DNA removal, reduction, alkylation, trypsin digestion, and cleanup without manual intervention. Peptides were analyzed by capillary flow LC-MS/MS on a Thermo Scientific Vanquish Neo UHPLC coupled to an Orbitrap Astral mass spectrometer. A 13-minute gradient plus 3-minute loading was applied at 2–2.5 µL/min through a 2 µm C18 column (150 µm×15 cm) at 50°C. Data-independent acquisition (199 windows spanning 380–980 m/z) generated MS1 scans every 0.6 seconds at 240k resolution. Data were saved to the Thermo Scientific Ardia platform and processed automatically via Proteome Discoverer 3.1 with the CHIMERYS 2.7.9 search algorithm against a comprehensive Feliformia database. Match Between Runs was enabled through a pilot inclusion list, and multiconsensus analysis combined all runs.

Main results and discussion

Across 1,072 injections from 540 samples, over 53,000 unique peptides and more than 7,000 protein entries were identified, with an average of 10,000 peptides and 1,500 proteins per sample. Protein abundance signals spanned over seven orders of magnitude, reflecting the Astral’s high dynamic range. No significant loss of sensitivity or shifts in protein groups were observed over storage time. Principal component analysis of peptide abundances revealed clear species-specific clustering, mirroring known phylogenetic relationships. These findings indicate proteome stability under standard refrigeration for up to 28 days.

Benefits and practical applications

• The automated AccelerOme workflow ensures reproducible sample preparation with minimal hands-on time.
• The Vanquish Neo UHPLC–Orbitrap Astral combination supports high-depth, high-throughput analysis (100+ samples/day).
• Automated data acquisition and processing via the Ardia platform reduces data management burden and accelerates results for large cohort studies.

Future trends and potential applications

Advances may include integration of cloud-based or AI-driven data analysis for real-time decision support in wildlife transfusion protocols. Expanded proteomic databases could improve species coverage and reduce redundancy. Further exploration of alternative storage conditions or additive solutions may enhance red cell preservation. The workflow could extend to other non-domestic taxa or to the study of other cell types.

Conclusion

This work demonstrates that automated, high-throughput proteomics can reliably profile red blood cell proteomes from a diverse cohort of non-domestic felids without significant degradation over 28 days of storage. The combined AccelerOme–Orbitrap Astral–Ardia platform offers an efficient and robust solution for large-scale studies, informing better blood storage and transfusion practices in wildlife medicine.

References

No formal literature references were provided in the source document.