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Quantitative Bioanalysis of Oligonucleotides
Laixin Wang, [email protected]
May 24, 2016Thermo BioSeparations Workshop
Innovative Mass Spectrometric Solutions
1
Outline
• Overview
– Thermo instruments at NovaBioAssays
– Bioanalysis of oligonucleotide therapeutics
• DNApac PA200 columns for hybridization based LC-fluorescence assays
• DNApac RP columns for ion-pair LC-MS/MS and LC-HRAM analysis
– Full Scan, target-SIM and PRM/DIA analysis on Q-Exactive
• Q & A
2
3
52 Dragon Court, Suite 3BWoburn, MA 01801, USAPhone: (781)933-3480
Where Are We
3
4
What Do We Have (1/3)
Bruker MicroTOFQ II(nano and ESI source)
(x2)
Thermo Q Exactive MS (Plus)(x2)
Waters Acquity UPLCWaters nanoAcquity UPLC
(x7)
Thermo TSQ Vantage(nano and ESI source)
AB SCIEX 5000 LC/MS/MS(X2)
Accela 1250 and CTC PAL Thermo Easy nLC
Thermo Vanquish Flex UHPLC System With Vanquish™ Fluorescence
Detector F (4 Excitation/Emission)
What Do We Have (2/3)
Waters Acquity UPLC with PDA
And Fluorescence Detectors
What Do We Have (3/3)
6
Thermo KingFiser Flex 96Magnetic Particle Processor
Thermo Versette™ Automated Liquid Handler
TomTec Quadra 3 SPEAutomated Liquid Handler
(x2)
PPE, LLE, SPE Affinity/Immunoaffinity Purification
Mass Spectrometric Immunoassay (MSIA)
Quantitative Bioanalysis (API5000 or Q-Exactive or FD/PDA)• Protein bioanalysis using LC-MS/MS or LC-HRAM
• Lipids/Polymers using LC-MS/MS or LC-HRAM
• Small molecule analysis using LC-MS or LC-HRAM
• Oligonucleotides bioanalysis using
LC-MS/MS or LC-HRAM
Hybridization UPLC fluorescence
UPLC-UV
Qualitative analysis (Q-Exactive and Q-tof)• Primary structural characterization using LC-HRAM and LC-MS/MS
• Tertiary structural characterization using H/D exchange LC-HRAM and LC-MS/MS
• Quant-Qual analysis using LC-Q Exactive
In Vitro Protein Binding and Stability/Metabolite ID• Rapid Equilibrium Dialysis(RED) and Ultracentrifugation
• Microsomal/Plasma/Whole blood stability
• Hepatocyte incubation
What Can We Do?
7
Quantitative Bioanalysis of Oligonucleotides
8
N
O B
PN O
O
O
OO
O B O
OHO
O B
LNA UNA
N
NH
O
B
O
PNA PMO
P
O
O OH PO OH
O
Chemical Structures of Oligonucleotides
9
O
O
O
O
O
O
R
PO
X-
B1
B2
R
X = O (Phosphodiester)S (Phosphorothioate)
R = -H (DNA)-OH (RNA)-OCH3 (2’-Methyl)-F (2’-Fluoro)-OCH2CH2OCH3 (2’-MOE)
Quantitative Bioanalysis of Oligonucleotides/mRNA
10
• Hybridization-based assay (indirect analysis)
– Northern blot/in situ hybridization
– Microarray
– Deep sequencing (NGS)
– RT-qPCR
– Hybridization ELISA
• Direct ELISA, Sandwich ELISA, etc
• Branched DNA (bDNA) signal amplification technology (QuantiGene)
• Chromatographic-based assay (direction analysis)
– HPLC-UV/Fluorescence
– Hybridization-based LC-fluorescence
– LC-MS/MS and LC-HRAM
Analyte Oligo
Detection Probe
Quantitative Bioanalysis of Oligonucleotides/mRNA
11
• Hybridization-based assay (indirect analysis)
– Northern blot/in situ hybridization
– Microarray
