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
Target Customer
Automated workflows designed for analytical chemists
Microchromatography
Cartridges
quantitative binding and
elution
Antibody (protein)
purification
• PA-W
• PG-W
• SA-W
Simple User Interface
Uses customer language - not
automation language
• Antibody (protein) Purification
• In-Solution Digestion
• Peptide Cleanup (desalting)
• Fractionation
• Liquid handling utilities
Cleanup:
• C18
• RP-S
Fractionate:
• SCX
• RP-S
• C18
Positive Displacement Pipetting
Syringes interface directly with cartridges and
enable precise, controlled liquid flow through
cartridges with no air bubbles to disrupt
binding
AssayMAP vs. pipette tips
Pipet Tip Column
AssayMAP Cartridge
• Multi-pass contact
(equilibrium adsorption)
• Single-pass contact
(chromatography!)
• N0 flow rate control
• Precise, controlled flow rate
• Non-quantitative extraction
• Quantitative binding & elution
Bath
vs.
Shower
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
AssayMAP cartridges are like microscale HPLC columns which mate to a 96-head
HPLC yielding chromatographic-like performance
AssayMAP tools for proteomicists
•
Intact molecular
weight
Peptide Mapping
Antibody Purification
•
•
PA-W, PG-W
Protein Purification
-
Antibody Purification
-
•
SA-W
•
PA-W, PG-W
Protein Purification
-
SA-W
•
In-Solution Digestion
•
Peptide Cleanup
-
•
Glycan Analysis
•
Antibody Purification
-
PA-W
-
PG-W
GlykoPrep Plus
C18, RP-S
Fractionation
-
SCX, C18, RP-S
4
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 interface
Peptide Sample Prep: Workflow Navigator
In-Solution Digestion
1-3 hrs
Input:
• Particulate-free, solutions of proteins
• Up to 384 samples, four 96-well plates
• Sample volume dependent on chemistry
conditions
2-18 hrs
Output:
• Sample plates contain digested
proteins = peptides
• Samples now ready for peptide
cleanup
In-Solution Digestion user interface
In-Solution Digestion user interface
Peptide Cleanup (reversed-phase)
C18 or RP-S
Input:
• Particulate-free, aqueous
solutions of peptides
• Up to 96 samples (1 plate)
• Up to 250 µL sample load
Up to 25x
concentration
factor!!!
Output:
• Salt-free, solutions of
peptides in organic
solvent
• Elution volume as
little as 10 µL
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
Day 1 cleanup = C18
Day 2 cleanup = RP-S
Low %CVs for both
intra- and interday
digestion and cleanup
using either urea or
guanidine
Reversed-phase cartridge characterization:
BSA load capacity study
RH = relative hydrophobicity
Reversed-phase cartridge characterization:
BSA peptide recovery
25 peptide summary
RH = relative hydrophobicity
Application Note 5991-2957EN
Peptide Fractionation
SCX, C18, or RP-S
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)
User interface for step-wise Fractionation
User interface for step-wise Fractionation
Multi-step sample preparations workflows for LC/MS
1290 Infinity LC with
AdvanceBio Peptide
Mapping Column
AssayMAP Bravo
6550 iFunnel
Q-TOF with
Dual Agilent
Jet Stream
ESI Source
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
PA-W
PG-W
Up to 100x
concentration
factor!!!
Output:
• Highly purified antibodies in
aqueous buffer
• Elution volume as little as
10 µL
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
Affinity purification of biotinylated proteins using SA-W
Sample matrix = PBS
Affinity purification of biotinylated proteins using SA-W
Sample matrix = PBS
AssayMAP Bravo – scalable sample preparation
for precision proteomics
Fully automated AssayMAP Bravo Workstation for
peptide sample prep
BenchBot Robot
AssayMAP Bravo
Plate Hub
PlateLoc
XPeel
Centrifuge
Multidrop Combi
INHECO
Incubator MP
Acknowledgements
AssayMAP R&D team
Steve Murphy
Zach Van Den Heuvel
Michael Bovee
Adam Krahenbuhl
Jennifer Reich