Discover New Proteins Using Immunodepletion

Transcription

1 Discover New Proteins Using Immunodepletion Proteomics Peter Mrozinski Application Scientist Agilent Technologies December 11, 2007

2 Introduction Complexity and dynamic range of protein concentrations present a major challenge Concentration - 10 orders of magnitude Complexity 10 6 different proteins can be addressed with a combination of prefractionation techniques that deplete highly abundant proteins and fractionate Page 2

3 Target Market for Multiple Affinity Removal System The Major Application is Biomarker Discovery Why Blood/Plasma/Serum? Easily available Believed to be the sample with the largest representation of the human proteome Believed to contain subsets of the tissue proteome and xenobiotics Other fluids such as cerebrospinal fluid, synovial fluid, urine and other body fluids can also be used for more specific applications. Challenges Dynamic range of protein amounts ( ) present Structural complexity of these proteins. Proteins present are not at equal concentration. Page 3 Month ##, 200X

4 Sample Processing Strategy The Magic Triangle Level 1 Selective filters Level 2 Selective filters Level 3 Selective filters Sample handling & sample clean-up Multidimensional separations Identification & quantitation by MS Target substances Page 4

5 Why the Interest in Analysis of Low Abundance Proteins? In human serum the concentrations of proteins are estimated to range over twelve orders of magnitude Level (mg/ml) (ug/ml) (Orders of Magnitude) (ng/ml) (pg/ml) (fg/ml) Level 1 Level 2 Level 3 MS Immunoaffinity + MS Tissue Biomarkers Number of Proteins With Immunodepletion Still Remaining Page 5

6 Why the Interest in Analysis of Low Abundance Proteins? In human serum the concentrations of proteins are estimated to range over twelve orders of magnitude Level (mg/ml) (ug/ml) (Orders of Magnitude) (ng/ml) (pg/ml) (fg/ml) Level 1 Level 2 Level 3 MS Number of Proteins MS Tissue Biomarkers With Multi-Level Immunodepletion Still Remaining Page 6

7 What is New in Protein Sample Preparation and Separation? Sample Prep & Fractionation Separation Analysis Protein Expression & Purification Protein Characterization High Abundant Protein Removal Protein Fractionation HPLC-Chip MS mrp Column Packing materials Multiple Affinity Removal System OGE Focus: Recovery of sample (fewest number of steps, return of sample) Selectivity Reproducibility (run to run, lot to lot of product) Reliability and increased productivity Page 7

8 The Multiple Affinity Removal System A polyclonal antibody based system to rapidly deplete multiple high abundant proteins in serum, plasma, CSF, and other biological fluids. H H L H L L H Launched in August 2003 L L H H Individual Ab Materials are mixed in selected percentages and packed into a column format Agilent continues to innovate and lead this market HH H H HH H H HL H L H Apply Crude Human Serum Unbound Fraction (low abundant proteins) Bound Fraction (high abundant proteins) Buffer A Buffer B L L L L L L H H H H HH H H Low-Abundant Proteins Free from Interferences High-Abundant Proteins Total Run Time (30 min) Page 8 Month ##, 200X

9 Importance of Orientation in Antibody Based Depletion Antigen Binding Site xx xx x x x x x x xx x x Porous Particle x x x x x x Fc Region x Specific Antibody Crosslinker Versus Specific Target Protein (eg. Albumin) x x x x x x x Page 9

10 Representative Chromatogram of 4.6 X 100 mm Column mau Simple two buffer system Automatable Reproducible Low collection volumes HPLC and FPLC compatible Robust min Page 10

