an introduction to high resolution mass spectrometry ... · an introduction to high resolution mass...

©2018 Waters Corporation 1 COMPANY CONFIDENTIAL

An Introduction to High Resolution Mass Spectrometry versus Tandem Quad Mass Spectrometry for Large Molecule Bioanalysis

Ian Edwards, PhD Business Development Manager


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


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


Peptide and Protein Bioanalysis Workflows

LC-MS

Tandem HRMS

?


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


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


Peptide and Protein Bioanalysis Workflows

LC-MS

Tandem HRMS


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


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


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


Peptide Selection

Precursor Selection

(~ 0.7 Da window)

Quadrupole #1


Stage 1

Fragmentation

Unique peptide fragmentation pattern

Collision Cell


Unique peptide fragments (products)

related to the precursor are filtered in the mass

analyzer

Stage 2

MS Filtering (~0.7 Da window)

Quadrupole #2




analyzer

Stage 2


Quadrupole #2


Stage 2


Quadrupole #2

MRM transitions

Fragment signal is collected in channels


Stage 2


Quadrupole #2

Chromatogram MRM transitions

Signal is then integrated across the elution profile

of the peptide


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


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


Peptide Selection

Quadrupole

Precursor Selection

(~ 1Da window)


Fragmentation

Unique peptide fragmentation pattern

Collision Cell


MS Separation

Fragments (products) are separated over time in the mass

analyzer

Time of flight mass analyzer


Fragments (products) are detected at high mass resolution with

accurate mass

MRM transition


(Extraction of signal using

mDa window & accurate mass)

Fragments (products) are detected at high mass resolution with

accurate mass

Chromatogram MRM transition


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)



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



HRMS (Q-Tof)

Tandem Quad MS


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


Case Study 3: Trastuzumab

Monoclonal Antibody, Molecular Weight: ~150 kDa


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


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


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

%

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100

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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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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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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




Mass Extraction Window: 20 mDa Mass Extraction Window: 20 mDa Mass Extraction Window: 20 mDa

Tof MRM (Precursor to Fragment)


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


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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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

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

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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

%

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

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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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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


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

©2018 Waters Corporation 35

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


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


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


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?

http://dmpk.waters.com/en/boot-camp

http://dmpk.waters.com/en/boot-camp


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

an introduction to high resolution mass spectrometry ... · an introduction to high resolution mass...

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