Use of a recombinant biomarker protein DDA library increases DIA coverage of low abundance plasma proteins

Seong Beom Ahn*, Karthik S. Kamath, Abidali Mohamedali, Zainab Noor, Jemma X. Wu, Dana Pascovici, Subash Adhikari, Harish R. Cheruku, Gilles J. Guillemin, Matthew J. McKay, Edouard C. Nice, Mark S. Baker

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)


Credible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). In this proof-of-concept study, we employed a mixture of selected recombinant proteins in DDA libraries to subsequently identify (not quantify) cancer-associated low abundance plasma proteins using SWATH/DIA. The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 recombinant human proteins that had been previously implicated as possible cancer biomarkers from both our own and other studies. The rPSL was then used to identify proteins from nondepleted colorectal cancer (CRC) EDTA plasmas by SWATH-MS. Most (32/36) of the proteins used in the rPSL were reliably identified from CRC plasma samples, including 8 proteins (i.e., BTC, CXCL10, IL1B, IL6, ITGB6, TGFα, TNF, TP53) not previously detected using high-stringency protein inference MS according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and postdigestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 could be identified using DDA-MS. The 20 additional proteins exclusively identified using the rPSL SWATH approach were almost exclusively lower abundance (i.e., <10 ng/mL) proteins. To mitigate justified FDR concerns, and to replicate a more typical library creation approach, the DDA rPSL library was merged with a human plasma DDA library and SWATH identification repeated using such a merged library. The majority (33/36) of the low abundance plasma proteins added from the rPSL were still able to be identified using such a merged library when high-stringency HPP Guidelines v3.0 protein inference criteria were applied to our data set. The MS data set has been deposited to ProteomeXchange Consortium via the PRIDE partner repository (PXD022361).

Original languageEnglish
Pages (from-to)2374-2389
Number of pages16
JournalJournal of Proteome Research
Issue number5
Publication statusPublished - 7 May 2021


  • recombinant protein spectral DDA library (rPSL)
  • low abundance plasma protein identification
  • cancer biomarkers


Dive into the research topics of 'Use of a recombinant biomarker protein DDA library increases DIA coverage of low abundance plasma proteins'. Together they form a unique fingerprint.

Cite this