11 1. Dilute and Filter Sample 2. Remove Buffer 3. Apply Sample 4. Wash and Collect Flowthrough F1 5. Wash and Collect Flowthrough F2 Dilute 6-8 μl human plasma sample to 200 μl with Buffer A. Consult cartridge certificate for true sample capacity. Filter through 0.22 μm spin filter. 6. Prepare for Elution Remove cartridge cap and plug and remove buffer from top of resin bed with transfer pipette. Never let frits or resin bed run dry. 7. Elute Bound Fraction F1 Add 200 μl diluted plasma sample. Cap cartridge loosely or leave open. Place in 1.5 ml collection tube labeled Flow-through fraction 1 (F1). Centrifuge 30 sec at 200 x g. 8. Re- Equilibrate F1 Add 400 μl Buffer A. Centrifuge 1 min at 200 x g. Collect in F1 tube. 9. Analyze F1 + F2 Place spin cartridge in new collection tube labeled Flowthrough fraction 2 (F2). Add 400 μl Buffer A. Centrifuge 1 min at 200 x g. Collect in F2 tube. F1 + F2 F2 Simple two buffer system 10 minute protocol Reproducible Low collection volumes Only need centrifuge Robust Remove spin cartridge from F2 tube and attach luer-lock adapter tightly to top of cartridge. Fill 5 ml Luer-Lok plastic syringe with 2 ml Buffer B and attach to Luer-Lok adapter. Slowly push Buffer B through cartridge to elute bound proteins into new collection tube. Save eluate with targeted high-abundance proteins for analysis or discard. Fill new 5 ml plastic syringe with 4 ml Buffer A and attach to Luer-Lok adapter. Slowly push Buffer A through cartridge to re-equilibrate the cartridge for the next sample or store wetted with Buffer A (at 4 C). Re-cap both ends for storage. Fractions F1 and F2 can be analyzed individually or combined. Concentrate and analyze these fractions containing low-abundance proteins. Page 11

12 MARS 14 Devices Offered Format Size Capacity Spin Cartridge 0.45 ml 10 ul Column 4.6 X 50 mm 20 ul Column 4.6 X 100 mm 40 ul Column 7.5 X 75 mm 100 ul Column 10 X 100 mm 250 ul Column Custom Any Bulk Media Any Any Custom Blend Any Any Page 12

13 The Agilent Multiple Affinity Removal System Selectivity Only native human plasma proteins are used as antigens. This ensures highest selectivity for epitopes in real samples. Our antibodies are so selective that species cross-reactivity is very low. Our buffers are specifically formulated to minimize protein-protein interactions resulting in highest possible selectivity of binding (minimize any possible protein-protein interactions, such as with albumin) Reproducibility Run to run: Coupling chemistry of antibodies to column beads is designed for longest possible lifetime of Antibodies resulting in excellent run to run reproducibility. Only native protein antigen is used for affinity purification resulting in reproducible antibody selection. Buffers for affinity purification of our polyclonal antibodies are designed to disruption unwanted protein-protein interactions (such as with albumin) resulting in reproducible epitope selection. Lot to lot: - Manufacturing processes have been engineered to provide excellent lot to lot reproducibility Page 13 Month ##, 200X

14 The Agilent Multiple Affinity Removal System Ease of Use LC column: Automated single pass, 2 buffer, minute total run time to deplete ul of human plasma/serum (4.6 x 100 mm column) at 98-99% efficiency. Larger column sizes available on request. Spin tube: 2-step re-usable system, 10 minute total run time to deplete ul of human serum/plasma Lifetime and Robustness Antibody Binding and Buffer formulation: Antibody resin and buffers are designed to work together the result is unmatched lifetime and robustness Compatibility with Downstream Analysis 1D gel: Proteins elute in buffer system immediately ready for application for 1DGE HPLC: Proteins can be simultaneously concentrated, desalted, and fractionated on our new mrp column MS: There are no detergents present in our buffers Page 14 Month ##, 200X

15 Why Deplete the High Abundant Proteins? pi 4-7 pi 4-7 Crude serum Removal of these proteins clearly improves the resolution in the albumin area and increases the intensity of low abundance proteins Depleted serum Courtesy of Dr. Tasso Miliotis, Karin Björnhall and Dr. Pia Davidsson, Experimental Medicine/Molecular Sciences, Astra Zeneca, Mölndal, SE Page 15

