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

The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based 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 ∼60 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 ∼100 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. The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based 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 ∼60 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 ∼100 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. There has been tremendous interest in using advanced proteomics technologies to analyze human bodily fluids such as plasma and serum for the purpose of discovering and verifying new candidate protein biomarkers applicable to different diseases (1Hu S. Loo J.A. Wong D.T. Human body fluid proteome analysis.Proteomics. 2006; 6: 6326-6353Crossref PubMed Scopus (431) Google Scholar, 2Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1367) Google Scholar). These technologies are challenged to detect low abundance physiologically relevant proteins with extremely wide dynamic ranges in concentrations (i.e., more than 10 orders of magnitude for protein concentrations reported in human plasma) (3Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects.Mol. Cell. Proteomics. 2002; 1: 845-867Abstract Full Text Full Text PDF PubMed Scopus (3551) Google Scholar, 4Qian W.J. Jacobs J.M. Liu T. Camp II, D.G. Smith R.D. Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications.Mol. Cell. Proteomics. 2006; 5: 1727-1744Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Despite significant recent advances, current proteomics technologies still fall short of being able to reliably detect in blood plasma low ng/ml to sub-ng/ml protein concentrations, a level of detection often required for discovering disease-specific biomarkers (4Qian W.J. Jacobs J.M. Liu T. Camp II, D.G. Smith R.D. Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications.Mol. Cell. Proteomics. 2006; 5: 1727-1744Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Many different fractionation/separation techniques have been developed and applied in a multidimensional fashion to enhance detection of low abundance proteins in human biofluids (5Lee H.J. Lee E.Y. Kwon M.S. Paik Y.K. Biomarker discovery from the plasma proteome using multidimensional fractionation proteomics.Curr. Opin. Chem. Biol. 2006; 10: 42-49Crossref PubMed Scopus (98) Google Scholar, 6Wang H. Clouthier S.G. Galchev V. Misek D.E. Duffner U. Min C.K. Zhao R. Tra J. Omenn G.S. Ferrara J.L. Hanash S.M. Intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids.Mol. Cell. Proteomics. 2005; 4: 618-625Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). One of the most commonly applied strategies to potentially alleviate the “masking” effect created by the presence of the highly abundant proteins is to remove them (7Liu T. Qian W.J. Mottaz H.M. Gritsenko M.A. Norbeck A.D. Moore R.J. Purvine S.O. Camp II, D.G. Smith R.D. Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 2167-2174Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In the human plasma proteome, 22 proteins are responsible for 99% of the bulk mass of the total protein content in human plasma; this leaves perhaps hundreds of thousands of other proteins in only 1% of the protein mass (3Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects.Mol. Cell. Proteomics. 2002; 1: 845-867Abstract Full Text Full Text PDF PubMed Scopus (3551) Google Scholar). As a result, effective strategies for removing these 22 proteins (and possibly other medium abundance proteins) should greatly enhance detection of low abundance proteins within this important biofluid proteome. Multicomponent immunoaffinity separation strategies are increasingly being applied in various biomarker discovery applications to remove the abundant proteins and achieve comprehensive surveys of the biofluid proteomes. In such approaches, affinity-purified polyclonal antibodies typically immobilized by cross-linking on either chromatographic matrices or microbeads are used as immunoaffinity reagents to specifically remove abundant proteins. An optimized mixture of different antibody-immobilized beads for targeting a of proteins within the column for and of proteins. The of immunoaffinity separation was demonstrated by R. Anderson N.L. S. immunoaffinity subtraction an a comprehensive of the human plasma PubMed Scopus Google a for removing 10 abundance proteins in a single are for removing abundant proteins, including the N. J. J. techniques for protein 2005; 5: PubMed Scopus Google IgY12 system R. separation of plasma proteins by the of sample and analysis.Proteomics. 