Sample Preparation : Combining specific affinity ligands with combinatorial libraries for improved d

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Sample Preparation Combining specific affinity
ligands with combinatorial libraries for improved
detection of trace proteins
Julia Tait Lathrop1 and David Hammond2 1American
Red Cross Holland Lab and 2Prolias
Technologies HUPO 5th International Congress,
Long Beach, CA Oct 30- Nov 2, 2006
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Proteins Measured Clinically in Plasma Span gt 10
Orders of Magnitude in Abundance- intractable to
analysis
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(Human male 50yr - from Specialty Laboratories
Books)
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Dynamic range of 2-D gels or LC/MS
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Normal Range Abundances Log10 Concentration in
pg/mL
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Total Cost 10,695 per sample (clinical assays)
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4
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The human plasma proteome History, character,
and diagnostic prospects. Anderson, N.L. and
Anderson, N.G., Molecular and Cellular
Proteomics, 1.11, 845-867 (2002)
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Strategies for Managing the Dynamic Range

• Immunoaffinity depletion
• Removes abundant, bound proteins and active
  antibodies
• Enrichment
• Digest proteins and analyze peptides- shot-gun
  proteomics
• Combination methods solid-phase
  microextraction/multistep elution/CE/tandem MS
• Analysis of specific sub-proteomes, classes of
  proteins

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FIoNA Libraries (including the Equalizer)An
Addition to the Proteomic Toolbox

• Decrease the concentration range of proteins in
  complex mixtures like plasma, serum, whole blood
• Enrich trace proteins, decrease concentration of
  abundant proteins
• Maintain concentration differentials of an
  analyte across samples
• No specific depletion of proteins
• Based on a combinatorial ligand library
  synthesized on custom-designed beads

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FIoNA Library
Technology Applications
2007
2006
2005
2004
2003
2002
2001
Polymer selection
Starting materials
Ligand libraries
Natural, unnatural, specialized and (tyrosine
phosphonates) and amino acid, analogs
Nucleic acids, carbohydrates, synthetic drugs
Base resin, linkers, spacers
Whole blood, plasma, environmental samples, etc.
Combinatorial Library Fractionation of Proteins
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FIoNA Library
Technology Applications
Microfluidics/ Arrays
Impurity product profiling
Prion reduction device CE marked
2007
Myocardial Infarction
Activity
Cytokines
Melanoma
2006
Therapeutic apheresis
ELISA
Pathogens, viruses
Live cell
Equalizer
OPases
Analyte recovery
Western blot
Dead cell
Capillaries
Specific protein reduction
Proteinases
2005
Formats
Enzymes
Protein Purification
Sample
Engineered Proteins
2004
FACS
Diagnostics
Cell based
Ligand identification
Proteins
Whole Organisms
characterization
2003
FIo
Drug/protein, protein/ protein interactions/ bioma
rkers
NA
Blot
2002
Bead
2001
Polymer selection
Starting materials
Ligand libraries
Natural, unnatural, specialized and (tyrosine
phosphonates) and amino acid, analogs
Nucleic acids, carbohydrates, synthetic drugs
Base resin, linkers, spacers
Whole blood, plasma, environmental samples, etc.
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Overview

• Basis of the Technology
• Use with Blood
• Sequestering highly interactive proteins

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Overview

• Basis of the Technology
• Use with Blood
• Sequestering highly interactive proteins

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FIoNA Library Ligands

• Peptide ligands containing 1-6 amino acids are
  synthesized onto chromatography resins- beads
• Combinatorial synthesis involves sequentially
  coupling amino acids to the beads, forming
  ligands of defined length
• This results in a population of millions of
  beads, or a library, each bead bearing many
  copies of a different ligand sequence

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Synthesis of Combinatorial Library
Diversity- function of aa and length of ligand-
186 34 x 106
O
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Sample Processing
Collect whole blood, plasma, environmental
samples etc.
Bind proteins to ligands on beads
Remove majority of abundant protein, blood cells
Elute sample enriched for trace proteins
Plasma Derivatives Holland Laboratory
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ProteinLigand Interactions Incubation of
library with AP-labeled polyclonal antibodies
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Sample Preparation
Elute sample enriched in trace proteins
Plasma Derivatives Holland Laboratory
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Moderation of the Concentration Range Human
Plasma Proteins Bound to Library
Abundant proteins found in starting material (2
9), non- (3 8) and weakly bound (4 7)
samples. More diversity found in (5) without,
and (6) plus, antibodies
Thulasiraman et al, 2005
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Overview

