Selective recovery of foreign gene transcripts as virus-like particles ...
From Human Nucleosomes to Virus-Like Particles ...
Transcript of From Human Nucleosomes to Virus-Like Particles ...
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CASSSMass Spec 2020September 17, 2020
Neil L. KelleherNorthwestern University
Multiplexing the Orbitrap to Readout Individual Ion Mass Spectra of Virus-Like Particles
Mass-to-Charge Ratio(M/Z)
Charge (Z)
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Executive Summary
Supporting COVID-19 Vaccine Development Individual Ion MS (I2MS or “i2MS”) Antibodies – Super ResolutionDeconvoluting Protein ComplexityProtein ComplexesVirus-like Particles
A New Sample Stream for Intacts and Complexes
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Novavax: Major Player in COVID-19 Vaccine Development
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Spike Protein
Vincent Racaniello: TWiV and Virology at Columbia
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Model of spike protein & the human ACE2 receptor (blue) and complex glycans (magenta)
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Pinto et al., Nature 583, 290–295 (2020)
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Pinto et al., Nature 583, 290–295(2020)
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Spike RBD Denatured
Mass (Da)
Ion
Cou
nts
Monomer
Viral complexity is astounding!!
Heald-Sargent and Gallagher, Viruses 4(4), 557-580 (2012)
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Spike RBD Native
Mass (Da)
Ion
Cou
nts
Monomer
Dimer
Trimer
Mass (Da)
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Spike RBD Native
Mass (Da)
Ion
Cou
nts
Monomer
Dimer
Trimer
Mass (Da)
**RBD region forms complexes under native conditions
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4 Recent Publication on Individual Ion MS (I2MS)
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Initiation
Oct 2017
Development
2017-2018
Applications
2019
Orbitrap-based Individual Ion Mass Spectrometry
Ion Charge Assignment and True Mass Determination
JaredKafader
RafaelMelani
KenDurbin
BryanEarly
RyanFellers
AlexanderMakarov
JoshuaMaze
DevenShinholt
PingYip
StevenBeu
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Assemblies Viruses Bacteriophages
Increasing Molecular Weight
Charge Detection Mass Spectrometry
Keifer et al., Analyst 142, 1654-1671 (2017)
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Charge Detection Mass Spectrometry
Kafader et al., Nat Methods 17, 391–394 (2020)Contino et al., Int J Mass Spectrom 345-347, 153-159 (2013)
Linear Ion Trap Orbitrap
ProsHigh Availability
High Resolution (m/z)High Mass Accuracy
Harmonic PotentialPrecise Kinetic Energies
100x Ion CollectionSpace Charge Tolerance
High VacuumOptimized Desolvation
ProsHigh Resolution (z)
Low CapacitanceDouble Measurement Frequency
Efficient Signal PickupIon trajectory Independent
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Orbitrap Single Ion Measurements
Makarov et al., JASMS 20, 1486 (2009)
Increase transient length
Increase Resolution
Orbitraps are sensitive enough to measure single ion events
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Charge DetectionUsing Only Intensity
Ion Intensity
Num
ber o
f Sig
nals
+21
+20
+19 Problems
Decayed ions
Large proteins
Not Filterable
Solution: Integration of ion signal over time (STORI plots)
Using Summation of Ion Signal Over TimeN
umbe
r of S
igna
ls
Charge
Kafader et al., Nat Methods 17, 391–394 (2020)
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Charge DetectionUsing Only Intensity
Ion Intensity
Num
ber o
f Sig
nals
+21
+20
+19 Problems
Decayed ions
Large proteins
Not Filterable
Solution: Integration of ion signal over time (STORI plots)
Using Summation of Ion Signal Over TimeN
umbe
r of S
igna
ls
Charge
Multiple Ions
Ions Died During Detection
Kafader et al., Nat Methods 17, 391–394 (2020)
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Charge DetectionIn
tens
ity (1
x105
)
Time (Sec)
Inte
grat
ed Io
n Si
gnal
(1x1
05)
B
A
Ions A & B are survivors
Frequency (Sec)
Kafader et al., J Am Soc Mass Spectrom 30, 2200 (2019)
