next gen trypsin: large molecule lc-ms/ms bioanalysis ... · -although tpev is an exception to the...
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Next Gen Trypsin: Large Molecule LC-MS/MS Bioanalysis Today, Not TomorrowDr Ashley S Phillips 18th November [email protected]
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Large Molecule Bioanalysis by LC-MS/MS
We are always in search of lower LLOQs
which pushes us towards long, complicated protocols
leading us to:
• Overnight digestion steps• Immunoaffinity enrichment• Solid Phase Extraction
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• Next Generation Trypsin reagents aim to speed up the digestion process:
• They are either:- Heat Stable Variants.- Bioreactor Style.- All in One Kits.
What is Next Generation Trypsin?
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The aim is to assess the next gen trypsin reagents for use in a quantitative bioanalytical method within a regulated laboratory environment.
Stringent Criteria:o No miscleavages.o No deamidation or oxidation modifications.o Confident assignment.o Reproducible!
Fast… but is it any good?
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Phase 2Top performing
trypsinsevaluated using
test sample.
Phase 1Assess trypsinsby digestion of
protein standards in buffer.
5
The Master Plan
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Promega Trypsin Gold
ThermoSmart Digest
Soluble
Thermo Magnetic
Smart DigestPreOmics
iST KitPromega Rapid
Digestion Trypsin
Type Traditional Heat Stable Heat Stable All in One Heat stable
Digest Overnight 1 hour 1 hour 3 hour 1 hour
Flexibility
Cost € €€ €€ €€€ €€
Our Candidates6
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Protein standards selected to represent potential future biotherapeutics or biomarkers and a range of structural complexities.
Phase 1 Overview
BSA3V03
Cytochrome CHorse Heart1AKK
SILuLiteSigmaMAbIgG1
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Protein standards selected to represent potential future biotherapeutics or biomarkers and a range of structural complexities.
Phase 1 Overview
SILuLiteSigmaMAbIgG1
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Standard Peptide Mapping Approach used for Phase 1 and Phase 2
> Gradient: 2-50% organic over 50 minutes> Phases: 0.1% FA in ACN and 0.1% FA (aq)> Column: Waters Acquity HSS T3 2.1x100 mm
Instruments:• Phase 1:
> Waters Synapt G2 equipped with a Waters Acquity H-Class Bio> Acquisition Type: MSe
> Analysis Software: Biopharmalynx
• Phase 2:> Sciex 6600 equipped with a Waters Classic Acquity> Acquisition Type: SWATH> Analysis Software: Biopharmaview
The Method
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Phase 1Digestion in buffer
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Heavy Chain
T001
T024
T027
T010
T013
T014
T038
T039
T004
T022
T031
T043
T006
T023
*
T015
*
T021
*
T002
*
T016
*
T011
*
T037
*
T042
*
Trypsin GoldRapid Digestion - Trypsin without R&A
Smart Digest (Soluble) without R&APreOmics iST Kit
Smart Digest (Magnetic) without R&A
Light Chain
T004
T008
T012
T014
T009
T002
*
T005
T011
T010
*
T017
*
T016
*
Trypsin GoldRapid Digestion - Trypsin without R&A
Smart Digest (Soluble) without R&APreOmics iST Kit
Smart Digest (Magnetic) without R&A
SILuLite SigmaMAb
Low ----------------> High
n/o - not observed* denotes carbamidomethyl modification
intensityn/o
Smart Digest Soluble and Rapid Digestion – Trypsin are the best performing candidates across all protein standards
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Reduction and Alkylation: a thing of the past?
Heat Stable Trypsins:Heat denaturation
vs. Chemical denaturation
But why?
