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An Introduction to High Resolution Mass Spectrometry versus Tandem Quad Mass Spectrometry for Large Molecule Bioanalysis
Ian Edwards, PhD Business Development Manager
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Goals of Presentation
Performance Comparison: Tandem Quad MS versus HRMS
Principles of Large Molecule Quantification: Tandem Quad MS versus HRMS
Mass Resolution & Mass Accuracy: Tandem Quad MS versus HRMS
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Large Molecule Bioanalysis
Ligand binding assay (LBA) is the preferred method for large molecule bioanalysis – However there are concerns around reagent specificity and cost, particularly in Discovery bioanalysis
LC-MS can serve as an alternative or complementary approach to LBAs, owing to its inherent capability for direct, highly selective, mass measurement
Endogenous interference can be a significant challenge when analyzing large molecules in biological matrix without an optimized workflow (sample preparation, LC separation & MS detection)
In this module, we’ll discuss the role of MS detection and the benefits of tandem quad MS versus HRMS for large molecule bioanalysis
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Peptide and Protein Bioanalysis Workflows
LC-MS
Tandem HRMS
?
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Mass Resolution & Mass Accuracy: Definitions
Mass Accuracy: The ability to accurately measure the mass of a molecule
x106
Mass Resolution:
The ability to discriminate molecules of similar mass
The ability to accurately
Mass Resolution (R) = Peak Mass / Peak Width at Half Height (FWHM)
> R >
Mass Accuracy (parts per million, ppm) = Measured Mass – Calculated Mass
Calculated Mass
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Tandem Quad MS versus HRMS: Mass Resolution & Mass Accuracy
Parathyroid hormone, human plasma, tryptic peptide Sequence: SLGEADKADVNVLTK [M+H]+
Molecular formula: C66H114N18O25 Theoretical isotopic distributions
Tandem Quad MS can measure the mass of a peptide to the nearest decimal
HRMS can measure the mass of a peptide to within a few ppm or 3 decimal places (accurate mass)
HRMS has higher mass resolution and mass
accuracy to discriminate molecules of similar mass
High mass resolution
(R = 40, 000)
Monoisotopic mass: 1559.828 [M+H]+
Unit mass resolution (0.7 Da FWHM)
(R = 2228)
Monoisotopic mass: 1559.8 [M+H]+
Tandem Quad HRMS
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Peptide and Protein Bioanalysis Workflows
LC-MS
Tandem HRMS
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Distinguishing features of Tandem Quad MS versus HRMS
Tandem Quad MS is the gold standard for high sensitivity quantification of peptides and digested proteins Workhorse for routine LC-MS assays Limited mass range for quantification of larger intact proteins
LC-MS
Tandem
Application Note: Accurate and Sensitive LC-MS/MS Quantification of Adalimumab in Serum/Plasma: Impact of Sample Preparation on Method Performance, March 2018 720006210EN
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Distinguishing features of HRMS versus Tandem Quad MS
Higher mass resolution & accurate mass can help to resolve interference when faced with a challenging sample Different mindset and skills to tandem quad MS Wide mass range for quantification of larger intact proteins Qualitative capability for biopharmaceutical characterization including biotransformation
LC-MS
HRMS
Application of high-resolution MS for development of peptide and large-molecule drug candidates, Matthew E Szapacs et al Bioanalysis, 8 (3), 169-177, 2016
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General Principle of Large Molecule Quantification using Tandem Quadrupole Mass Spectrometry
Peptides
LC separation
Quadrupole Mass Analyzer
LC-MS
Tandem
Quadrupole Mass Analyzer Collision Cell Detector
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Peptide Selection
Precursor Selection
(~ 0.7 Da window)
Quadrupole #1
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Stage 1
Fragmentation
Unique peptide fragmentation pattern
Collision Cell
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Unique peptide fragments (products)
related to the precursor are filtered in the mass
analyzer
Stage 2
MS Filtering (~0.7 Da window)
Quadrupole #2
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Unique peptide fragments (products)
related to the precursor are filtered in the mass
analyzer
Stage 2
MS Filtering (~0.7 Da window)
Quadrupole #2
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Unique peptide fragments (products)
related to the precursor are filtered in the mass
analyzer
Stage 2
MS Filtering (~0.7 Da window)
Quadrupole #2
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Stage 2
MS Filtering (~0.7 Da window)
Quadrupole #2
MRM transitions
Fragment signal is collected in channels
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Stage 2
MS Filtering (~0.7 Da window)
Quadrupole #2
MRM transitions
Fragment signal is collected in channels
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Stage 2
MS Filtering (~0.7 Da window)
Quadrupole #2
Chromatogram MRM transitions
Signal is then integrated across the elution profile
of the peptide
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Peptide Selection Fragmentation MS Filtering (~0.7 Da window)
Key point Multi-stage process with high selectivity
Fragments are detected in channels at unit mass resolution
