waters oligonucleotide analysis solutions
TRANSCRIPT
©2017 Waters Corporation 1COMPANY CONFIDENTIAL
Oligonucleotide Analysis SolutionsFrom Characterization to QC
©2017 Waters Corporation 2COMPANY CONFIDENTIAL
Optimized LC-MS Workflows for Oligonucleotide Characterization and QC Analysis
SEPARATIONS DETECTION & INFORMATICS
MassPREP StandardQuality reference material
Oligonucleotide Columns with BEH TechnologySuperior temperature and pH stability; exceptional sample resolution and column life
Bio-inert flow path and the benefits of UPLC resolution, sensitivity, and throughput for optimal performance
Characterization and High Res Monitoring: ACQUITY UPLC H-Class Bio with TUV and Xevo G2-XS QTof
+ProMass HR
+ProMass or
ProMass HR
UPLC-MS
UPLC-HRMS
QC Analysis with Mass Detection: ACQUITY UPLC H-Class Bio with TUV and ACQUITY QDa Mass Detector
UPLC-HDMS
+ProMass HR
Characterization with ion mobility: ACQUITY UPLC H-Class Bio with TUV and SYNAPT G2-Si
©2017 Waters Corporation 3COMPANY CONFIDENTIAL
Tools for Achieving Optimal Oligonucleotide Separations
©2017 Waters Corporation 4COMPANY CONFIDENTIAL
ACQUITY H-Class Bio UPLC Liquid Chromatography System
Unrivaled UPLC resolution, sensitivity and throughput for optimum peak clarity and selectivity
Quaternary Solvent Manager (QSM) with Auto ●Blend Plus™ for fully automated multi-solvent blending
Bio-inert flow path with excellent corrosion resistance - uniquely suited for the high ionic strength aqueous conditions commonly used for biomolecule separations
*Note: The ACQUITY H-Class Bio is our recommended LC system for oligonucleotide analysis, though other Waters LC systems may be used
©2017 Waters Corporation 5COMPANY CONFIDENTIAL
Industry Leading Waters Oligonucleotide Columns with BEH* Technology
ACQUITY UPLC columns for optimum speed, performance and throughput – BEH, C18, 130Å, 1.7µm Particles:
o 50 x 2.1 mm, 1.7 µm columno 100 x 2.1 mm, 1.7 µm column
Xbridge™ HPLC/UHPLC columns for routine analysis and lab scale purification– BEH, C18, 130Å, 2.5µm Particles:
o 50 x 2.1 mm, 2.5 µm columno 50 x 4.6 mm, 2.5 µm columno 50 x 10 mm, 2.5 µm column
Unique Patented *Bridged Ethyl Hybrid (BEH) Technology delivers N-1 oligo resolution and superior column life at elevated pH values and temperatures – widely considered the industry gold standard
©2017 Waters Corporation 6COMPANY CONFIDENTIAL
Longevity of BEH Particles vs. Silica for Oligo Separations at High pH & Temp.
Accelerated High pH Stability Test of Competitive Columns
©2017 Waters Corporation 7COMPANY CONFIDENTIAL
Waters MassPREP™ Oligonucleotide Standard
Note: Every batch of ACQUITY and XBridge Oligonucleotide column packing material is QC Tested with this standard to ensure performance and consistency
High quality oligonucleotide reference material for calibration, troubleshooting and system suitability.
P/N 186004135
©2017 Waters Corporation 8COMPANY CONFIDENTIAL
++++ -
--
-
- ---+C18 sorbent
Ion Pairing Reverse Phase(IP-RP) Chromatography for Optimal Separations
Reversed Phase interaction with ion-pairing reagent- Hydrophobicity of the nucleobases +charge-charge interaction (oligo backbone)
Reversed-phase interaction without Ion-Pairing reagent- hydrophobicity of the nucleobases
0 minutes 100 minutes 10
1520
2535
3015 20
25
35
30
TEA+
TEA+ layer on the column surface
©2017 Waters Corporation 9COMPANY CONFIDENTIAL
Ion Pairing Reagents for Oligonucleotide LC-MS Analysis
Gong, L. and J. S. O. McCullagh (2014). "Comparing ion-pairing reagents and sample dissolution solvents for ion-pairing reversed-phase liquid chromatography/ electrospray ionization mass spectrometry analysis of oligonucleotides." Rapid Communications in Mass Spectrometry 28(4): 339-350.