2005; 5: PubMed Scopus Google and that and human plasma proteins, respectively. These separation have been demonstrated to highly for removing the specifically proteins as as being both and (7Liu T. Qian W.J. Mottaz H.M. Gritsenko M.A. Norbeck A.D. Moore R.J. Purvine S.O. Camp II, D.G. Smith R.D. Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 2167-2174Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, T. of immunoaffinity of human serum 2005; 4: PubMed Scopus Google Scholar, J. T. S. efficiency and recovery of from a 2006; 6: PubMed Scopus Google Scholar). the current immunoaffinity technologies for to abundance proteins have coupling immunoaffinity with used macrophage colony-stimulating factor Protein matrix used macrophage colony-stimulating factor Protein matrix or LC-MS/MS analysis to plasma proteins at concentrations of ng/ml or a challenge (4Qian W.J. Jacobs J.M. Liu T. Camp II, D.G. Smith R.D. Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications.Mol. Cell. Proteomics. 2006; 5: 1727-1744Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). this challenge an effective fractionation to the dynamic range and detection of the low abundance proteins of In this we present a new immunoaffinity separation system for ability to enhance detection of low abundance proteins in human plasma. The new SuperMix system has been to applied in tandem with the IgY12 system for abundant proteins in to the most abundant proteins in plasma. we present results from this that illustrate the potential for enhanced detection of low abundance proteins as as the reproducibility of the SuperMix for LC-MS/MS plasma proteome The human blood plasma sample by the of was from a for the of this was from the of the of and the in with The protein was as by protein protein sample processing was at an immunoaffinity column with a mixture of antibodies that to abundant proteins in human plasma, a plasma sample was of the abundance proteins using an IgY12 The flow-through medium or low abundance proteins was used as a mixture of for and a mixture of polyclonal The flow-through was used as and to for an which was used to the antibodies from the total from the with the fraction. The mixture of antibodies from the column was to and into an immunoaffinity column the SuperMix The plasma samples were to the separation of abundance proteins and using a IgY12 column with a column of of plasma using an The were in these separations, which were to (7Liu T. Qian W.J. Mottaz H.M. Gritsenko M.A. Norbeck A.D. Moore R.J. Purvine S.O. Camp II, D.G. Smith R.D. Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 2167-2174Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The three were 10 100 100 in a separation that of sample by a for a total of The flow-through and were the IgY12 separations, the flow-through were in mass by to Protein was by protein The IgY12 flow-through were using a SuperMix immunoaffinity column on the The separation using the SuperMix column were to that for the IgY12 separation (7Liu T. Qian W.J. Mottaz H.M. Gritsenko M.A. Norbeck A.D. Moore R.J. Purvine S.O. Camp II, D.G. Smith R.D. Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 2167-2174Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). the flow-through and from the SuperMix column were and as with to and protein was using the protein the of differential protein six non-human standard proteins were into three human plasma samples at and concentrations, respectively. The six proteins into plasma were and plasma sample with protein was to tandem IgY12-SuperMix immunoaffinity separations with of plasma for the SuperMix flow-through and for of the were as The protein samples from IgY12 SuperMix and SuperMix were and in 10 for 1 at Protein were with for at The protein mixture was with was at a of The sample was at for The sample was a column and with of were from the column with 1 of and was by protein samples were at of peptides from the IgY12 flow-through and SuperMix flow-through were in of 10 and by on a A column by a column using an system at a of used were 10 and was the column and with A for 10 were by a to by a from to 10 was for A total of were with being to LC-MS/MS samples were analyzed using a system to a mass an The column was by into a The of in and in of peptides the the was at A for was by the from to the the mass was in a in which a was by 10 The 10 most were selected in the of to and to using a of and a dynamic of 1 The was at the was at The LC-MS/MS were into a by in and the was used to independently the the human Protein with a total of total protein The used were as for and 1 for with and a of three of and dynamic of were used the are a in proteomics we developed and applied a of based on the for the to to at the level as W.J. Liu T. Jacobs J.M. Camp D.G. Smith R.D. of and protein from tandem mass and the human 2005; 4: PubMed Scopus Google Scholar, T. Qian W.J. Gritsenko M.A. S.M. Moore R.J. Purvine S.O. Camp II, D.G. Smith R.D. High dynamic range of the plasma Cell. Proteomics. 