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Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer
Published on: August 2, 2018
An efficient method for native protein purification in the selected range from prostate cancer tissue digests
Rumana Ahmad1, Carrie D Nicora2, Anil K Shukla2
1Department of Urology, University of Washington, Seattle, WA 98195, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA. rumana_ahmad@yahoo.co.in.
Researchers developed a new method to isolate native prostate cancer proteins for biomarker discovery. This approach purifies proteins directly from tumor tissue, yielding anterior gradient 2 (AGR2) and avoiding common contaminants, facilitating antibody development for cancer diagnostics.
Area of Science:
- Biochemistry
- Proteomics
- Cancer Biology
Background:
- Prostate cancer (CP) cells exhibit distinct gene expression profiles compared to normal cells.
- Secreted or extracellular proteins with elevated expression in CP are potential diagnostic biomarkers.
- Developing clinical assays requires high-quality monoclonal antibodies, ideally generated against native proteins.
Purpose of the Study:
- To develop an efficient method for isolating native proteins from prostate cancer tissue for use as immunogens.
- To overcome limitations of recombinant protein synthesis and improve antibody generation for biomarker discovery.
Main Methods:
- Prostate cancer tissue specimens were processed to obtain single cells.
- Proteins in the 10-30 kDa molecular weight range were isolated using sonication, dialysis, and ultrafiltration.
- Western blotting and mass spectrometry (MS) proteomics identified proteins within the selected fraction.
Main Results:
- Anterior gradient 2 (AGR2), a cancer-specific protein, was identified.
- The absence of prostate-specific antigen (PSA)/KLK3 was confirmed.
- Proteomics analysis detected AGR2 and other proteins, including some outside the target molecular weight range.
Conclusions:
- The optimized isolation method effectively removed contaminants like collagenase, ACPP/PAP, and AZGP1, preventing unwanted antibody responses.
- This technique provides a rapid and convenient way to isolate native proteins within specific size ranges for immunogen preparation.
- The study presents a valuable scheme for generating specific antibodies against native cancer biomarkers.