TEA-HFIP was first ion pairing system for high efficiency LC/MS analysis of oligonucleotidesApffel, A., et al. (1997). "Analysis of Oligonucleotides by HPLC−Electrospray Ionization Mass Spectrometry." Analytical Chemistry 69(7): 1320-1325.
Waters scientists expanded on these capabilities over the following decade + of research
Apffel, A., et al. (1997). "New procedure for the use of high-performance liquid chromatography–electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides." Journal of Chromatography A 777(1): 3-21.
Gilar, M., et al. (2002). "Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction." Journal of Chromatography A 958(1–2): 167-182.
Fountain, K. J., et al. (2003). "Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry." Rapid Communications in Mass Spectrometry 17(7): 646-653.McCarthy, S. M., et al. (2009). "Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA." Analytical Biochemistry 390(2): 181-188.
©2017 Waters Corporation 10COMPANY CONFIDENTIAL
Range of Ion-Pairing Reagents Used Today for Oligonucleotide Analysis
Ion Pairing Agent Buffering Acid AbbreviationTriethylammonium Acetate TEAATriethylammonium Bicarbonate TEABDimetylbutylammonium Acetate DMBAATributylammonium Acetate TBAATripropylammonium Acetate TPAAHexylammonium Acetate HAATriethylammonium Hexafluoroisopropanol TEA-HFIP
Selectivity and resolution are impacted by choice We have found TEA-HFIP to be the most effective for single-stranded
oligo analysis in terms of achieving optimal LC-MS sensitivity and resolution, and it is used throughout this presentation
©2017 Waters Corporation 11COMPANY CONFIDENTIAL
Triethylammonium HFIP Reagent
Triethylammonium Acetate Reagent
0 30Minutes
13 14 15 1618
20
21 22 26
29
25
28
30
27
24
23
19
171211
UV 2
60 n
m
10 32
24
11 12 13 14 15 16+17
18+19
20
21 2223
25
28
29
30
Minutes
UV 2
60 n
m
T
G
GG
C
G
A
TTT
C
C
TGTTAAGT
TEAA vs. TEA-HFIP for IP-RP Separation of an Oligo Mixture
©2017 Waters Corporation 12COMPANY CONFIDENTIAL
Recent work: Alternative HFIP LC/MS Buffer systems
400 mM HFIP, pH 8.0
25 mM HFIP, pH 9.5
50 mM HFIP, pH 9.0
Triethylamine
Butylamine
Dibutylamine
Cost
$
$$$$$
$$$ 10x less
20x less
“standard”
©2017 Waters Corporation 13COMPANY CONFIDENTIAL
Injection # 48 98 153 203 248 317 365 429Peak 4/5Peak 3/5Peak 2/5
Peak 1/5
BEH Columns Perform well under High pH
OST BEH, C18, 135Å, 1.7µm50 x 2.1 mm column
buffer exchange
Peak 4/5Peak 3/5Peak 2/5
Peak 1/5
Relative retention time to peak 5 (35 nt)15mM BA:50mM HFIP, pH 9.0
1 2 3 4 5
Stable chromatography over 400 injections using butylamine/ 50mM HFIP buffer
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Peak Width (W50) Inter assay Intra assay
Mean < 0.08 min 0.08 minS.D. < 0.003 0.002R.S.D (%) < 3.5 2.49
Hour 29, inj # 53
Hour 124, inj # 243
Hour 193, inj # 421
Ruggedness of BEH Columns
Average Peak width (W50) vs. Hours at pH 9.0
stable column performance in harsh conditions
15nt 20nt 25nt 30nt 35nt
N-1
N-1
N-1
Highly Reproducible and robust column performance over 200 hours at pH 9.0, 60 C using butylamine/ 50mM HFIP buffer
©2017 Waters Corporation 15COMPANY CONFIDENTIAL
Troubleshooting Oligonucleotide LC-MS- Quality of Mobile Phase Matters!
