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.

Figure 1

The HAP and MAP distributions in the human blood plasma proteome. The top 14 HAP are targeted by the IgY14 column (A) and ~50 MAP are targeted by the SuperMix column. The percentage of protein abundances are based on spectral count data from LC-MS/MS experiments. In this context, HAP are defined as 14 proteins captured by the IgY14 column, and MAP are those ~50 proteins captured by the SuperMix column as listed in Table 1 except C3. All other proteins are considered as LAP.

Figure 2

The tandem IgY14-SuperMix immunoaffinity separation strategy. The SuperMix column is generated by using the protein mixture from IgY14-depleted human blood plasma as mixed antigens. For the tandem separations, plasma or other biofluid samples are initially separated by IgY14 column. The flow-through from IgY14 column is further separated by the SuperMix column into the flow-through and eluted fraction. The typical recoveries are indicated assuming ~150 μL plasma is loaded.

Figure 3

The IgY14-SuperMix separation strategy with the tandem connection mode. (A) IgY14 and SuperMix columns are connected to ports 1 and 2 of a six-port valve. Port 1 is connected to port 2 when human blood plasma sample is injected into the columns. The flow-through is collected as SuperMix flow-through (SuperMix_FT). (B) Port 1 is connected to port 6 following (A), and the elution buffer is injected into the IgY14 column. The eluted sample is collected as IgY14 bound (IgY14_B). (C) After IgY14_B collection, port 1 is again connected to port 2. Elution buffer is sent through the SuperMix column and the eluted SuperMix bound fraction (SuperMix_B) is collected. (D) An example of LC chromatograms of 3 QC experiments of the tandem IgY14-SuperMix separations during the initial, middle and late stage of the column life.

Figure 4

The 40 low-abundance plasma proteins detected in 2D-LC-MS/MS analyses of SuperMix flow-through. The protein concentration is plotted on a log scale. All proteins were reported to have a normal concentration <100 ng/mL; only 21 detected by IgY-12. The protein concentrations of the two highlighted proteins, M-CSF and MMP8, were validated by ELISA assays for the same plasma sample.

Figure 5

Reproducibility of the tandem IgY12-SuperMix separations evaluated by LC-MS/MS. Reproducibility of three replicates of the tandem IgY12-SuperMix separations were assessed based on spectral count information of identified proteins from individual LC-MS/MS.