STORI analysis1) Correctly assigns slopes to +20 charge state2) Higher precision3) Filterable Results
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Charge Detection
STORI analysis1) Correctly assigns slopes to +20 charge state2) Higher precision3) Filterable Results4) Rescues decayed signals5) Identifies events
Inte
nsity
(1x1
05)
Time (Sec)
Inte
grat
ed Io
n Si
gnal
(1x1
05)
B
A
C
D
Ions A & B are survivors
Ions C & D are victimsFrequency (Sec)
Kafader et al., J Am Soc Mass Spectrom 30, 2200 (2019)
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Individual Ion Workflow
Analyze individual ion signals to determine:
Mass-to-charge ratio (m/z)Normal FT-MS analysis
Charge value (z)Newly developed STORI analysis
Generate true MASS spectra1) Mass determination for large native complexes
Kafader et al., Nat Methods 17, 391–394 (2020)
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STORI Slope to Charge Calibration
DenaturedProteins
NativeComplexes
Kafader et al., Nat Methods 17, 391–394 (2020)
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Deconvolution: 4 Protein Mix
Single Ion hCDMS Analysis Solves Charge State Congestion Problem
Kafader et al., Nat Methods 17, 391–394 (2020)
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Deconvolution: Reduced Ab
Standard MS hCDMS
+GdHClTCEP - HCl
Kafader et al., Nat Methods 17, 391–394 (2020)
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Single Ion MS: Resolution Gain
m/z
Rel
ativ
e In
tens
ity
Ensemble ion measurement
Single ion measurement
1Scan
234
Charge State
Resolution
Isotopic
Resolution
~20x Resolution Gain
Kafader et al., Anal Chem 91(4), 2776-2783 (2019)
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Individual Ion MS: Resolution Gain
m/z
Rel
ativ
e In
tens
ity
Ensemble ion measurement
Single ion measurement
1Scan
234
Charge State
Resolution
Isotopic
Resolution
~20x Resolution Gain
Kafader et al., Anal Chem 91(4), 2776-2783 (2019)McGee et al., Submitted (2020)
Pyruvate Kinase (Tetramer)
Mass (Da)
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Individual Ion MS: Resolution Gainβ-Galactosidase (Tetramer)
Mass (Da)
Isotopic Confirmation
Jack McGeeMcGee et al., Submitted (2020)
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Deconvolution: Extreme Proteoform Complexity
5-30 kDa mix of human proteoforms
Kafader et al., Nat Methods 17, 391–394 (2020)
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Deconvolution: Extreme Proteoform Complexity
I2MS Measurement
5-30 kDa mix of human proteoforms
Kafader et al., Nat Methods 17, 391–394 (2020)
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Top Down MS: Triosephosphate Isomerase
Luis Schachner
Kafader et al., J Proteome Res 19(3), 1346-1350 (2020)
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Top Down MS: Triosephosphate Isomerase
+39
Kafader et al., J Proteome Res 19(3), 1346-1350 (2020)
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Top Down MS: Fragment Matches
Mass (Da)
hCDMS
B97 B154
Kafader et al., J Proteome Res 19(3), 1346-1350 (2020)
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Top Down MS: Fragment MatchesI2MS2 Standard MS2
Kafader et al., J Proteome Res 19(3), 1346-1350 (2020)
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Top Down MS: Fragment Matches
Kafader et al., J Proteome Res 19(3), 1346-1350 (2020)
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Mass Determination: Virus-like Particles
27 nmT= 3 Geometry
Standard MS
Heterogeneous DNA and RNA cargo
Kafader et al., Nat Methods 17, 391–394 (2020)
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Mass Determination: Virus-like ParticlesI2MS
27 nmT= 3 Geometry
Standard MS
Heterogeneous DNA and RNA cargo
Kafader et al., Nat Methods 17, 391–394 (2020)
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Mass Determination: Virus-like Particles
27 nmT= 3 Geometry
Standard MS
Heterogeneous DNA AND RNA
cargo Capsid 2.47 Cargo(DNA) 0.58Cargo(RNA) 0.15_________________TOTAL ~3.20
MDa
Kafader et al., Nat Methods 17, 391–394 (2020)
I2MS
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Virus-like Particles: WT vs MUTANT
Dr. Danielle Tullman-Ercek
Kafader et al., Nat Methods 17, 391–394 (2020)
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Virus-like Particles: WT vs MUTANT180 Coat Proteins
