Rapid High-Throughtput Biomarker Discovery

A novel, high-throughput method for the identification of low-abundance biomarkers in human plasma samples.

March 31, 2022

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Tuberculosis (TB) is still a major threat to health on a global scale. In 2020, a year that saw several new variants of SARS-CoV-2 sweep across the globe TB was still the second most deadly infectious disease on the planet.  As with many diseases, early diagnosis of TB is critical to successful treatment, and with an infection as serious as TB early detection is critical to stop the infection from spreading any further. However, rapid and accurate high-throughput testing from easily obtained samples such as blood or plasma has remained a missing piece in the fight against TB and a critical unmet need.

 

Finding biomarkers in human plasma

 

Plasma samples are easy to obtain and have been successfully used in testing for a variety of disease states, including AIDS, Hepatitis C, and more recently COVID-19. The search for suitable disease biomarkers, however, remains a time and resource-intensive process due to the complexity of the samples involved. Blood plasma contains many thousands of proteins and peptides, and the disease state biomarkers are typically present at very low concentrations. Current methods to identify biomarkers often involve either low-throughput pre-fractionation techniques or lengthy mass spectrometry (MS) data acquisition approaches to aid the discovery of low-abundance disease markers.

 

In a recent publication[1] a San Francisco-based research team have designed an unconventional plasma analysis protocol for rapid, high-throughput disease biomarker discovery. The scientists chose to base their method on DIA-MS (Data independent acquisition-mass spectrometry) analysis. An analytical method designed to permit scrutiny of both low and high abundance peptides present in a sample. While DIA-MS has been used previously to examine relative protein abundance, most studies have been limited to identifying up to about 300 proteins, mostly due to the lack of comprehensive mass spectral libraries which can be used to identify peptides.  To address this issue, the researchers chose to combine the DIA-MS approach with both a novel peptide library generation method and a peptide digest strategy designed to provide better overall sequence coverage of the plasma peptides.

 

High-throughput analysis of case-control samples

 

Plasma from three adults with TB and three without TB were used to generate peptide libraries. Individual samples were depleted of the most abundant plasma peptides using a standard depletion kit, and the remaining sample was concentrated, then subjected to proteolysis using one of three orthogonal proteases: trypsin, Glu-C, or Asp-N. Following digestion, the samples were fractionated via C18 tip-based fractionation with fractions eluted using increasing acetonitrile concentration. The fractions were then subjected to proteomic analysis, and the combined results were used to generate spectral libraries that were specifically TB positive or TB negative.

 

96-well filter plates enable high-throughput analysis

 

To carry out the biomarker studies researchers used a high-throughput approach. 5 mL plasma samples from individuals with and without TB were separated into three equal portions, then loaded into AcroPrepTM Advance 96-well 3K MWCO filter plates for sample clean-up and differential peptide digestion (trypsin, Glu-C, or Asp-N).

 

AcroPrep 96-well filter plates are designed to meet the stringent regulatory requirements for high-throughput applications and are ideal for manual, semi-automated, and automated processes.

 

The Omega ultrafiltration membrane used in this study is available in AcroPrep 24, 96, and 384-well filter plates and in a number of molecular weight cut-offs (MWCOs) including 3K, 10K, 30K, and 100K.

 

After washing and an overnight digest, peptide samples were prepared for MS analysis based on the previously generated MS spectral libraries. This strategy resulted in the identification of more than 30,000 peptides mapping to 3,309 proteins.

 

High-throughput identification of low abundance biomarkers

 

Tuberculosis peptides have typically been difficult to detect in plasma due to their low abundance. The streamlined approach described in this paper allows for rapid and sensitive high-throughput peptide identification from TB plasma samples. The new strategy successfully identified more than 400 differentially expressed plasma proteins in 50 minutes of MS acquisition, including known TB diagnostic proteins as well as novel proteins that had not previously been identified.

 

In summary, the strategy of combining DIA-MS analysis with high-throughput sequence library generation and an alternative protease digest methodology should prove to be a significant advancement in the proteomic profiling of clinical specimens for biomarker discovery.

 

Pall offers a wide range of high-throughput filtration products designed for analytical sample preparation. You can learn more about the AcroPrep filter plates including those used in this study on the Pall website.

 

References

 

 

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