1. 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

2. 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

3. 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

4. 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

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

6. 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

7. “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

8. 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

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

10. 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

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

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

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

14. 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

15. 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

16. 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

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

18. 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

19. 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

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

21. 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

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

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

24. 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

25. 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

26. 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

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

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

29. 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

30. 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