Protein biomarkers discovery of lead (Pb) toxicity and adaptive response in House Sparrows from Australian Mining Towns

Introduction: Lead (Pb) toxicity is a persistent issue in Australian mining and smelting towns like Broken Hill, Port Pirie and Mount Isa. While high lead exposure poses significant health risks to humans and wildlife, low doses are not lethal but subtly affect fitness and reproduction. House Sparrows (Passer domesticus) withstand high levels of lead in Australian mining towns with minimal adverse signs on fitness and reproduction. Genomics studies previously done in sparrows have indicated the genetic basis of resilience to lead in these birds. We are interested in protein networks that may be involved in the adaptive resistance in birds. We have conducted untargeted proteomics studies to find the biomarkers of adaptation in birds.

Methods: Lead affects red blood cells, immune cells and circulating proteins in blood, but blood proteomics is challenging due to the dominance of highly abundant proteins like haemoglobin and albumin. We overcame this issue using Mitra VAMS micro-sampling tips, which effectively remove the most abundant proteins from avian whole blood, frozen blood pellets, and plasma. We optimized method to work with as little as 1 µL of blood or pellet without compromising protein IDs. Using this approach, we collected and prepared sparrow blood samples from Australian mining towns and analysed them on Thermo Exploris-480 connected to Vanquish-Neo LC. Data was collected in DIA mode and searched using DIA-NN against House Sparrow protein database. Statistical analysis and modelling were performed using R.

Preliminary Data: Avian red blood cells are nucleated, which interferes with protein extraction and the effective removal of highly abundant proteins. Our optimized method overcomes this challenge, efficiently depleting abundant proteins and enabling deeper proteome analysis. With just 1 µL of blood, we quantified over 2,000 unique protein IDs in a single run. The method shows low technical variability (<10% CV) and has few missing values.
Preliminary results from sparrows in different Australian mining towns of Broken Hill and Port Pirie reveal significant differences in their proteomes, suggesting distinct adaptive responses to lead exposure. These populations have been geographically separated for over 100 years, leading to independent evolutionary pathways to counteract the same environmental stressor. Our analysis identified key proteomic shifts related to lead resistance, including the activation of protein renewal pathways and enhanced glutathione-mediated protection against reactive oxygen species.

Interestingly, one population exhibited a significant upregulation of immune response proteins, despite no known infections in sampled individuals. This suggests a potential link between lead exposure and immune system activation. These findings highlight the complexity of proteomic adaptations in response to long-term environmental lead exposure and provide insight into how different populations develop unique resistance mechanisms over time. We have recently completed a dosing study, where sparrows from these towns were challenged with lead along with controls from non-leaded towns. The longitudinal samples were collected and currently being analysed. The results from this, which will further illustrate the adaptive proteomics of sparrows from these towns, will be presented.

Novel Aspect: Our method outperforms existing approaches and enables minimally-invasive sample collection, providing new opportunities for field researchers in wildlife studies.

Conflict of Interest Disclosure: The authors declare no competing financial interest.