LC-MS grade solvents and additives should be used MS-grade TEA purchased fromSigma (Fluka 65897-50ML-F)
MS-grade HFIP purchased from Sigma (Fluka 42060-50ML)
©2017 Waters Corporation 16COMPANY CONFIDENTIAL
Troubleshooting Oligonucleotide LC-MS- Mobile Phases Need to be Fresh!
We recommend mobile phases be prepared daily to prevent MS signal loss
8.84E7
0.2556
5.87E7
0.2563
1.13E7
0.2609
1st injection
Base-line
+ 9 hours
~30% drop in MS response
+ 2.5 days
~87% drop in MS response
Remake Buffer
Recover MS response
1.43E8
0.213
TUV
MS
TIME
©2017 Waters Corporation 17COMPANY CONFIDENTIAL
Troubleshooting Oligonucleotide LC-MS- System Cleanliness Matters!
Contaminatedsystem
Clean system
Contaminatedsystem
Clean system
Systems previously used for aqueous based separations may require a multi-step cleaning protocol
LC-HRMS/HDMS systems will require extended flushing with 50:50 H2O:MeOH, 0.1%FA to reduce phosphate adduct from cleaning procedure
©2017 Waters Corporation 18COMPANY CONFIDENTIAL
Recent Work: A simple, highly effective protocol for reducing the accumulation of metal adducts
Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/ mass spectrometry analysis
Robert E. Birdsall, Martin Gilar, et.al. Volume 30, Issue 14 | 30 July 2016 (pages 1667–1679)
This manuscript by Robert Birdsall, et.al., was featured on the cover of RCM in July, 2016. It presents a simple, highly effective solution to a historic problem with oligonucleotide analysis - the accumulation of metal adducts over time, which degrade the MS signal and lead to inconsistent performance.
©2017 Waters Corporation 19COMPANY CONFIDENTIAL
Waters Oligo Applications Notebook
Download @ waters.com/oligos
Contains 24 application notes illustrating the oligonucleotide separations performance and productivity that can be achieved using Waters column chemistries, reagent standards, HPLC & UPLC systems, and LC-MS workflows:
• UPLC-MS• UPLC-HRMS• UPLC-HDMS
©2017 Waters Corporation 20COMPANY CONFIDENTIAL
UPLC-MSScreening for Oligo ID and Purity
with the ACQUITY QDa Mass Detector
Our most deployable and scalable solution Easy-to-use with a low maintenance requirement
Quickly confirm product ID & assess purity
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The ACQUITY® QDa™ Mass Detector
Revolutionary innovative design focused on ease of use for analysts
Empowering analytical chemists everywhere with orthogonal mass detection – added information with every sample
Compact, robust and affordable - built for constant use with a wide variety of conditions
Seamlessly integrates with Empower and MassLynx based HPLC and UPLC™ systems
©2017 Waters Corporation 22COMPANY CONFIDENTIAL
The QDa is Easily Deployed – Like adding an optical detector
+ QDa
QDa Mass Detector
Empower® and MassLynx™ Control Minimal operator training requiredQualification available110/220V operationWorkhorse - minimal maintenance
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Automated Start Up and CalibrationEnsures Reproducible Performance
Automated resolution and calibration with each start-up ensures mass information is accurate, precise and reproducible– No tuning required!
ESI source optimized for UPLC ensures chromatographic resolution, sensitivity and throughput is preserved
Graphic QDa™ monitor display enables easy viewing and adjustment of system parameters
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Familiar Graphical User Interfaces for Ease-Of-Use and Fast Adoption
Interface identical to that of a PDA for setup, data viewing and reporting
Very little training needed Quick addendum to current lab
SOP
MassLynx delivers additional functionality
Ability to deconvolute data Export to ProMass HR for
assignments
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Disposable Sample Aperture and Capillary for Easy Maintenance
Sample Aperture:As simple as replacing a detector lamp
Capillary:
No cutting or assembly required
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ACQUITY QDa in Summary
A breakthrough mass detector that enables analytical chemists / chromatographers to collect mass data on a routine basis
Delivers greater insight into every peak; enhances monitoring and streamlines workflows for improved productivity
Easily deployed, simple to maintain, compact, robust and affordable!