2006; 5: Full Text Full Text PDF PubMed Scopus Google Scholar). The human protein was created by the of the for and the for was by the of peptides identified from the by the of peptides identified from the normal the and with for the data. A of was observed at the level following such for this used for in a new was used as a to a of proteins or protein R. A for proteins by tandem mass Chem. PubMed Scopus Google Scholar). that the were the of 1 and into the for to a of One protein was selected to protein that of a of proteins or protein with two or more were as protein The plasma protein concentrations for macrophage colony-stimulating factor 1 and matrix were in using ELISA following the sample was analyzed at three different to the The used to the SuperMix column is in The the SuperMix immunoaffinity separation is that antibodies such as the proteins present in human plasma. the to the abundance and the of the proteins N.G. Anderson N.L. techniques for and PubMed Scopus Google the is that abundant proteins in human plasma to than abundance proteins. As a result, a mixture of these antibodies used to a of medium abundance proteins especially applied in tandem with a abundance protein strategy such as the SuperMix column antibodies medium abundance proteins from the plasma it applied in tandem with the IgY12 column as a separation strategy to significantly low abundance proteins by a of or medium abundance proteins the IgY12 and SuperMix 1 and for the separation for ∼100 of total proteins is to from of plasma As separations and sample processing are for quantitative applications in clinical we the reproducibility of tandem IgY12-SuperMix separations the reproducibility based on in terms of sample for of separation and sample of proteins in the IgY12 and SuperMix and SuperMix as as the peptides in the SuperMix flow-through are the reproducibility of both tandem separations and and protein The for peptides in the SuperMix flow-through is than the protein level of from the following for tandem IgY12 and SuperMix of plasma was used with the total protein of are as the of proteins or peptides with the of with the standard flow-through of proteins or peptides for of the IgY12 flow-through was used for SuperMix flow-through of the IgY12 flow-through was used for SuperMix flow-through of the IgY12 flow-through was used for SuperMix of plasma was used with the total protein of are as the of proteins or peptides with the of with the standard The of proteins or peptides for of the IgY12 flow-through was used for SuperMix in a new the three of samples from the IgY12 SuperMix and SuperMix were selected for LC-MS/MS An of proteins were identified from of IgY12 SuperMix and SuperMix respectively. The of that identified a protein was used to (7Liu T. Qian W.J. Mottaz H.M. Gritsenko M.A. Norbeck A.D. Moore R.J. Purvine S.O. Camp II, D.G. Smith R.D. Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 2167-2174Abstract Full Text Full Text PDF PubMed Scopus (192) Google the reproducibility count has been demonstrated as a for samples as as for protein abundances within a sample H. A for and of protein abundance in Chem. PubMed Scopus Google Scholar, W.J. Jacobs J.M. Camp II, D.G. Moore R.J. Gritsenko M.A. Smith R.D. proteome of human plasma following in using multidimensional separations with tandem mass 2005; 5: PubMed Scopus Google Scholar, of abundance from and for quantitative analysis using Chem. 2005; PubMed Scopus Google Scholar). the of spectral to identified proteins for of IgY12 flow-through samples and SuperMix flow-through samples reproducibility was observed for both SuperMix and IgY12 flow-through suggesting that the reproducibility of the tandem IgY12-SuperMix separations is to that of the single IgY12 A standard using six non-human standard proteins into the plasma at three different and was to the ability for of protein abundances the tandem separation immunoaffinity separations, both the SuperMix flow-through and from the three plasma samples were analyzed using LC-MS/MS to spectral count for The proteins were in the SuperMix for which was observed at the level in the with an spectral count of The spectral count for the six standard proteins as in the flow-through samples are in As spectral count within the were observed for six proteins at of the three levels with the of which was at the level These results illustrate the reproducibility of the tandem separations with LC-MS/MS as as the in that of six proteins were reliably at the 1 level with single LC-MS/MS The ability of this separation to abundance is demonstrated based on the observed increase in spectral with the increase of protein six proteins, the abundance between