Assay MAP Bravo for Automated Sample Preparation Yanan

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

Overview Peptide Quantitation • • 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 • • • Assay. MAP 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

Assay. MAP Technology Components Target Customer Automated workflows designed for analytical chemists Microchromatography Cartridges Quantitative binding & elution 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 Peptide Quant Protein purification • PA-W (protein A) • PG-W (protein G) • SA-W (streptavidin) Reversed-phase cleanup: • C 18 (peptide) • RP-S (peptide) • RP-W (reduced m. Abs) Peptide Fractionation: • SCX • RP-S • C 18 Phosphopeptide enrichment: • Ti. O 2 Positive Displacement Pipetting Syringes interface directly with cartridges and • Fe(III)-NTA enable precise, controlled liquid flow through cartridges with no air bubbles to disrupt binding For Research Use Only. Not for Use in diagnostic procedures

Assay. MAP Technology Each Assay. MAP cartridge is slurry packed, back-pressure tested, and inspected for voids Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

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

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

“Apps”-Based Solution User Interface Workflow-specific control parameters 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 Standardized deck layout “Pushbutton” run controls

Assay. MAP Peptide Sample Prep Workflow Feature Set In-Solution Digestion Peptide Cleanup Fractionation Automated protocol with minimal manual steps, that processes up to four 96 well plates with usersupplied reagents. Cleanup one 96 well plate of peptide samples using reversed phase cartridges. Elute in 10 μL volume, allowing minimal to no dry down times. Simplify one 96 well plate of complex samples, offline from LCMS, using stepwise p. H or salt elution to generate up to 6 fractions. Labware is provided in starter kits Peptide Quant Uses Assay. MAP C 18 or RP-S cartridges For Research Use Only. Not for Use in diagnostic procedures Uses Assay. MAP C 18 or RP-S cartridges

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

Assay. MAP Digestion and Cleanup Workflow Total Workflow Reproducibility Total workflow CVs < 4% • Agilent Assay. MAP Peptide Sample Prep • Agilent Advance. Bio Peptide Mapping column • Agilent 1290 Infinity LC • Agilent 6550 i. Funnel Q-TOF mass spectrometer The Assay. MAP 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 n. L of plasma SISCAPA enrichment of targeted peptides Greater specificity and analytical sensitivity – plus faster analysis! 10 µL of plasma >1000 x 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) 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 Assay. MAP Bravo Fe(III)-NTA Ti. O 2 Aqueous solution of peptides Phosphopeptide solutions Up to 96 samples (1 plate) Elution volume ≥ 10 µL Up to 1 m. L sample load m. Ab Reduced sample complexity Up to 100 x concentration factor Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Phosphopeptide Enrichment Portfolio Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

Peptide Fractionation SCX, C 18, 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 Peptide Quant Output: • Solutions of peptides in various solvents/salts • As many as 6 fractions can be collected • Can collect small volume fractions (~ 5 µL) 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

Assay. MAP SCX Fractionation Workflow - E. coli Lysate Each step in the workflow was performed using the Assay. MAP 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

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

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

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

Assay. MAP SCX Fractionation Workflow - E. coli Lysate Elution by increasing p. H Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

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

Assay. MAP Sample Prep Solution HPLC-like performance on a liquid handling platform Feature Set 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 The Agilent Assay. MAP platform is an easy to use yet flexible automation solution for preparing peptide samples for LC/MS analysis With the Assay. MAP 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