©2017 Waters Corporation 27COMPANY CONFIDENTIAL
TUV
15 nt20 nt 25 nt
30 nt35 nt
QDa Proof-of-Principle with Waters MassPREP™ OST Standard
nt Avg. MW [M-4H]-4 [M-5H]-5 [M-6H]-6 [M-7H]-7 [M-8H]-8 [M-9H]-9 [M-10H]-10 [M-11H]-11 [M-12H]-12 [M-13H]-13 [M-14H]-14 [M-15H]-15 [M-16H]-16 [M-17H]-17
15 4,500.9 1124.2 899.2 749.1 642.0 561.6 499.1 449.1 408.2 374.1 345.2 320.5 299.1 280.3 263.820 6,021.9 1504.5 1203.4 1002.6 859.3 751.7 668.1 601.2 546.4 500.8 462.2 429.1 400.5 375.4 353.225 7,542.9 1884.7 1507.6 1256.1 1076.5 941.9 837.1 753.3 684.7 627.6 579.2 537.8 501.9 470.4 442.730 9,063.8 2265.0 1811.8 1509.6 1293.8 1132.0 1006.1 905.4 823.0 754.3 696.2 646.4 603.2 565.5 532.235 10,584.8 2645.2 2116.0 1763.1 1511.1 1322.1 1175.1 1057.5 961.2 881.1 813.2 755.0 704.6 660.5 621.6
green = observable charge states
QDa50 pmol on-column
Multiple charge states observed per oligonucleotide
Detection readily observed with low pmol column loads
Quantification based on UV Channel with MS providing mass confirmation P/N 186004135
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Robust Reproducible Performance
15 nt 20 nt25 nt
30 nt35 nt
646.5
696.2
754.4
823.1
905.41006.2 1131.8
n=3 [M-8H]-8 [M-9H]-9 [M-10H]-10 [M-11H]-11 [M-12H]-12 [M-13H]-13 [M-14H]-14 [M-15H]-15
Expected m/z 1132.0 1006.1 905.4 823.0 754.3 696.2 646.4 603.2
Average m/z 1132.1 1006.1 905.4 823.1 754.3 696.2 646.5 603.4
Delta +0.1 0.0 0.0 +0.1 0.0 0.0 +0.1 +0.2
%RSD 0.01 0.01 0.00 0.00 0.01 0.01 0.02 0.01
All data within QDa mass accuracy specification: +/- 0.2
TIC Trace:50 pmol on-column
Observed charge states for 30nt oligo: (-8 to -14)
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MaxEnt1
Deconvolution
Manual Deconvolution with MaxEnt1
30 nt35 nt
Calculated Average Mass of 30nt oligo = 9,063.8 Da
9,064.0 Da
+Na
Deconvoluted Spectrum
9,086.0 Da
Raw ESI Spectrum
646.5
696.2
754.4
823.1
905.41006.2 1131.8
MaxEnt1 deconvolution is available within MassLynx, and provides for manual deconvolution of both nominal mass and high resolution data.
©2017 Waters Corporation 30COMPANY CONFIDENTIAL
Enabling Automated Data Workflowswith ProMass and ProMass HR
“Automated” deconvolution and reporting package Supports both nominal mass and high resolution mass spec (HRMS) data Enables higher throughput workflows
+
Znova™ deconvolution for nominal mass data (Quadropole)
Respect™ deconvolution for high res data (QTof) – only available with Promass HR
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ProMass Workflow
MassLynxAcquisition
• Acquisition of LC-MS(/MS) data
• Automatically calls ProMass Bridge
ProMass“Bridge”
• Informatics tool that enables the transfer of data from MassLynx to ProMass for automated batch processing
ProMassProcessingand Reporting
• Automated deconvolution
• Assesses / confirms target mass and purity
• Assignment of impurity peaks
• Report Generation
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ProMass: Deconvolution Parameters
Peak width is set to encompass the width at the base of most peaks from the input ESI mass spectra. A typical Peak Width value is 2-3 m/z units.
