IgY14 and SuperMix immunoaffinity separations coupled with liquid chromatography–mass spectrometry for human plasma proteomics biomarker discovery

Abstract

Interest in the application of advanced proteomics technologies to human blood plasma- or serum-based clinical samples for the purpose of discovering disease biomarkers continues to grow; however, the enormous dynamic range of protein concentrations in these types of samples (often >10 orders of magnitude) represents a significant analytical challenge, particularly for detecting low-abundance candidate biomarkers. In response, immunoaffinity separation methods for depleting multiple high- and moderate-abundance proteins have become key tools for enriching low-abundance proteins and enhancing detection of these proteins in plasma proteomics. Herein, we describe IgY14 and tandem IgY14-Supermix separation methods for removing 14 high-abundance and up to 60 moderate-abundance proteins, respectively, from human blood plasma and highlight their utility when combined with liquid chromatography-tandem mass spectrometry for interrogating the human plasma proteome.

Precise quantitation of 136 urinary proteins by LC/MRM-MS using stable isotope labeled peptides as internal standards for biomarker discovery and/or verification studies

“…Because screening multiple markers confers greater accuracy in patient diagnostics than using a single biomarker alone [47], verification of this type of extended panel signifies a marked improvement in non-invasive pre-clinical testing at the discovery and verification stages of the protein biomarker pipeline [4]. Furthermore, as evidenced by our quantified panel (see Supplemental Table 2), the less complex urine matrix enabled lower-abundance plasma proteins (e.g., macrophage colony-stimulating factor 1, P09603 [48]; osteopontin, P10451 [49]; protein DJ-1, Q99497 [50]), to be detected without multidimensional LC fractionation [51] or immunoaffinity-based depletion [52]. The cause is attributed to the lower concentration range of proteins in urine (approximately 6 orders of magnitude [53,54] vs. >10 in plasma [55]), which allows the high abundance plasma proteins to be quantified at significantly lower levels in urine.…”

Clinical translation of MS-based, quantitative plasma proteomics: status, challenges, requirements, and potential

“…One strategy for improving the sensitivity of detection involves removing a subset of the top 22 proteins through immunoaffinity depletion [39][40][41][42]. In one example, differentially expressed proteins from discovery experiments were quantified in a plasma pancreatic cancer cohort (n = 60) using a bottom-up LC-MRM/MS method that involved up-front depletion of albumin and immunoglobulin G (IgG, via a ProteoPrep ® kit, Sigma-Aldrich) [43].…”

Contributions of immunoaffinity chromatography to deep proteome profiling of human biofluids

“…Compared to spin columns, the LC column-based products utilizing automated LC systems provide a number of advantages in effective removal of targeted proteins, such as minimal carryover, good reproducibility, and minimal nonspecific binding [38]. Besides these single-stage depletion systems, an IgY-based SuperMix depletion column has been developed to enable the removal of ~50 MAPs by applying it with IgY12 or IgY14 column in tandem to further enrich LAPs prior to follow-up analysis [21, 39]. In our experience, a typical LC depletion column will offer reproducible depletion for 100–200 biological samples with a shelf life for several years, which provides a great potential for large-scale biomarker discovery and verification studies.…”

“…The IgY12 system was later improved to IgY14 for removing 14 HAPs in human plasma and the product is now commercialized as Seppro® IgY14 from Sigma Aldrich [39, 60–62]. Both IgY14 and MARS Hu-14 are very popular depletion products for proteomics applications since the performance characteristics of MARS Hu-14 and IgY14 are very comparable with both products offering options of multiple loading capacities (customization options available as well) [63]; however, the IgY antibodies appeared to display the least nonspecific binding [64].…”

“…The concept of a SuperMix column was later developed to be applied in tandem with IgY12 or IgY14 so that the detection of LAPs can be further enhanced by depleting an additional number of MAPs [21, 39, 71]. The SuperMix column was developed by immunizing chickens with the protein mixture from IgY14-depleted human blood plasma as mixed antigens, and the purified antibodies were further immobilized and packed into the SuperMix column to target a relatively large number of MAPs [21].…”