16 Why Multiple Affinity Removal System? α γ β Plasma Plasma after Depletion Data: Dr. Y.K. Paik Page 16 Month ##, 200X

17 Agilent Multiple Affinity Removal System: Where Are We? & What is Next? Original Top-6 Human Serum High Capacity Top-6 Human Serum High Capacity Top-7 Human Plasma Level-II Human-14 Spin Tube format Spin Tube format Mouse-3 While Maintaining our Focus: Recovery of sample (fewest number of steps, return of sample) Selectivity Reproducibility (run to run, lot to lot of product) Reliability and increased productivity Page 17

18 MARS Human 14 Anti HSA Anti IgG Anti Transferrin Anti Haptoglobin Anti Alpha1-antitrypsin Anti IgA Anti Fibrinogen Anti Alpha2-Macroglobulin Anti Alpha1-Acid Glycoprotein Anti IgM Anti Apolipoprotein AI Anti Apolipoprotein AII Anti Complement C3 Anti Transthyretin Proteomic Sample High Abundance Protein Low Abundance Protein Proteomic Sample with Low Abundance Proteins In Column Flow Through Page 18

19 MARS Human 14 H H L H L L H L L H H H L H L H L L L L L L L IgG Transferrin Fibrinogen IgA Haptoglobin α1-antitrypsin α1-acid Glycoprotein Complement C3 Apolipoprotein AII Transthyretin 15% Apolipoprotein AI 6% Remaining for Analysis IgM Albumin α2-macroglobulin Page 19

20 Overlay of Chromatograms From Run 1, 50, 100, 150 and 200 on a Human 14 Column No1rm mm ID x 100 mm column Bound Fraction 2000 Flow-through Fraction Column performs reproducibly for 200+ runs Plasma Injection ml/min Elution 1 ml/min Re-equilibration min Figure 3 Page 20

21 SDS-PAGE Analysis of Human14 Reproducibility kda Runs 1, 50, 100, 150, Mark12 Standards 2 - Flow-through from Run Flow-through from Run Flow-through from Run Flow-through from Run Flow-through from Run 200 The correct binding and elution formulations are required for reproducible long life use. Reproducible depletion of target proteins from human serum as indicated by constant gel pattern of the depleted serum. Protein content of flow-through fractions remains consistent during 200 runs.

22 SDS-PAGE Analysis of Fractions from MARS14 Column Serum Plasma kda MultiMark Standards 2 - Serum 3 - Serum Flow-through Fraction 4 - Serum Bound Fraction 5 - MultiMark Standards 6 - Plasma 7 - Plasma Flow-through fraction 8 - Plasma bound fraction 9 - MultiMark Standards * For 4.6x100 mm column 40μl sample loading results in the 94% total protein depletion from serum (~194µg of protein in the flow-through fraction) and 92% of total protein depletion from plasma (~270μg of protein in the flow-through fraction). Page 22

23 ELISA Results for the Depletion Efficiency After 200 Runs * Target Protein Depletion in Serum Depletion in Plasma Albumin 99.9% 99.6% Haptoglobin 99.0% 98.9% Transferrin 98.6% 99.5% IgG 99.6% 99.5% IgA 99.2% 99.5% α1- Anti-trypsin 98.0% 99.5% α2-macroglobulin 99.6% 99.3% α1-acid Glycoprotein 99.6% 99.5% Apolipoprotein AI 99.2% 98.4% Apolipoprotein AII 96.0% 95.0% Complement C3 96.0% 98.6% IgM 99.3% 99.5% Transthyretin 99.3% 97.5% Fibrinogen N/A 97.6% Page 23