• Basis of the Technology
• Use with Blood
• Sequestering highly interactive proteins

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Moderation of the Concentration Range Whole
Blood and Human Plasma
Blood Plasma
1 2 3 4 5 6 7 8

• Whole blood before contact
• Whole blood non-bound (FT)
• Plasma of 2.
• Bound proteins from whole blood
• Bound proteins from plasma
• Plasma non-bound (FT)
• Plasma before contact
• MWM

188
98
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Albumin
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38
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Hemo- globin
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Effect of Anti-coagulants and Thrombin Inhibitors
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Hemocompatibility of Base Resin with Whole Blood
Also, no evidence of hemolysis, clogging, no
effect on red cell stability
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Enrichment of Trace Analytes
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(No Transcript)
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Enhanced Detection of Analytes in Diagnostic
Assays
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Overview

• Basis of the Technology
• Use with Blood
• Sequestering highly interactive proteins

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Over-representation of Highly Interactive Proteins
Flow Through
Plasma
LDS Eluates 1,2,3
188 92 62 49 38 28 17
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Sequestering Highly Interactive Proteins
Concept

• Sequester highly interactive proteins on specific
  affinity ligands
• co-incubated with library so complexes are
  available
• Push equilibrium toward affinity resin
• Free up ligands for trace proteins, improve their
  detection by sensitive methods
• Identify affinity ligands using FIoNA libraries
  in the Bead Blot to specifically sequester
  proteins
• Faster than antibodies, more robust
• Selected ligands for commercial purification
  processes
• Ligands biocompatible

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Sequestering Highly Interactive Proteins
Approaches
Affinity resin
library
Co-incubated, separated by compartments, mesh
Affinity ligand on magnetic beads
Affinity ligand on dipstick
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Sequestering of Fibrinogen Co-incubation with
Fibrinogen Affinity Resin
1 2 3 4 5 6 7
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1 MWM 2 Plasma 3 FT, Library alone 4 FT,
Library CG1358 5 FT, CG1358 alone GuHCl
eluates 6 Library alone- GuHCl 7 Library
CG1358 8 CG1358
188 92 62 49 38 28 17
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Sequestering Co-incubation with Fibrinogen and
HDL Resins
1 MWM Library eluates 2Plasma alone 3
library alone 4 Library CG1358 5 Library
CG1358HDL lig Affinity resin eluates 6
CG1358 7 CG1358 HDL lig
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Summary

• Use of libraries of combinatorial ligands to
  decrease concentration range, enrich trace
  proteins
• Libraries are compatible with and can be used
  with whole blood to speed sample handling,
  decrease artifacts, improve cost effectiveness
• Sequestering highly interactive proteins may
  further improve detection of trace proteins from
  complex samples

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Acknowledgements

• ARC
• Liliana Gheorghiu
• Diane Nelson
• Melanie Poncheri
• Serguei Soukharev
• Xiaguo Zhan
• ProMetic Biosciences
• Kevin Carrick
• Ferdinand Camps
• Tim Hayes

PRDT (Prion Removal Project) Peter Edwardson
(ProMetic) Patrick Gurgel (ProMetic) Luisa
Gregori (BREF) Viterose Wiltshire (NCSU) Steve
Burton (ProMetic) Ruben Carbonell (NCSU) Robert
Rohwer (U Maryland)
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HDL Affinity Ligand Binds HDL Complex
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Affinity Bioseparation of Proteins via
Combinatorial Ligands
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Affinity Binding of Proteins to Library
Diverse Ligand Library
Bind
Wash away Excess

• Each bead may bind a unique protein, protein
  complex
• With sufficient diversity, there will be a ligand
  to most, if not all, proteins in the mixture

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Protein Identified From Affinity Resin Elution
FIoNALibrary
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