27 nm Diameter
10,300 nm3 Volume
60 Coat Proteins
17 nm Diameter
2,600 nm3 Volume
Dr. Danielle Tullman-Ercek
Kafader et al., Nat Methods 17, 391–394 (2020)
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Virus-like Particles: WT vs MUTANT180 Coat Proteins
27 nm Diameter
10,300 nm3 Volume
60 Coat Proteins
17 nm Diameter
2,600 nm3 Volume
Dr. Danielle Tullman-Ercek
Kafader et al., Nat Methods 17, 391–394 (2020)
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Mass Determination: Virus-like Particles
17 nmT= 1 Geometry
Heterogeneous DNA and RNA cargo
Standard MS
Kafader et al., Nat Methods 17, 391–394 (2020)
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Mass Determination: Virus-like Particles
17 nmT= 1 Geometry
I2MSHeterogeneous DNA and RNA cargo
Capsid 0.82 Cargo(DNA) 0.24Cargo(RNA) 0.03_________________TOTAL ~1.09
MDa
Standard MS
Kafader et al., Nat Methods 17, 391–394 (2020)
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Mass Determination: Virus-like Particles
17 nmT= 1 Geometry
Heterogeneous DNA OR RNA cargo
Capsid 0.82 Cargo(DNA) 0.24Cargo(RNA) 0.03_________________TOTAL ~1.09
MDa
Standard MS
Kafader et al., Nat Methods 17, 391–394 (2020)
I2MS
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Improved sample handling for native and denatured top-down MS
New SampleStream tech: Automated Buffer Exchange 20-100x Protein Concentration Detergent Removal nanograms to low micrograms
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SampleStream Operating Mode 1: Focusing
membrane
Frit
Flows from Inlet Ports
Sample and Buffer
Buffer and small molecules pass through membrane to waste port (buffer exchange)
Sample introduced to one or both flows entering upper ports
Analytes retained by MWCO membrane accumulate above membrane where flows cancel (concentration)
Frit
Analyte
Membrane
Proprietary and Confidential
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Membrane
Frit
SampleStream Operating Mode 2: Elution
To Analysis
Elution buffer delivered to waste port
Retained sample band lifted off membrane and transported out top port to analysis
Generates elution profile reminiscent of liquid chromatography
Proprietary and Confidential
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Comparison of SS to LC
• 500 ng Herceptin analyzed via optimized LC method provided by Genentech (red) and comparable SampleStream method (black)
• Identical mass spectrometer and tune file• LC loading volume: 5 µL• SS Loading volume: 100 µL• LC run time: 6 min• SS run time: 2.5 min
Key Point: Equivalent data from 20x more dilute samples with over 2x the throughput
Proprietary and Confidential
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SS & Native Mass Spectrometry
• 3.3 pmol loaded for various molecules• SampleStream module operated with native
buffer• Total method time: 80 sec
Key Point: Simple buffer change enables sensitive native analysis on the same platform can rapidly switch between native and denaturing modes
Proprietary and Confidential
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Sample Preparation in the SS Channel
ReductionDigestion
Key Point: Can perform enzymatic digestions and chemical transformation inside the channel
Proprietary and Confidential
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The SampleStream™ Platform
Philip Compton, Ph.D. CEO [email protected]
Jared Drader, Ph.D. CTO [email protected]
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Concluding Remarks Orbitrap analyzers are capable of multiplexing
CDMS analysis
I2MS can quickly produce native mass spectra (even isotopically resolved up to 466 kDa)
Orbitrap-based I2MS has many applications for proteins in the kDa to MDa range, e.g., COVID-19 vaccine candidates with high glycosylation
SampleStream for improved sample handling of intact proteins
New paradigm for MASS spectrometry
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Acknowledgements
Jared KafaderPhilip Compton
Jack McGeeRafael Melani
Ken DurbinBryan EarlyRyan Fellers
Luis Schachner
Michael SenkoVlad Zabrouskov
Steven BeuJoshua Maze
Deven ShinholtPing Yip
Danielle Tullman-Ercek
Bon Ikwuagwu
VLP Engineering