I’ll do it anyway
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Heavy Chain
T001
T024
T027
T010
T013
T014
T038
T039
T004
T022
T031
T043
T006
T023
*
T015
*
T021
*
T002
*
T016
*
T011
*
T037
*
T042
*
Trypsin Gold
Rapid Digestion - Trypsin with R&ARapid Digestion - Trypsin without R&A
Smart Digest (Soluble) with R&ASmart Digest (Soluble) without R&A
Smart Digest (Magnetic) with R&ASmart Digest (Magnetic) without R&A
Light Chain
T004
T008
T012
T014
T009
T002
*
T005
T011
T010
*
T017
*
T016
*
Trypsin Gold
Rapid Digestion - Trypsin with R&ARapid Digestion - Trypsin without R&A
Smart Digest (Soluble) with R&ASmart Digest (Soluble) without R&A
Smart Digest (Magnetic) with R&ASmart Digest (Magnetic) without R&A
The impact of reduction and alkylation
Low ----------------> High
n/o - not observed* denotes carbamidomethyl modification
intensityn/o
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SILuLite SigmaMAb ± Reduction & Alkylation
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A word of warning: its subject specific
assess during development
Cytochrome C T016
T008
T004
T010
T013
T020
T015
T012
Trypsin Gold
Rapid Digestion - Trypsin with R&ARapid Digestion - Trypsin without R&A
Smart Digest (Soluble) with R&ASmart Digest (Soluble) without R&A
Smart Digest (Magnetic) with R&ASmart Digest (Magnetic) without R&A
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Our Candidates16
Weigh up requirements
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Phase 2Digestion in matrix
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• Test sample: SILuLite SigmaMAb was reconstituted in Rat K2 EDTA plasma.- Samples run in triplicate.- Reduction & Alkylation performed prior to digestion
Phase 2 Overview
Promega Rapid Digestion -
TrypsinPromega
Trypsin GoldThermo
Smart Digest Soluble
Time 1, 2, 3 hours Overnight 1, 2, 3 hours
Ratio 50:1, 100:1, 500:1 100:1 50:1, 100:1, 700:1 (MRR)
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Overview statistics do not tell the whole story…
A Bottom Up Protein LC-MS/MS Bioanalytical Assay will typically focus on a single surrogate peptide for quantitation.
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• The most intense charge state for each peptide was selected and averaged amongst replicates to enable comparison.
• Heavy Chain- STS GGTAALGCLV K
Does not feature any residues susceptible to modification (M,N,Q)
- T PEVTCVVVDV SHEDPEVK Does not feature any residues susceptible to modification (M,N,Q)
• Light Chain- TVAPTECS
Observed in all reagents including Trypsin Gold
Selection of Surrogate Peptides
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Next Gen Trypsins perform much better than traditional overnight digestion
TVAPTECS Surrogate Peptide
Potential issues with peptide degradation.Rapid Digestion suggested protein:enzyme ratios are overkill
- Higher protein:enzyme ratios give comparable data
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STSG Surrogate Peptide
Rapid Digestion Trypsin generates a far greater peptide intensity in a fraction of the time!
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The traditional overnight digest far outperforms the next gen trypsins
TPEV Surrogate Peptide
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• Promega Rapid Digestion – Trypsin is our preferred next generation trypsin- It performs as comparably or better than Trypsin gold, whilst requiring a
shorter digestion time.
- Although TPEV is an exception to the rule – sensitivity demand will decide whether next gen trypsins are a viable option.
Phase 2 Outcomes
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Is this the end of two day protein LC-MS/MS
bioanalysis?
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But what are the benefits?
• Improved Sensitivity**not guaranteed
• Quicker Study Turnaround• Faster Method Development
• Rapid Troubleshooting
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Is this the end of two day protein LC-MS/MS?
Almost…
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Will a 1 hour digest actually save me any
time?
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Limited selection of enzymesSometimes trypsin just isn’t suitable
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Optimisation is Key!
Protein:Enzyme Ratio, Incubation Time and Pre-Digestion Processing all impact performance.
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Acknowledgments
• Curiosity Grant Scheme• Team Hows Large- Rob Wheller- Simon Noble- Szabolcs Szarka- Emma Duvall- Richard Lucey