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Optimizing red blood cell protein extraction for biomarker quantitation with mass spectrometry

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Abstract

Red blood cells (RBC) are the most common cell type found in blood. They might serve as reservoir for biomarker research as they are anuclear and lack the ability to synthesize proteins. Not many biomarker assays, however, have been conducted on RBC because of their large dynamic range of proteins, high abundance of lipids, and hemoglobin interferences. Here, we developed a semiquantitative mass spectrometry–based assay that targeted 144 proteins and compared the efficiency of urea, sodium deoxycholate, acetonitrile, and HemoVoid™ in their extraction of the RBC proteome. Our results indicate that protein extraction with HemoVoid™ led to hemoglobin reduction and increased detection of low abundance proteins. Although hemoglobin interference after deoxycholate and urea extraction was high, there were adequate amounts of low abundance proteins for quantitation. Extraction with acetonitrile led to an overall decrease in protein abundances probably as a result of precipitation. Overall, the best compromise in sensitivity and sample processing time was achieved with the urea–trypsin digestion protocol. This provided the basis for large-scale evaluations of protein targets as potential blood-based biomarkers. As a proof of concept, we applied this assay to determine that alpha-synuclein, a prominent marker in Parkinson’s disease, has an average concentration of approximately 40 μg mL−1 in RBC. This is important to know as the concentration of alpha-synuclein in plasma, typically in the picogram per milliliter range, might be partially derived from lysed RBC. Utilization of this assay will prove useful for future biomarker studies and provide a more complete analytical toolbox for the measurement of blood-derived proteins.

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Abbreviations

AcN:

Acetonitrile

AD:

Alzheimer’s disease

ALS:

Amyotrophic lateral sclerosis

APOE:

Apolipoprotein E

CV:

Coefficient of variation

DOC:

Sodium deoxycholate

HV:

HemoVoid™

LT:

LysC + Trypsin

MRM:

Multiple reaction monitoring

PD:

Parkinson’s disease

PET:

Positron emission tomography

RBC:

Red blood cell

SRM:

Selected reaction monitoring

T:

Trypsin

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Acknowledgments

We would like to acknowledge the research team of The Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL; a complete list of researchers can be found at www.aibl.csiro.au) and the volunteers and their families. We would also like to thank the Victorian Government’s Operational Infrastructure Support Program, the Wicking Trust and The Florey Institute of Neuroscience and Mental Health Neuroproteomics Facility. Furthermore, we would like to thank the Melbourne Mass Spectrometry and Proteomics Facility of The Bio21 Molecular Science and Biotechnology Institute (University of Melbourne) for the support of mass spectrometry analysis. We also thank Alicia Siew for assistance in testing protocols used for the development of these assays and Oliver R. B. Thomas for his comments and critical review of the manuscript.

Funding

We acknowledge funding and support; BR receives partial support from the National Health and Medical Research Council (1061550 & 1138673), and Motor Neuron Disease Research Institute of Australia and the Neuroproteomics Facility. BR and SK receive support from the Michael J. Fox Foundation (MJFF, Parkinson’s Research). BR receives support from Alzheimer’s Disease Drug Discovery Foundation. BR receives research support from Agilent Technologies and e-MSion, Inc.

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Authors and Affiliations

  1. The Florey Institute of Neuroscience and Mental Health, Melbourne Dementia Research Centre, The University of Melbourne, 30 Royal Parade, Parkville, Victoria, 3052, Australia

    Stephan Klatt, Anne Roberts, Amber Lothian, Colin L. Masters, Christopher Fowler & Blaine R. Roberts

  2. Cooperative Research Centre for Mental Health, Parkville, Victoria, 3052, Australia

    Stephan Klatt, Anne Roberts, Amber Lothian, Colin L. Masters, Christopher Fowler & Blaine R. Roberts

  3. Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, Victoria, 3010, Australia

    Roberto Cappai

  4. Department of Biochemistry, Department of Neurology, School of Medicine, Emory University, Atlanta, GA, 30322, USA

    Blaine R. Roberts

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  1. Stephan Klatt
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  2. Anne Roberts
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  3. Amber Lothian
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  4. Colin L. Masters
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  5. Roberto Cappai
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  6. Christopher Fowler
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  7. Blaine R. Roberts
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Contributions

S. Klatt designed the research, performed the experiments, optimized the mass spectrometry-based assay, analyzed the results and wrote the paper. A. Roberts and A. Lothian co-developed RBC protein extraction and mass spectrometry-based assay. C. Masters and C. Fowler identified blood samples from the AIBL study and were involved in the experimental design. R. Cappai designed the alpha-synuclein plasmid and designed the expression and purification protocols. B. Roberts designed the research, co-developed the mass spectrometry-based assay, analyzed the results and wrote the paper.

Corresponding author

Correspondence to Blaine R. Roberts.

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BR receives research support from Agilent Technologies, Biosensis and eMSion. All other authors declare to have no conflict of interest.

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Klatt, S., Roberts, A., Lothian, A. et al. Optimizing red blood cell protein extraction for biomarker quantitation with mass spectrometry. Anal Bioanal Chem 412, 1879–1892 (2020). https://doi.org/10.1007/s00216-020-02439-5

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