Tandem Quadrupole Mass Spectrometry - Multiple Reaction Monitoring (MRM)
Quadrupole #2 Quadrupole #1 Collision Cell Chromatogram MRM transitions
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Peptides
General Principle of Large Molecule Quantification using High Resolution Mass Spectrometry
LC separation
Quadrupole Collision Cell Time of Flight Mass Analyzer
LC-MS
HRMS
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Peptide Selection
Quadrupole
Precursor Selection
(~ 1Da window)
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Fragmentation
Unique peptide fragmentation pattern
Collision Cell
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MS Separation
Fragments (products) are separated over time in the mass
analyzer
Time of flight mass analyzer
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Fragments (products) are detected at high mass resolution with
accurate mass
MRM transition
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(Extraction of signal using
mDa window & accurate mass)
Fragments (products) are detected at high mass resolution with
accurate mass
Chromatogram MRM transition
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MS Separation Peptide Selection Fragmentation
Key Point Multi-stage process with high selectivity
MS separation results in high mass resolution detection of fragments with accurate mass measurement
High Resolution Mass Spectrometry - Time of Flight - Multiple Reaction Monitoring (TOF-MRM)
Collision Cell Quadrupole Time of Flight Mass Analyzer
(Extraction of signal using
mDa window & accurate mass)
Chromatogram MRM transition
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Leveraging HRMS Selectivity
An endogenous species that co-elutes with a molecule of similar mass can interfere with signal measurement
High resolution mass spectrometers have
more resolving power to discriminate molecules of similar mass (e.g. peptide fragment and interference)
– Measurement of accurate mass enables
the precise extraction of analyte signal
Chromatogram MRM transition
Chromatogram MRM transition
HRMS (Q-Tof)
Tandem Quad MS
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Leveraging HRMS Selectivity to Decrease Endogenous Interference
MS signal: Parathyroid hormone, human plasma, tryptic peptide: LQDVHNFVALGAPLAPR [M+3H]3+ y5
HRMS (50 mDa window)
0.5 ng/ml
Tandem Quad MS (~ 0.7Da window)
1 ng/ml
5 ng/ml
0.5 ng/ml
1 ng/ml
Courtesy of GSK
Blank Blank
5 ng/ml
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Case Study 3: Trastuzumab
Monoclonal Antibody, Molecular Weight: ~150 kDa
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Protein Bioanalysis Workflow: Surrogate Peptide Approach
Identify unique peptides and
transitions
Optimize / fine-tune
MS conditions
WORKFLOW
LC-MS
Protein digestion
Results
Protein clean-up (optional)
Peptide clean-up (optional)
Data processing
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Trastuzumab Method Development: Sample Preparation Affinity Purification, Sample Digestion, and Peptide Purification
Protein Level Clean-Up
• Protein A Affinity Clean-up • Wash resin to equilibrate • Load/Incubate mAb spiked rat plasma • Wash away plasma interferences • Elute mAb from resin and neutralize for protein digestion
Protein Digestion
• ProteinWorks™ eXpress Digest Kit • Denature mAb with Rapigest™ • Reduce (DTT), Alkylate (IAM), and Digest (Trypsin) mAb • Quench to stop the digestion and centrifuge to remove Rapigest and
other impurities
Peptide Level Clean-Up
• OASISTM MCX µElution SPE Clean-up • Condition and equilibrate SPE resin • Load digested mAb peptides • Wash away interferences • Elute mAb peptides • Dilute with water to improve chromatography
Trastuzumab +
Rat Plasma
Protein A – Affinity Clean-up
ProteinWorks eXpress Digest Kit
OASIS MCX µElution SPE
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FTISADTSK
Acquisition Mode Curve (µg/mL) Log10 Range Weighting Linear Fit
(R2) % Accuracy
Range Tof MRM (Precursor to Fragment) 0.025 – 500 4.3
1/X2 0.991 89.2 – 114.4
Tof MRM (Precursor to Precursor) 0.250 – 100 2.6 0.991 91.9 – 109.5 Tof MS (Full Scan) 0.250 – 100 2.6 0.997 97.3 – 102.7
HRMS Trastuzumab Quantification Performance: All Modes
Blank Rat Plasma - Rep 1
Time5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
0
100
5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
0
100
5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
0
100
5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
0
100
Blank Rat Plasma - Rep 1
Time5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
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100
5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
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5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
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5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
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Blank Rat Plasma - Rep 1
Time5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
0
100
5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
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5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
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5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50
%
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Blank
1.00 µg/mL
0.50 µg/mL
0.25 µg/mL
Tof MRM (Precursor to Precursor) Tof MS (Full Scan)
485.25 > 721.37 Peak Area: 62.8
485.25 > 721.37 Peak Area: 30.0
485.25 > 721.37 Peak Area: 10.4
