Reducing dynamic range with Proteominer

Transcription

1 Reducing dynamic range with Proteominer

2 Dynamic Range of the Plasma Proteome Albumin Apolipo-A1 Apolipo-B AGP Lipoprotein A IgG Transferrin Fibrinogen IgA α2 macroglobulin IgM α1-at C3 Comp factor Haptoglobulin 10% Factor H Ceruloplasmin C4-Comp factor Comp factor B Pre-albumin C9-Comp factor C1q-Comp factor C8-Comp factor 1% Deep Proteome Large number of low-abundance proteins H.J. Issaq, 2003 M.F. Lopez, 2000

3 Immunodepletion Antibody-based identification is limited to known species Albumin (30-40mg/ml) Removal of high abundance-proteins may remove associated species IgG Factor VIII (150 ng/ml) vwf (5-10 µg/ml) No concomitant concentration of low abundance species IL6 (<1 ng/ml) Overall dynamic range extends over ten orders of magnitude.

4 Proteominer TM : a Way to Detect Low Abundance Proteins Decrease the dynamic range - dilute abundant and concentrate trace proteins Based on a Combinatorial Ligand Library unique peptide ligands One unique ligand per bead Highly specific affinity interactions between proteins and ligands ensure binding the greatest number and variety of proteins Limited bead capacity allows maximum concentration of rare species while dilution of high abundant species Equalized High Abundance Sample Intermediate Abundance Low Equalized Abundance Sample Amenable to Conventional Biomarker Discovery Methodologies

5 Model for ligand library construction Split, Couple, Recombine Split" "Couple" POOL "Recombine" POOL Number of ligands = N n where N = number of building blocks and n = number of cycles; When N=20 and n=6: 64,000,000 possible structures

6 Principle of dynamic range compression with Proteominer TM Technology Diverse Ligand Library 94 Yellow 5 Blue 3 Green 1 Red 1 Pink Bind Wash away Excess Elute Each bead may bind a unique protein, or protein complex With sufficient diversity, there will be a ligand to most, if not all, proteins in the mixture 4 Yellow 4 Blue 3 Green 1 Red 1 Pink

7 ProteoMiner Example Serum Surface enhanced laser desorption/ ionization (SELDI) MS Before After Before After Molecular Mass (M/Z) IMAC-Cu ProteinChip Arrays 14

8 Plasma treatment with proteominer Flow Through Elution Plasma protein recovery after bead treatment (%) CV (%) Sihlbom & al. J Proteome Research 2008

9 Plasma treatment with proteominer Sihlbom & al. J Proteome Research 2008

10 Red-Blood cell lysates Concentration Equalized Initial sample Concomitant Dilution of Hemoglobin and Concentration of Low abundance Proteins Proteins Untreated RBC sol proteins (load 1300 µg) Ebeads-treated RBC lysate (load 600 µg) 81 spots 950 spots Roux-Dalvai & al. Mol Cell Proteomics 2008

11 Roux-Dalvai & al. Mol Cell Proteomics 2008

12 Relative protein quantitation after equalization Roux-Dalvai & al. Mol Cell Proteomics 2008

13 Relative protein quantitation after equalization 1000 pmol/100pmol Ratios of XIC areas pmol/100pmol C4/C1 C5/C2 C6/C3 C7/C1 C8/C2 C9/C3 Ratios are close to what s expected Relative protein quantification after equalization is possible if the protein does not saturate the beads Roux-Dalvai & al. Mol Cell Proteomics 2008

14 Differential quantitation after Proteominer treatment Human serum samples (1 ml, Millipore) were spiked with 0, 1, 5, 10, 20, 40, 80, and 160 ng/µl SAA and applied to the columns.

15 CONCLUSION A promising novel prefractionation approach As an alternative to immunodepletion methods, ProteoMiner beads are used to reduce high-abundance proteins and enrich medium- and low-abundance proteins High potential to explore proteomes with a large dynamic range Low-abundance proteins will not saturate their binding sites; therefore, a true representation of the relative concentrations of these proteins in different samples is retained. Proteominer treatment generates a reasonable number of fractions for downstream analysis The ProteoMiner protein enrichment kit can be used upstream of multiple protein separation workflows, including SDS-PAGE, 2DGE, SELDI, as well as LC-based methods

16 Proteominer Beads ProteoMiner Kit # Reagent for 10 samples 1 elution step (acetic Acid) Compatible with 2D electrophoresis and other downstream application ProteoMiner Introductory Kit # Same as # but for 2 samples ProteoMiner Sequential Elution Kit # For SELDI Users (NOT compatible with 2D) Reagents for 10 Samples 4 different Elution Steps (Sodium chloride, Glycine, Ethylene Glycol, Organic)