Leading Proteomics into the Age of Automation: High-throughput Protein Sample Preparation for Mass Spectrometry Applications Enabled by the AssayMAP Bravo Automation Platform

Jason D. Russell, Ph.D.LC/MS Scientist – AssayMAP

MSACL – San Diego, 2014

AssayMAP technology components

Microchromatography Cartridgesquantitative binding and elution

Antibody (protein) purification• PA-W• PG-W• SA-WCleanup: • C18• RP-SFractionate:• SCX• RP-S• C18

Positive Displacement PipettingSyringes interface directly with cartridges and enable precise, controlled liquid flow through cartridges with no air bubbles to disrupt binding

Simple User InterfaceUses customer language - not automation language• Antibody (protein) Purification• In-Solution Digestion• Peptide Cleanup (desalting)• Fractionation• Liquid handling utilities

Target Customer

Automated workflows designed for analytical chemists

AssayMAP vs. pipette tipsAssayMAP CartridgePipet Tip Column

• Single-pass contact (chromatography!)

• Precise, controlled flow rate• Quantitative binding & elution

• Multi-pass contact (equilibrium adsorption)

• N0 flow rate control• Non-quantitative extraction

Benefit Pipet Tip AssayMAP

Washing • Less efficient removal of contaminants

• More efficient removal of contaminants due to unidirectional, chromatographic-like flow

Elution • < 100 % of capture• Diluted >10 X

• 100% of capture• Minimal volume (10 µL)

Bias • Unknown or variable • Minimized, defined, measureable

Bath vs. Shower

AssayMAP cartridges are like microscale HPLC columns which mate to a 96-head HPLC yielding chromatographic-like performance

4

Peptide Mapping Glycan Analysis

• Antibody Purification- PA-W, PG-W

• Protein Purification- SA-W

• Antibody Purification- PA-W, PG-W

• Protein Purification- SA-W

• In-Solution Digestion

• Peptide Cleanup- C18, RP-S

• Fractionation- SCX, C18, RP-S

• Antibody Purification- PA-W- PG-W

• GlykoPrep Plus

Intact molecular weight

AssayMAP tools for proteomicists

AssayMAP Launchpad 3.0

Flexible and intuitive protocols to enable assay customization without learning automation software

Peptide Sample Prep – 3 primary tools

In-Solution Protein Digestion Peptide Cleanup Peptide

Fractionation

Software-user interfacePeptide Sample Prep: Workflow Navigator

In-Solution Digestion

Input:• Particulate-free, solutions of proteins• Up to 384 samples, four 96-well plates• Sample volume dependent on chemistry

conditions

Output:• Sample plates contain digested

proteins = peptides• Samples now ready for peptide

cleanup

1-3 hrs 2-18 hrs

In-Solution Digestion user interface

In-Solution Digestion user interface

Peptide Cleanup (reversed-phase)

Input:• Particulate-free, aqueous

solutions of peptides• Up to 96 samples (1 plate)• Up to 250 µL sample load

Output:• Salt-free, solutions of

peptides in organic solvent

• Elution volume as little as 10 µL

Up to 25x concentration

factor!!!

C18 or RP-S

Peptide Cleanup user interface

Peptide Cleanup user interface

Stringing together a common LC/MS workflow: Performance metrics of In-Solution Digestion and Peptide Cleanup

BSA digestion and cleanup - reproducibility

Low %CVs for both intra- and interdaydigestion and cleanup using either urea or guanidine

Day 1 cleanup = C18Day 2 cleanup = RP-S

Reversed-phase cartridge characterization:BSA load capacity study

RH = relative hydrophobicity

25 peptide summary

Reversed-phase cartridge characterization:BSA peptide recovery

RH = relative hydrophobicity

Application Note 5991-2957EN

Peptide Fractionation

Input:• Particulate-free, solutions

of peptides• 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)

SCX, C18, or RP-S

User interface for step-wise Fractionation

User interface for step-wise Fractionation

AssayMAP Bravo

1290 Infinity LC with AdvanceBio Peptide

Mapping Column

6550 iFunnel Q-TOF with Dual Agilent Jet Stream ESI Source

Multi-step sample preparations workflows for LC/MS

E. coli fractionation by SCX

Experiment repeated on two different days with 4 replicates each day.

E. coli fractionation by SCX

Experiment repeated on two different days with 4 replicates each day. Elution with increasing ionic strength. There are 8 overlays for each peptide.

E. coli fractionation by SCX

Elution by increasing ionic strength (KCl)

E. coli fractionation by SCX

Same AssayMAP Fractionation protocol, same SCX cartridges…different chemistry

Comparison of Day 1, replicate 1, samples for each SCX elution condition

Application Note 5991-3602EN

AssayMAP tools for antibody purification: PA-W/PG-W

Input:• Particulate-free, solutions

containing antibodies to be purified

• Up to 96 samples (1 plate)• Up to 1 mL

Output:• Highly purified antibodies in

aqueous buffer• Elution volume as little as

10 µL

Up to 100x concentration

factor!!!

PA-WPG-W

Antibody Purification user interface

Antibody Purification user interface

Antibody purification using PA-W

Sample matrix = PBS

AssayMAP tools for biotin-streptavidin chemistry: SA-W

Affinity purification of biotinylated proteins using SA-W

Sample matrix = PBS

Sample matrix = PBS

Affinity purification of biotinylated proteins using SA-W

Sample matrix = PBS

Affinity purification of biotinylated proteins using SA-W

AssayMAP Bravo – scalable sample preparation

for precision proteomics

AssayMAP BravoBenchBot Robot

Multidrop CombiINHECO

Incubator MP

XPeel

Fully automated AssayMAP Bravo Workstation for peptide sample prep

Plate Hub

PlateLoc

Centrifuge

AcknowledgementsAssayMAP R&D team

Steve Murphy

Zach Van Den Heuvel

Michael Bovee

Adam Krahenbuhl

Jennifer Reich