Enhanced Detection of Low Abundance Human Plasma Proteins Using a Tandem IgY12-SuperMix Immunoaffinity Separation Strategy

Abstract

The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MSbased proteomics technologies as it makes it especially difficult to detect low abundance proteins in human biofluids such as blood plasma, which is an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low abundance proteins in human plasma. The tandem IgY12-SuperMix system separates ϳ60 abundant proteins from the low abundance proteins in plasma, allowing for significant enrichment of low abundance plasma proteins in the SuperMix flow-through fraction. High reproducibility of the tandem separations was observed in terms of both sample processing recovery and LC-MS/MS identification results based on spectral count data. The ability to quantitatively measure differential protein abundances following application of the tandem separations was demonstrated by spiking six non-human standard proteins at three different levels into plasma. A side-by-side comparison between the SuperMix flow-through and IgY12 flow-through samples analyzed by both one-and two-dimensional LC-MS/MS revealed a 60 -80% increase in proteome coverage as a result of the SuperMix separations, suggesting significantly enhanced detection of low abundance proteins. A total of 695 plasma proteins were confidently identified in a single analysis (with a minimum of two peptides per protein) by coupling the tandem separation strategy with two-dimensional LC-MS/MS, including 42 proteins with reported normal concentrations of ϳ100 pg/ml to 100 ng/ml. The concentrations of two selected proteins, macrophage colony-stimulating factor 1 and matrix metalloproteinase-8, were independently validated by ELISA as 202 pg/ml and 12.4 ng/ml, respectively. Evaluation of binding efficiency revealed that 45 medium abundance proteins were efficiently captured by the SuperMix column with >90% retention. Taken together, these results illustrate the potential broad utilities of this tandem IgY12-SuperMix strategy for proteomics applications involving human biofluids where effectively addressing the dynamic range challenge of the specimen is imperative.

Advances in purification and separation of posttranslationally modified proteins

“…Further, moderately abundant proteins can be further depleted by antibodies obtained from immunizing chickens with plasma proteins from which the highly-abundant proteins have been removed. This two-step procedure theoretically provides a fraction of low-abundance proteins (in the flow-through) and one of moderately abundant proteins (in the eluted fraction) [40]. Of course, this approach has limits and the price of mixed antibody columns is quite considerable.…”

Alterations in Protein Regulators of Neurodevelopment in the Cerebrospinal Fluid of Infants with Posthemorrhagic Hydrocephalus of Prematurity

“…The samples were run through an autosampler at 4°C during MAF. Albumin, IgG, IgA, IgM, ␣ 1 -antitrypsin, transferrin, haptoglobin, apolipoprotein A1, apolipoprotein A2, fibrinogen, ␣ 1 -acid glycoprotein, ␣ 2 -macroglobulin, complement C3 and LD were removed with the first column (IgY-14) and the second in-line column (Supermix LC5 (12.7 ϫ 39.5 mm), Sigma) removed the moderately abundant plasma proteins (14). The LC configuration (supplemental Fig.…”

“…From Preterm Infants with PHH-Our first goal was to determine if a multi-affinity approach developed for abundant proteins in plasma (14) would enable us to reliably identify and quantify brain proteins in blood-containing CSF. We used two types of MAF columns, IgY-14 and Supermix, assembled in series to remove high-and moderate-abundance plasma proteins, respectively, from CSF.…”

“…S1), we analyzed the peptides from the unretained proteins using LC-MS. We identified 438 pro-teins after searching the human protein database, using Protein Prophet algorithm (supplemental Table S1). After removal of hemoglobin, plasma proteins that are efficiently removed by the two MAF columns (14), and contaminant proteins (e.g. keratin), we uploaded the proteins into Ingenuity Pathway software (IPA) for knowledge-based software analysis.…”

Impact of the depletion of high-abundance proteins in blood plasma/serum on the proteome of these media in growing farm animals

“…Utilization of immobilized antibodies enables selective isolation of target molecules from complex mixtures (21). Quian et al (22) depleted human serum/plasma proteins with chicken IgY antibodies. Immunoaffinity-based depletion methods are rarely used to prepare samples from animal body fluids for 2DE because commercially available kits are predominantly dedicated to human or laboratory animals serum/plasma proteins (23).…”