Merge Width determines the m/z window around the detected peaks where intensities are summed (merged) to compute a centroided value. Typically, this is set to 10 - 25% of the Peak Width parameter (e.g., 0.2-0.5 for 2 m/z unit peaks)
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ProMass: Batch Processing Parameters
Results Tab Reporting Tab
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ProMass: Report Generation
Summary Report
ESI Mass Spectrum
Deconvoluted Spectrum
Magnified Deconvolution
Deconvolution Peak Reports
Note:• Requires dongle to process
(can not remote in)• PDF also available
• A top-level summary page that shows results from multiple runs and provides hyperlinks that allows users to quickly drill down into individual results
• Target mass features allows one to automatically search for one or more target masses from the LC-MS data
©2017 Waters Corporation 35COMPANY CONFIDENTIAL
ProMass: ZNova Mass Accuracy based on Waters MassPREP™ Oligonucleotide Standard
n=3 nt 15 nt 20 nt 25 nt 30 nt 35Expected Mass (Da) 4,500.9 6,021.9 7,542.9 9,063.8 10,584.8
Observed Mass (Da) 4,500.8 6,022.0 7,543.3 9,063.8 10,585.3
mass -0.1 0.1 0.4 0.0 0.5
SD 0.06 0.17 0.20 0.10 0.06
15 nt20 nt
25 nt
30 nt35 nt50 pmol
on-column Triplicate Analysis
2 minute window centered around each peak used for combined spectra
QDa mass data deconvoluted with Promass Znova™ algorithm
Mass errors within 1.0 Da of expected
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Demonstrating QDa Enabled Mass Confirmation & Impurity Profiling
Upper Strand (50 pmol)
TUV, 260 nm QDa, 410-1250 m/z
Upper Strand, MW 6693.1 Da5’-UCGUCAAGCGAUUACAAGGTT-3’
Lower Strand, MW 6607.0 Da5’-TTCCUUGUAAUCGCUUGACGA-3’
Sample: Two distinct 21nt ssRNA oligos representing the complementary upper and lower strands of a siRNA duplex (10 pmol/uL)
Time (min)
Flow (ml/min)
A B C D
Initial 0.200 82.0 18.0 0.0 0.0
4.00 0.200 80.0 20.0 0.0 0.0
4.01 0.200 50.0 50.0 0.0 0.0
6.00 0.200 50.0 50.0 0.0 0.0
6.01 0.200 82.0 18.0 0.0 0.0
4 Minute Gradient: 0.5%/min
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Summary Report of Batch Processing Results with Interactive Display
Target Mass: 6693.1 Da (Upper Strand)
Data for 48 samples processed in < 10 min
100% match with expected results -displayed in easy-to-use color-coded format
Multiple target masses can be searched
96-well plate format
Click for interactive sample report (following slide)
©2016 Waters Corporation 38
Interactive ProMass Sample Report
Total Ion Chromatogram
Blue Text = Hyperlinks to data views
Raw ESI Spectrum Deconvoluted Spectrum
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Data Analysis (upper strand): Retention Time and Mass Accuracy
2
2.1
2.2
2.3
2.4
2.5
2.6Retention Time (min)
Vial position
Rete
ntio
n tim
e (m
in)
N=42 Retention time (min)
Average 2.44S.D. 0.01
%RSD 0.57
A1 A3 A5 A8 B2 B4 B7 C1 C3 C6 C8 D2 D5 D7 E1 E4 E6 E8 F3 F5 F7-1
-0.8-0.6-0.4-0.2
00.20.40.60.8
1Mass Error (Da)
Vial position
Mas
s er
ror
(Da)
N=42 Mass (Da)Expecte
d 6693.1Average 6693.3
S.D. 0.3
Consistent retention times across all 42 samples
All data within 1 Dalton of expected mass
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ProMass: Spectral Analysis for Purity
A1 A3 A5 A8 B2 B4 B7 C1 C3 C6 C8 D2 D5 D7 E1 E4 E6 E8 F3 F5 F780828486889092949698
