AssayMAP Bravo for Automated Sample Preparation Yanan Yang, PhD LC/MS Application Scientist January 2018 Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Overview Peptide Quantitation Break Automated sample preparation Jet Stream proteomics - description and performance Agilent 6495 Triple Quadrupole MS system – features design to enhance peptide quantitation Software tools Application examples Break Automated sample preparation AssayMAP Bravo system – description Peptide Sample Prep workflow SISCAPA workflow Phosphopeptide enrichment Fractionation workflow Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP Technology Components Target Customer Automated workflows designed for analytical chemists Microchromatography Cartridges Quantitative binding & elution Protein purification PA-W (protein A) PG-W (protein G) SA-W (streptavidin) Reversed-phase cleanup: C18 (peptide) RP-S (peptide) RP-W (reduced mAbs) Peptide Fractionation: SCX RP-S C18 Phosphopeptide enrichment: TiO2 Fe(III)-NTA Simple User Interface Uses customer language - not automation language Affinity (protein) Purification In-Solution Digestion Peptide Cleanup (desalting) Phosphopeptide Enrichment IMAC Cartridge Customization Fractionation Sample Normalization Liquid handling utilities Positive Displacement Pipetting Syringes interface directly with cartridges and enable precise, controlled liquid flow through cartridges with no air bubbles to disrupt binding Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP Technology Presentation Title 11/8/2018 Confidentiality Label Regulatory statement (if applicable) Each AssayMAP cartridge is slurry packed, back-pressure tested, and inspected for voids Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP® Microchromatography Cartridges any resin 15 – 100 µm Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP Cartridges Packed pipet tips vs AssayMAP cartridges Pipet Tip Column AssayMAP Cartridge Multi-pass contact (equilibrium adsorption) NO flow rate control Non-quantitative extraction Recover < 100% of capture Large elution volume (diluted >10x) Bias is unknown or variable Single-pass contact (chromatography!) Precise, controlled flow rate Quantitative binding & elution Recover 100% of capture Minimal elution volume (10 µL) Minimized, defined bias Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

“Apps”-Based Solution User Interface Workflow-specific control parameters Standardized deck layout “Pushbutton” run controls Application-specific interface VWorks automation software Standardized system configuration Protocol validated to run the chemistry Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP Peptide Sample Prep Workflow Feature Set In-Solution Digestion Automated protocol with minimal manual steps, that processes up to four 96 well plates with user- supplied reagents. Labware is provided in starter kits Peptide Cleanup Cleanup one 96 well plate of peptide samples using reversed phase cartridges. Elute in 10 μL volume, allowing minimal to no dry down times. Uses AssayMAP C18 or RP-S cartridges Fractionation Simplify one 96 well plate of complex samples, offline from LCMS, using stepwise pH or salt elution to generate up to 6 fractions. Uses AssayMAP C18 or RP-S cartridges Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Peptide Sample Prep Workflow Navigator Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Built-in Reagent Calculators Calculator input: Desired reaction conditions Calculator output: Stock solution recipes, reactant concentrations, user interface input values and master mix plate set up Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Standardized User Interfaces: Digest Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Standardized User Interfaces: Cleanup Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Standardized User Interfaces: Fractionate Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP Digestion and Cleanup Workflow Presentation Title 11/8/2018 Total Workflow Reproducibility Confidentiality Label Regulatory statement (if applicable) Total workflow CVs < 4% Agilent AssayMAP Peptide Sample Prep Agilent AdvanceBio Peptide Mapping column Agilent 1290 Infinity LC Agilent 6550 iFunnel Q-TOF mass spectrometer The AssayMAP workflow took 2 hours hands-on time each day (n = 128) Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

SISCAPA: Enrich Target Peptides and Decrease Sample Complexity Direct injection of unfractionated protein digest 10 nL of plasma SISCAPA enrichment of targeted peptides Greater specificity and analytical sensitivity – plus faster analysis! 10 µL of plasma >1000x fold increased MRM analytical sensitivity Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Targeted Peptide Enrichment With Anti-peptide Antibodies (SISCAPA) Presentation Title 11/8/2018 Confidentiality Label Regulatory statement (if applicable) Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Targeted Peptide Enrichment With Anti-peptide Antibodies N = 4, each condition Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Increasing Throughput for SISCAPA With Faster Chromatography 3 min 20 min 3 minute MRM analytical method Normal flow (400 μL/min) Quantitation of >400 samples/day Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Phosphopeptide Enrichment with AssayMAP Bravo Presentation Title 11/8/2018 Confidential Fe(III)-NTA TiO2 Aqueous solution of peptides Up to 96 samples (1 plate) Up to 1 mL sample load Phosphopeptide solutions Elution volume ≥ 10 µL Reduced sample complexity Up to 100x concentration factor mAb Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Phosphopeptide Enrichment Portfolio Presentation Title 11/8/2018 Confidential Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Peptide Fractionation SCX, C18, or RP-S Input: Particulate-free, solutions of peptides Sample plates from Peptide Cleanup protocol Up to 96 samples (1 plate) Up to 250 µL sample load Output: Solutions of peptides in various solvents/salts As many as 6 fractions can be collected Can collect small volume fractions (~ 5 µL) Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Fractionation User Interface Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP SCX Fractionation Workflow - E. coli Lysate Each step in the workflow was performed using the AssayMAP Bravo Sample input = 10 μL E. coli lysate @ 10 μg/μL (100 μg) Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP SCX Fractionation Workflow - E. coli Lysate Elution by increasing ionic strength (KCl) Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP SCX fractionation workflow - E. coli lysate Elution by increasing ionic strength (KCl) Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP SCX fractionation workflow - E. coli lysate Elution by increasing pH using volatile buffers Not necessary to do another round of sample cleanup Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP SCX Fractionation Workflow - E. coli Lysate Elution by increasing pH Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP Bravo – Scalable Sample Preparation for precision proteomics Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

AssayMAP Sample Prep Solution Feature Set HPLC-like performance on a liquid handling platform Total workflow reproducibility Parallel processing for rapid method development Dramatically reduces human variability, hands on time Increases Mass Spec throughput Standardized interfaces are easy to use Out-of-the box solution – no automation engineer Integrated workflows, simple method transfer Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Summary Presentation Title 11/8/2018 Confidentiality Label Regulatory statement (if applicable) The Agilent AssayMAP platform is an easy to use yet flexible automation solution for preparing peptide samples for LC/MS analysis With the AssayMAP Bravo system, you can Increase the precision of your LC/MS workflows by reducing the variability in your sample prep. Reduce tedious manual steps with walk-away automation so you can do more value-added work. Increase throughput without increasing variability or labor costs. Simplify your most challenging workflow using a broad range of chemistries and laboratory-tested protocols that you can combine to meet your needs. Peptide Quant For Research Use Only. Not for Use in diagnostic procedures