Blood plasma represents an important minimally invasive human biopsy to diagnose and study diseases, and follow therapeutic interventions. Although the plasma has a high protein content, achieving high coverage of the plasma proteome is challenged by the dynamic range of protein abundances from 20-30 mg/ml to few pg/ml. The PreOmics ENRICH-iST technology is an on-bead protein enrichment/digestion protocol, that reduces the dynamic range in plasma by unspecifically enriching low abundant proteins.
Two plasma studies including patients diagnosed with non-small cell lung cancer (NSCLC, N=10) patients and colorectal cancer (CC, N=8) as well as equal N of matched healthy control were prepared with the ENRICH-iST kit (PreOMics). The NSCLC study was also prepared with the iST protein digestion kit (PreOmics) to test if the enrichment step distorts protein abundances between the two study groups. The NSCLC study was separated on a nanoElute HPLC system equipped a Bruker Xtreme column using a 22 min gradient. Eluting peptides were detected on a TimsTOF HT operated in dia-PASEF mode. Data were analyzed with Spectronaut 18 in directDIA+ mode against the human fasta database. CC samples were analyzed on a nanoElute system equipped with a IonOpticks column (Aurora3, 25cm) using a 60 min separation gradient.
ENRICH-iST sample from the NSCLC study reported 1450 protein groups, whereas only 650 proteins were observed in the comparable neat plasma sample, applying a sample specific spectral library, more than 2000 proteins were observed in the ENRICH plasma. In the CC study over 3500 proteins were observed in the Enrich plasma samples, in comparison to 910 proteins in the neat plasma sample. The variation of protein abundances from proteins found in ENRICH plasma from all healthy patients in the NSCLC study was reported to be around 20%, demonstrating high reproducibility. Statistical analysis for significantly enriched protein showed a high overlap between neat and ENRICH plasma from NSCLC samples. The ENRICH plasma sample reported 21 significantly upregulated proteins in cancer samples, of which 7 were also reported in neat plasma.
ENRICH-iST sample preparation was optimized and evaluated for high plasma proteome coverage in cancer studies.