100% Abundance in Spectrum
Vial position
Spec
tral
abu
ndan
ce (
%)
N=42 % Abundance
Average 94.11S.D. 1.76
R.S.D. 1.87
A5
E6
N=40 % Abundance
Average 94.46S.D. 0.79
R.S.D. 0.84
Non target masses other than predicted adducts are calculated as impurities
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UPLC-HRMSCharacterization and HRMS Monitoring
with the Xevo G2-XS Qtof
Robust High Resolution MS Enhanced Sensitivity for Characterization Work
MS/MS for Sequence Confirmation
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Demonstrating High Res Performance using the MassPREP™ Oligonucleotide Standard
Instrumentation LC: Waters ACQUITY® H-Class Bio MS: Xevo G2-XS Qtof
Column Chemistry ACQUITY UPLC OST BEH C18 Column 130Å, 1.7
µm, 2.1 mm X 50 mm (P/N: 186003949)
Waters MassPREP Oligonucleotide Standard Reconstituted to 5 pmol/µL in pure H2O 1 µL of sample injected /LC-MS analysis
Method 15 min Ion Pairing RP gradient (TEA/HFIP) Data collected by TUV and by ESI- MS
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MassPREP™ Oligonucleotide Standard:TUV and TIC comparisonLoad: 5 pmol on-column
TUV
TIC
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MassPREP™ Oligonucleotide Standard: Raw ESI Spectra for 25nt Oligo
4-
3-5-6-7-8-
9-
10-
11-
12-
M3- charge state isotopic distribution
Resolution: > 30K
5 pmol on-column
+Na+
2508 2530m/z
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Promass HR: Respect™ Deconvolution Mass Accuracy based on MassPREP Standard
Deconvoluted spectrum for 30nt
OST Standard
Expected Mass
Observed Mass
Mass Accuracy
(ppm)
15nt 4498.7348 4498.7300 -1.07
20nt 6018.9650 6018.9730 1.33
25nt 7539.1952 7539.1990 0.50
30nt 9059.4254 9059.4210 -0.49
35nt 10579.656 10579.6260 -2.79
Avg. 1.24
Mass accuracy: < 5 ppm
Promass HR includes the Respect™ deconvolution algorithm for processing high resolution MS data
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MassPREP Oligonucleotide Standard:-Determining Limit of Detection (LOD)
TUV
TIC
Load: 20 fmol on-column
Detection limited by UV signal, not MS sensitivity
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Evaluating performance with complementary ssRNA strands
Upper Strand, MW 6693.1 Da5’-UCGUCAAGCGAUUACAAGGTT-3’
Lower Strand, MW 6607.0 Da5’-TTCCUUGUAAUCGCUUGACGA-3’
1 µL injected for LC-MS analysis.
Sample: Two distinct 21nt ssRNA oligos representing the complementary upper and lower strands of a siRNA duplex (10 pmol/uL). 10 minute gradient for high resolution chromatogram
Oligo sourced from IDT
©2017 Waters Corporation 49COMPANY CONFIDENTIAL
Upper Strand:10 Min High-Resolution Chromatogram
N
N-1 N+1
N-1 = 5’-rUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGrTT-3’Average mass = 6386.9 Da; ProMass HR observed mass:6386.8 Da
N = 5’-rUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGrTT-3’Average mass = 6693.1 Da; ProMass HR observed mass:6693.0 Da
N+1 = 5’-rGrUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGrTT-3’Average mass = 7038.3 Da; ProMass HR observed mass:7038.7 Da
A ssRNA sequence 5’-UCGUCAAGCGAUUACAAGGTT-3’ with a double thymine overhang was separated from the base deletion (N-1) and base insertion (N+1) forms using a 10 min high resolution separation gradient from 13% B to 21% B.
