Application of a targeted method for amino acid analysis to SARS-CoV2:
The Australian National Phenome Centre is constantly advancing the technology platforms used in metabolic phenotyping with the development of high-throughput and robust methods for the accurate measurement of metabolites associated with disease. A key platform in the laboratory is mass spectrometry (MS). As metabolic phenotyping evolves there becomes an increasing demand for applying the approach to ever increasing sample numbers in clinical and epidemiology studies.
An important technique in our portfolio is the ability to quantify the exact concentrations of known metabolites from key metabolic pathways linked to disease. This is commonly referred to as targeted or quantitative metabolic phenotyping. Traditionally, quantitative assays have utilized a type of mass spectrometry called triple quadrupole (QQQ) or tandem mass spectrometry (MS-MS) based methods.
The drawback of such methods focused methods is the removal of the ability to perform hypothesis generating discovery metabolic phenotyping that is untargeted in its nature. However, at the ANPC we developed an integrated workflow that collects targeted data, resulting in the quantification of 34 amino acids, whilst simultaneously collecting full scan high-resolution accurate mass (HRAM) exploratory metabolic phenotyping. Using this HRAM-MS data, retrospective phenotypic data mining can now be performed, allowing widening the ability to detect novel biomarkers of disease.
The approach was evaluated using a variety of detector platforms in house at the ANPC including a Waters TQ-XS (QQQ) and Bruker Impact II QToF (HRAMS-MS) and three human biofluids (plasma, serum and urine). Intra- and inter-day accuracy and precision were comparable between the QQQ and QToF instruments (<15%), with excellent linearity (R2 > 0.99) over the quantification range of 1–400 μmol L−1.
Importantly, at the ANPC we strive to ensure all of our assays are performing to expectation. One such way to evaluate performance is to run against external reference standards. In this instance we performed the assay on NIST1950 – a pool of plasma that has been well characterised and comes with certified reference concentrations. Our measurements were in agreement with the certified values.
The assay has now been applied to projects throughout the ANPC including COVID-19 and studies collaborating with an intensive care unit and a paediatric burns clinics.
Application of a targeted method for amino acid analysis to SARS-CoV2:
The Australian National Phenome Centre is constantly advancing the technology platforms used in metabolic phenotyping with the development of high-throughput and robust methods for the accurate measurement of metabolites associated with disease. A key platform in the laboratory is mass spectrometry (MS). As metabolic phenotyping evolves there becomes an increasing demand for applying the approach to ever increasing sample numbers in clinical and epidemiology studies.
An important technique in our portfolio is the ability to quantify the exact concentrations of known metabolites from key metabolic pathways linked to disease. This is commonly referred to as targeted or quantitative metabolic phenotyping. Traditionally, quantitative assays have utilized a type of mass spectrometry called triple quadrupole (QQQ) or tandem mass spectrometry (MS-MS) based methods.
The drawback of such methods focused methods is the removal of the ability to perform hypothesis generating discovery metabolic phenotyping that is untargeted in its nature. However, at the ANPC we developed an integrated workflow that collects targeted data, resulting in the quantification of 34 amino acids, whilst simultaneously collecting full scan high-resolution accurate mass (HRAM) exploratory metabolic phenotyping. Using this HRAM-MS data, retrospective phenotypic data mining can now be performed, allowing widening the ability to detect novel biomarkers of disease.
The approach was evaluated using a variety of detector platforms in house at the ANPC including a Waters TQ-XS (QQQ) and Bruker Impact II QToF (HRAMS-MS) and three human biofluids (plasma, serum and urine). Intra- and inter-day accuracy and precision were comparable between the QQQ and QToF instruments (<15%), with excellent linearity (R2 > 0.99) over the quantification range of 1–400 μmol L−1.
Importantly, at the ANPC we strive to ensure all of our assays are performing to expectation. One such way to evaluate performance is to run against external reference standards. In this instance we performed the assay on NIST1950 – a pool of plasma that has been well characterised and comes with certified reference concentrations. Our measurements were in agreement with the certified values.
The assay has now been applied to projects throughout the ANPC including COVID-19 and studies collaborating with an intensive care unit and a paediatric burns clinics.