two levels were observed to significant based on analysis of the the tandem IgY12-SuperMix separations with IgY12 separations using both and LC-MS/MS to the potential in detection of low abundance proteins. LC-MS/MS IgY12 and SuperMix flow-through samples from three separations were LC-MS/MS, was used to the of peptides from IgY12 and SuperMix flow-through samples into to proteins identified with two peptides from the were as a side-by-side comparison of proteome coverage for IgY12 and SuperMix flow-through samples in and LC-MS/MS LC-MS/MS of SuperMix flow-through an in proteome coverage with an in proteome coverage was using SuperMix for LC-MS/MS with IgY12 that the SuperMix in proteome coverage was significantly only a separation was In LC-MS/MS a of low ng/ml level proteins on concentrations reported in the were identified in the SuperMix flow-through proteins observed in the IgY12 flow-through were in the low to ng/ml such as factor and which is in with recent (4Qian W.J. Jacobs J.M. Liu T. Camp II, D.G. Smith R.D. Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications.Mol. Cell. Proteomics. 2006; 5: 1727-1744Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The of proteins and peptides identified using SuperMix and IgY12 are as 1 and with spectral count The enhanced detection of low abundance proteins using SuperMix is in which protein concentrations in the range of pg/ml to 100 ng/ml on for low abundance proteins in the flow-through analyzed by The concentrations of a and in the plasma sample were validated as 202 pg/ml and 12.4 ng/ml, based on by the protein was confidently identified by different peptides as in In comparison only of the proteins were observed in the IgY12 The results in and that the of tandem IgY12-SuperMix separations significantly the detection of low abundance proteins and the proteome this tandem separation strategy with LC-MS/MS has identification of a of physiologically relevant such as and and on the that were identified in this of which only three were observed in the IgY12 identification for two selected low abundance proteins reported to at sub-ng/ml in a new selected of or identified from LC-MS/MS of IgY12 and SuperMix flow-through and protein colony-stimulating factor factor factor factor factor in a new The observed significant in proteome coverage by the SuperMix separation is of binding of a of abundant proteins to the to the enrichment of abundance proteins. As a result, we the binding efficiency of the SuperMix column for abundance proteins. The binding efficiency was on the of observed spectral from LC-MS/MS of SuperMix and flow-through quantitative the and flow-through samples are different in this still to proteins that are efficiently captured by the column either or a minimum of spectral should observed in the 45 medium abundance proteins that were captured with >90% efficiency by the SuperMix proteins were captured with the SuperMix column a mixture of antibodies that were on the of the of medium abundance proteins that were present in the plasma that the of antibodies to low binding for of the proteins. this we the SuperMix column with the of sample flow-through from of of the SuperMix column significantly the total of proteins identified in the flow-through proteins proteins in The of proteins efficiently captured by the SuperMix column was from 45 to These results that of the SuperMix column enhance detection of low abundance proteins as a result of more abundant of abundant proteins that to the SuperMix column with efficiency is used to the for a protein to captured by the SuperMix it was for protein by the spectral count for protein from the by the of spectral from the and flow-through The efficiency for proteins with only a spectral count was observed in the it a by the these proteins are as factor factor factor of of to protein protein factor protein factor protein factor protein protein The efficiency is used to the for a protein to captured by the SuperMix it was for protein by the spectral count for protein from the by the of spectral from the and flow-through The efficiency for proteins with only a spectral count was observed in the it a by the these proteins are as in a new abundant proteins were efficiently by the SuperMix column of These proteins proteins, and of which were captured with efficiency abundances based on spectral count were the proteins present in the plasma. efficiency are in One of the challenges for advanced MS-based proteomics to clinical biomarker discovery the ability to effectively detect low abundance proteins in Many different fractionation/separation techniques have been developed and applied in a multidimensional fashion at both the protein and levels to this dynamic range challenge (5Lee H.J. Lee E.Y. Kwon M.S. Paik Y.K. Biomarker discovery from the plasma proteome using multidimensional fractionation proteomics.Curr. Opin. Chem. Biol. 2006; 10: 42-49Crossref PubMed Scopus (98) Google Scholar, H. Hanash S. based sample strategies for proteome analysis in with mass 2005; 24: PubMed Scopus Google Scholar, S. liquid separation of proteins by chromatographic and of the human serum proteome: and protein Cell. Proteomics. 