©2016 Waters Corporation 50
Upper Strand: Extracted TIC (XIC) of N (Target), N+1 and N-1 species
N+1
N (Target)
N-1XIC’s
time
©2016 Waters Corporation 51
Upper Strand:QTof ESI Raw Spectrum
4-
3-
5-6-
7-
8-
9-
M3- charge state isotopic distribution.
2199 2205m/z
©2016 Waters Corporation 52
Summary Report of Batch Processing Results with Interactive Display
Promass HR Respect™ algorithmn used
Target Mass: 6689.9550 Da (Upper Strand)
Data processed for all 48 samples in < 10 minutes
100% match with expected results -displayed in easy-to-use color-coded format
©2016 Waters Corporation 53
Interactive ProMass Sample Report
Total Ion Chromatogram Raw ESI Spectrum Deconvoluted Spectrum
Blue Text = Hyperlinks to Data Views
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Oligo MS/MS Sequence Confirmation
Collision of oligonucleotides with gas molecules at elevated energies produces characteristic fragment ions at each residue
McLuckey Fragmentation Schemec, y, & w ions predominate with QTof fragmentation
©2016 Waters Corporation 56
LC-MS/MS Sequence Confirmation - Upper Strand (M4- Peak Deconvoluted*)
y2
c2
w2
y3
c3
w3
y4
c4
w5
y5
c5w4
y6
c7
w7
y7
w6
y8c9
y9
c8
y11 y12y10
c10
c16
w13
c15
c12 y14c14
Y17y19c18
y20
-G
-G
M
y16
y18
c6
w8
w9 w10
Y13
-G
w14
U C G U C A A G C G A U U A C A A G G T T5’- -3’
5 pmol on-column
*Deconvolution using MaxEnt-3, which is available within Mass Lynx
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UPLC-HDMSCharacterization with Ion Mobility using the
SYNAPT G2-Si Qtof
Ion Mobility brings an added dimension of separation that translates into greater spectral clarity and enhanced
selectivity and sensitivity, especially for low level impurities
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Charge State / Drift Time Stripping – Reduces spectral complexity; makes data more suitable for deconvolution– Observe fragment ion series for each IMS resolved charge state
Wideband Enhancement– Improve the resulting signal intensity (ca. 5-10 fold) of MS/MS spectra for low
level impurities
Time Aligned Parallel (TAP) Fragmentation– Separation of primary fragment ions via ion mobility enables selection of
specific primary fragment ions for secondary fragmentation - Pseudo MS3
3 Ways MS/MS - Ion Mobility Spectrometry (IMS) Can Enhance Selectivity and Sensitivity
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Charge State / Drift Time Stripping Improves Clarity of Resulting MS/MS Spectra
Analysis of PEG 4450 via HDMS
DriftScope™ data showing the gas-phase separation power of the SYNAPT HDMS System. Components with different charge states are separated via ion mobility, thus enabling the examinations of different (minor) components in the PEG material.
Waters Application Note:Characterizing Polyethylene Glycol (PEG) by SYNAPT High Definition Mass Spectromet ry (HDMS).Petra Olivova, Weibin Chen, and John C. Gebler
Mass spectrum showing the ions with +2 charge state. Improved mass spectral clarity makes data more suitable for charge state deconvolution algorithms.
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Wideband Enhancement
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Time Aligned Parallel (TAP) Fragmentation
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In Summary - Our LC-MS Workflows for Oligonucleotide Analysis:
UPLC-MS ACQUITY QDa
HPLC-HRMSXevo G2-XS QTof
UPLC-HDMSSYNAPT G2 Si QTof
Mass Accuracy – Deconvoluted Data +/- 1.0 Dalton < 5 PPM
(~0.038 Da)< 5 PPM
(~0.038 Da)Typical Column Load 50 pmol 5-10 pmol 5-10 pmolLimit of Detection < 10 pmol 20 fmol 20 fmolMass Confirmation Impurity Profile Impurity ID MS/MS Sequencing
(in source)
Cost $ $$$ $$$$
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To Learn More Visit: WWW.Waters.com/oligos
or Contact your local Waters Account Representative
Thank You