MSACL 2024 Abstract

Accurate Quantification of Viral Protein to Support Reliable Viral Load Calculations Yan Yan Beer (1,2), Melanie M. Brinkmann (2,3), Gavin O´Connor (1,2) (1) Physikalisch-Technische Bundesanstalt, Braunschweig, Germany (2) Technische Universität Braunschweig, Braunschweig, Germany (3) Helmholtz Centre for Infection Research, Braunschweig, Germany Yan Yan Beer, Master of Science (Presenter) Physikalisch-Technische Bundesanstalt >> POSTER (PDF)

Presenter Bio: Education
2009-2012:
Training to Biological Laboratory Assistant at the Helmholtz Centre for Infection Research in Braunschweig, Germany
2016-2018:
Master Study Molecular Life Sciences at the HAN University in Nijmegen, Netherlands

Professional Experience
2012-2020:
Biological Labortaory Assitant in the fields of Infection Biology, Molecular Biology, Microbiology, Cell Biology and Immunology (Helmholtz Centre for Infection Research, Braunschweig, Germany)
2021 until now:
PhD Student at the Physikalisch-Technische Bundesanstalt in Braunschweig, Germany

INTRODUCTION: The monitoring of viral load is important to inform on disease progression and successful medical treatment. However, variabilities in quantitative measurement results may reduce the effectiveness of clinical intervention and disease management.
The ability to determine the number concentration of viral particles present in a biological sample, known as viral load, is essential in our continual quest to reduce the burden of viral infections on society. However, viral load is indirectly measured via the quantification of sequence specific viral nucleic acids, or proteins, in a representative sample. Quantitative PCR is the preferred analytical method for most viral infections. The quantification of viral DNA in a sample is nevertheless a non-specific incomplete indicator for active replication of the virus, while the quantification of viral proteins complements the information about the replication of the virus.
Combining DNA and protein measurements will provide a more complete picture of virus replication, while protein quantification will improve the measurement accuracy of infectious viral particles.

OBJECTIVES: We intend to assess the comparability of viral load measurements using DNA and protein measurement results to reduce the measurement uncertainty of viral load measurements and improving overall measurement comparability.

METHODS: The Human Cytomegalovirus (HCMV) served as a model organism. HCMV virions were produced in human fibroblasts and purified through a tartrate-glycerol gradient.
HCMV DNA was extracted using a viral DNA extraction kit. Digital droplet PCR of the HCMV gene UL54 was used to quantify the DNA and to calculate the genome copy number concentration within a sample.
Bottom-up proteomics was applied for the quantification of viral proteins. To identify suitable proteins and signature tryptic peptides for quantification, a peptide mapping approach was used. Time course experiments were run for selected signature peptides to confirm complete digestion of targeted proteins, confirmation of completeness was assumed when the ratio to isotopically labelled to natural protein plateaued and equimolar release of the peptides was observed.
For quantification, isotope dilution mass spectrometry (IDMS) was performed. To this end, synthetic labelled peptides served as internal standards and natural synthetic peptides were used as references for calibration. To ensure SI-traceability, amino acids analysis was performed on the synthetic peptides, using an exact matching IDMS calibration protocol and purified certified reference material (CRM) amino acids to prepare the calibration standards. The measurement uncertainty of the protein quantification was estimated according to the “Guide to the Expression of Uncertainty of Measurement” (GUM).

RESULTS:
Three viral capsid proteins, Major capsid protein (MCP), Triplex capsid protein 1 (Tri1), and Triplex capsid protein 2 (Tri2), were chosen for viral protein quantification. For each protein, 2-4 signature peptides were selected. Calibration curves ranging from 1-200 pmol/ml were established for all peptides to estimate the limit of detection. The limit of detection is below 88 pmol/ml. For all the selected signature peptides, the time course experiments revealed that a digestion plateau was reached within 8-10 hours of initiating the tryptic digestion.

CONCLUSION: Protein measurements provide complementary information about replication of infectious viral particles. Amino acid analysis, complete digestion, equimolar release of the target peptides and calibration using isotope dilution approaches ensure measurement results are traceable to the SI. This overall reference measurement improves viral load measurements and should be adaptable to different viruses. Therefore, it could play an important role in providing information and/or reference measurement procedures to monitor viral load for disease progression and successful medical treatment.