2006; 5: Full Text Full Text PDF PubMed Scopus Google Scholar). of the separation and fractionation of enrichment of a of low abundance proteins to achieve with antibodies that abundant proteins is a commonly used for human biofluid proteome profiling N. J. J. techniques for protein 2005; 5: PubMed Scopus Google Scholar, R. separation of plasma proteins by the of sample and analysis.Proteomics. 2005; 5: PubMed Scopus Google Scholar). In immunoaffinity separations an effective strategy for abundance from low abundance proteins in a highly fashion (7Liu T. Qian W.J. Mottaz H.M. Gritsenko M.A. Norbeck A.D. Moore R.J. Purvine S.O. Camp II, D.G. Smith R.D. Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 2167-2174Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, J. T. S. efficiency and recovery of from a 2006; 6: PubMed Scopus Google the effect of abundance proteins. in recent immunoaffinity separation strategies have been to the of antibodies that on the column to In this we a novel tandem IgY12-SuperMix immunoaffinity separation strategy that effective binding of or medium abundance proteins the IgY12-SuperMix of abundant proteins from low abundance proteins has to significant as by LC-MS/MS in plasma proteome coverage and detection has identification of a of low proteins, including and from a normal human and of the has been to present at pg/ml based on ELISA These results that tandem IgY12-SuperMix immunoaffinity separations with reproducibility an effective to into biofluid proteomes for quantitative clinical other fractionation that typically the immunoaffinity separations only two flow-through and the are a significant in the separations on an system with only a increase in sample processing of the of and immunoaffinity typically more than 100 separations per the of of a biological typically using a single One of the in immunoaffinity is the of demonstrated that the of or binding to a column is for a protein (7Liu T. Qian W.J. Mottaz H.M. Gritsenko M.A. Norbeck A.D. Moore R.J. Purvine S.O. Camp II, D.G. Smith R.D. Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 2167-2174Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). we of binding on the SuperMix column in this we the most of the SuperMix column as a such that both the and flow-through are analyzed to achieve more proteome profiling of using it as a for removing and medium abundance proteins. The observed reproducibility by both and flow-through the for quantitative proteomics The ability to abundance the tandem separations was demonstrated in standard protein spiking The spiking low binding as only protein of six proteins was in the at the A potential of the SuperMix column is that it an mixture of antibodies that are present at various on proteins to the the SuperMix column should as a fractionation with both the and flow-through analyzed for of potential abundance between The protein binding to the column the abundance on the of binding the of these abundance for a protein present at 1 in sample and at in sample (i.e., a and for a column that has an to of the protein for both a between the two samples observed following the SuperMix the reproducibility of the SuperMix separations, such in should have a effect on ability to significant abundance for candidate to in that observed of protein the quantitative in samples is protein of the SuperMix column is based on from human plasma, the of antibodies for protein on the of the proteins in the In we observed abundance proteins that were effectively by the SuperMix antibodies of these proteins developed and to enhance the of the SuperMix antibodies are of the sample more proteins to effectively the proteome detection of low abundance proteins, and ability for the of tandem IgY12-SuperMix immunoaffinity separations for clinical biomarker discovery applications with human These tandem separations with either or with or that this tandem IgY12-SuperMix strategy significantly enhance the of in the for candidate biomarker H. T. Carr S.A. for low abundance proteins in plasma by mass and Cell. Proteomics. 6: Full Text Full Text PDF PubMed Scopus Google Scholar, mass for plasma Cell. Proteomics. 2006; 5: Full Text Full Text PDF PubMed Scopus Google Scholar, J. V. R. R. High detection of plasma proteins by of Cell. Proteomics. 6: Full Text Full Text PDF PubMed Scopus Google Scholar). The was in the a by the of and at which is by for the with