24 Selectivity of Human-14 Column Proteins identified in the serum bound fraction by LC/MS/MS * 15* 16* 17* Human Serum Albumin Immunoglobulin G Immunoglobulin M Immunoglobulin A Haptoglobin Transferrin Alpha1-Anti-trypsin Alpha2-Macroglobulin Complement C3 Alpha1-Acid Glycoprotein Apolipoprotein AI Transthyretin (prealbumin) Apolipoprotein AII Apolipoprotein B-100 Plasma protease C1 inhibitor Zinc-alpha-2-glycoprotein Apolipoprotein L1 * Untargeted proteins not quantitatively removed Page 24

25 1D Removal of the 14 most abundant proteins with the Agilent Human-14 column Immunodepletion Low Abundance proteins High Abundance proteins HPLC-Chip / XCT Ultra Trap Digest 2D 2D mrp Column Simultaneously: Concentrate Desalt Fractionate mrp Column Proposed Workflow: 1. Depletion 2. Protein Fractionation 3. Peptide Fractionation 4. Peptide Separation Robust integrated nanospray system 3D + 4D 32 mrp fractions analyzed after tryptic digestion - 14 salt steps per fraction HPLC-Chip: Spectrum Mill processed the resulting 448 data files as one data set Enrichment column RP Separation Column 10-port valve Nano-electrospray tip Page 25

26 Human Plasma Immunodepletion of 14 high-abundant proteins on Human-14 column Sample Denaturation Reversed-phase separation of the depleted plasma on mrp-c18 column TFE-based tryptic digestion of RP fractions Peptide analysis by 2D HPLC-Chip LC/MS/MS system Page 26

27 Partial List of Low Level Protein Identified from Plasma Number Protein Name Insulin-like growth factor binding protein 3 (ng/ml) Transitional endoplasmic reticulum ATPase (TER ATPase) Intercellular adhesion molecule-2 precursor (ICAM-2) (ng/ml) Mitogen-activated protein kinase 3 (MAPK) Polycystic kidney and hepatic disease 1 precursor Dopamine beta-monooxygenase precursor ADAM 10 precursor (ng/ml) Integrin alpha-1 (CD49a) Peripheral-type benzodiazepine receptor-associated protein 1 (PRAX-1) Insulin-like growth factor binding protein 4 precursor (IGFBP-4) (ng/ml) 6-phosphofructokinase, liver type cgmp-dependent protein kinase 1, beta isozyme Cadherin-13 precursor (ng/ml) Serine/threonine-protein kinase MAK Insulin-like growth factor binding protein 6 precursor (IGFBP-6) (ng/ml) Tyrosine-protein kinase JAK1 Aminopeptidase N Tumor necrosis factor, alpha-induced protein 2 (pg/ml) Protein kinase C, mu type (npkc-mu) Interleukin-1 receptor-like 1 precursor (pg/ml) Cyclin T2 Interleukin-27 beta chain precursor (IL-27B) (pg/ml) LIM domain kinase 1 (LIMK-1) Protein-tyrosine phosphatase precursor Accession # P17936 P55072 P13598 Q8IVH8 Q8TCZ9 P09172 O14672 P56199 O95153 P22692 P17858 P14619 P55290 P20794 P24592 P23458 P15144 Q03169 Q15139 Q01638 O60583 Q14213 P53667 Q12913 Distinct Peptide # Page 27

28 Outlook Human-14 Multiple Affinity Removal Column depletes the top 14 abundant proteins from human serum, plasma, CSF, and other biological fluids. The Multiple Affinity Removal System provides ~94% total protein mass removal from plasma/serum and longest column lifetime when compared to other products. This translates to the lowest cost per ml of depleted sample. The Multiple Affinity Removal System also provides the greatest ease of use, highest selectivity and reproducibility (run to run, lot to lot) compared to any equivalent product. Protein prefractionation by immunodepletion, mrp and OGE enables one to dive deeper into the plasma proteome and provides methods compatible with LC-MS based analysis. All prefractionation methods and tools integrate well together minimizing sample loss due to excessive sample manipulations Page 28