485.25 > 721.37 Peak Area: 0.11
485.25 > 485.25 Peak Area: 654
485.25 > 485.25 Peak Area: 417
485.25 > 485.25 Peak Area: 224
485.25 > 485.25 Peak Area: 76
485.25 XIC Peak Area: 1477
485.25 XIC Peak Area: 969
485.25 XIC Peak Area: 626
485.25 XIC Peak Area: 150
Mass Extraction Window: 20 mDa Mass Extraction Window: 20 mDa Mass Extraction Window: 20 mDa
Tof MRM (Precursor to Fragment)
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Trastuzumab Quantification Performance: HRMS versus Tandem Quad MS
HRMS LLOQ within 2X of TQ-XS
FTISADTSK
Acquisition Mode Curve (µg/mL) Log10 Range Weighting Linear Fit
(R2) % Accuracy
Range Xevo TQ-XS (MRM) 0.010 – 250 4.4 1/X2 0.988 85.0 – 111.6
Xevo G2-XS (Tof-MRM) 0.025 – 500 4.3 0.991 89.2 - 114.4
Blank Rat Plasma - Rep 2
Time4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40
%
0
100
4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40
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4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40
%
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4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40
%
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100
Blank Rat Plasma - Rep 2
Time4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40
%
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100
4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40
%
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4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40
%
0
100
4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40
%
0
100
0.010 µg/mL
0.050 µg/mL
0.025 µg/mL
Blank
Xevo TQ-XS (MRM) Xevo G2-XS (Tof-MRM) 485.25 > 721.37 Peak Area: 3847
485.25 > 721.37 Peak Area: 2016
485.25 > 721.37 Peak Area: 1129
485.25 > 721.37 Peak Area: 95
485.25 > 721.37 Peak Area: 3.48
485.25 > 721.37 Peak Area: 1.55
485.25 > 721.37 Peak Area: 1.30
485.25 > 721.37 Peak Area: 0.11
LLOQ
LLOQ
Mass Extraction Window: 20 mDa
App note: Comparison of Tandem and High Resolution Mass Spectrometry for the Quantification of the Monoclonal Antibody, Trastuzumab in Plasma, March 2018 720006207EN
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Tandem Quad MS – The go-to when the highest levels of sensitivity are required – Workhorse for routine LC-MS assays (peptides, small proteins and digested proteins)
HRMS
– Challenging samples that require higher selectivity – Wide mass range for intact quantification of larger proteins – Quantitative & qualitative characterization of biopharmaceuticals (including variants & biotransformation)
Tandem Quad MS and HRMS are complementary technologies for large molecule quantification and together provide the capability to tackle the most challenging and diverse bioanalytical samples
Summary
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Tof-MRM
Precursor to Fragment Monitor selected fragments of
selected peptides
Tof-MRM Precursor to Precursor
(similar to SIM) Monitor selected peptides
MS/MS (similar to PRM)
Monitor all the fragments of selected peptides
MS (Full Scan) Monitor all peptides or all proteins in a sample
MSE (Full Scan)
Monitor all peptides and proteins and
all fragments in a sample
Five major modes of operation for quantification of peptides and proteins
Low to medium complexity samples, monitor many targets at once
Complex samples, highly selective, highly sensitive, monitor specific targets of interest
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1
2
Modes explained
MS MSE MS/MS Tof-MRM
Align Peptides with their Fragments
Detection of all Fragments from a Peptide
Detection of Specific Fragment/s + Target Enhancement to Boost Signal
Q1 Collision Cell Tof
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What’s Target Enhancement?
Waters QTofs have the ability to preferentially ‘push’ molecules into the Tof
With Tof-MRM we know which mass we are interested in, so we can maximize time spent pushing the molecule of interest into the mass detector
More pushes on target molecule = More detections Ultimate sensitivity
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For peptides, small proteins and digested proteins – Tof-MRM and MS/MS based approaches work well – You can maximize sensitivity/selectivity against the matrix similar to SRM
based approaches – See Case Studies in HRMS for Large Molecule Bioanalytical
Quantification to learn more (http://dmpk.waters.com/en/boot-camp)
For larger peptides and proteins (including species that don’t fragment very well) – MS (Full Scan) is recommended, flexibility to choose best charge states
and m/z regions which are less interfered with by matrix – See HRMS for Intact Protein Quantification to learn more
(http://dmpk.waters.com/en/boot-camp)
What mode should I choose?
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Tandem Quad MS
– The instrument of choice when the highest levels of sensitivity are required – Workhorse for routine LC-MS assays (peptides, small proteins, and digested proteins)
HRMS – Comparable sensitivity to tandem quad MS for large molecule bioanalytical quantification – Mass resolution & accurate mass can help to resolve interferences
o Tof-MRM (Precursor to Precursor) is quick to set up and useful for poorly fragmenting peptides o Tof-MRM (Precursor to Fragment) yields ultimate selectivity
HRMS Large Molecule Bioanalytical Quantification Summary
Tandem Quad MS and HRMS are complementary technologies for large molecule quantification and together provide the capability to tackle the most challenging and diverse bioanalytical samples