Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

SWATH library construction including recombinant proteins allows identification and quantification of lower abundance human plasma cancer biomarkers (#421)

Seong Beom Ahn 1 , Karthik Kamath 2 , Abidali Mohamedali 3 , Zainab Noor 3 , Dana Pascovici 2 , Jemma Wu 2 , Subash Adhikari 1 , Matthew J. McKay 2 , Mark S. Baker 1
  1. Department of Biomedical Sciences, Macquarie University, North Ryde, NSW, Australia
  2. Australian Proteome Analysis Facility, Macquarie University, North Ryde, NSW, Australia
  3. Department of Molecular Sciences , Macquarie University, North Ryde, NSW, Australia

Human plasma is the most informative, accessible biofluid for assessing the status of human health. However, detection and quantification of low abundance cancer-related proteins (e.g., CEA, cytokines, shed proteins) remains one of the principal challenges in proteomic biomarker discovery - due to high abundance plasma proteins obscuring biomarkers. Antibody technologies suffer from batch variation and non-specific detection, therefore, quantitative MS techniques such as SWATH-MS and similar DIA methods are an attractive alternative to assess plasma cancer biomarkers. These approaches rely on prior library construction using IDA/DDA data and the challenge of detecting low abundance biomarkers can be hampered by the same dynamic range issues which limit the proteome discovery space for plasma proteomics.

Here, we report on the use of a SWATH mini-library comprised of 32 previously-reported cancer biomarkers for the quantitative assessment of non-depleted pooled human plasma cohorts from clinically-staged (20 healthy or 20 stages I-IV) CRC patients by SWATH-MS. To ensure validity, we employed two independent SWATH analysis software (Skyline and PeakView) to identify quantifiable peptides. Of the 32 cancer biomarkers used to construct the SWATH mini-library, we reliably identified and quantified 25 proteins in human plasma of CRC patients. In all cases, a significantly higher peptide count for each protein allowed better quantification (e.g., 12 for CEA, 8 for IL-6) compared to prior results. In CRC, CEA expression was significantly upregulated, recapitulating many prior studies. Similarly, we recapitulated our observation that plasma ADAMDEC1 is upregulated in early-stage CRC. We propose the use of expanded recombinant protein SWATH libraries for the discovery of diagnostic, prognostic and theranostic protein biosignatures for cancers and other diseases.