– Deep sequencing (NGS)
– RT-qPCR
– Hybridization ELISA
• Direct ELISA, Sandwich ELISA, etc
• Branched DNA (bDNA) signal amplification technology (QuantiGene)
• Chromatographic-based assay (direction analysis)
– HPLC-UV/Fluorescence
– Hybridization-based LC-fluorescence
– LC-MS/MS and LC-HRAM
Analyte Oligo
Detection Probe
12
Ion-Pair (IP)-UPLC-UV Assays
• Simple sample preparation (single LLE or SPE)
• High reproducibility (15/20% acceptance criteria)
• Relatively selective (primarily by length/charge)
• Estimation of metabolite concentrations
• Reasonable run time (<10 minutes/sample)
• Low sensitivity (~100 ng/mL LLOQ)
Minutes
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
mA
u
0
100
200
300
mA
u
0
100
200
300
A
S1
411
Po
lyT
-30
Multi-Chrom 1 (1: 260 nm, 4 nm)10 44 S07169 20071690120 1004 AS1411 Day 6 URN-1 1
Name 26-mer
n-1
PolyT-30 Mobile Phase:20 mM tetrabutylammoniumsulfate in water/MeCNpH=9
Jian Chen, et. al., "Quantitative determination of AS1411 oligonucleotide in monkey plasma using UPLC-PDA," ISSX, 2008
Strong Anion Exchange (SAX)-HPLC-UV
• Relatively selective(primarily by length/charge)
• High reproducibility(15/20% acceptance criteria)
• Simple sample preparation (single LLE or protease k digest)
• Estimation of metabolite concentrations
• Low sensitivity(~200 ng/mL LLOQ)
• Only works for charged oligos
• Long run time (20-35 min)
Sandberg, et al. Antisense & Nucleic Acid Drug Development 2000; 10:153-162.
Analyte: 35-mer modified ribozyme RNA
IS: 43-mer analog RNALC column: DNAPac® PA100MP: (50 mM Tris pH=8.0)/Ethanol80/20 (v/v) with 50-250 mM NaClO4
Hybridization-based HPLC-Fluorescence Assays
14
• High sensitivity (~ 1 ng/mL LLOQ )
• Good specificity (Hybridization and LC separation)
• Large dynamic range (1000x)
• Simple sample preparation (protein digestion or LLE)
• Good reproducibility (± 15% accuracy/precision)
• Tolerance to structure modifications Base, sugar and phosphate backbone modifications
3’
5
’
Sense
Anti-sense
Fluoro-probe
+3’
3
’5’ 5’
5’3’
1) 95ºC
2) 20ºC
HPLC
(SAX)
Quantify siRNA in HuPl Using Fluorescence Labeled DNA Probe (1.00 – 500 ng/mL)
15Hutabarat R, et al. AAPS Annual Meeting 2011, poster presentation.
3’
5
’
Sense
Anti-sense
Fluo-probe
+3’
3
’5’ 5’
5’3’
1) 95ºC
2) 20ºC
Anti-
probe HPLC
(SAX)
Analyte: 21-mer siRNAProbe: 21-mer Atto dye labeled DNA oligoAnti-probe: Cholesterol conjugated DNA oligo
Sample Prep: Protease K digestionColumn: DNA Pac-PA-200, 4x250 mmMPA: 1 mM EDTA in 25 mM Tris-HCl (pH 8.0) with 10% MeCNMPB: 1 mM EDTA and with 800 mM NaCIO4 in 25 mM Tris-HCl
with 10% MeCN
Typical Chromatogram of 22-mer siRNA in HuPl Samples (>24 Hours)
16
22-mer siRNA
n-1
n-2
n-3
Quantification of a Neutral 20-mer PMO Using a 15-mer Fluorescein-DNA
(40.0-4,000 ng/mL)
Arora V, et al. J of Pharmaceutical Sciences 2002; 91(4):1009-1018.
AVI-4126: 5’-ACGTTGAGGGGCATCGTCGC-3’ PMO (Phosphorodiamidate Morpholino Oligomer)
LC column: Dionex DNA Pac PA-100 (4 x 250 mm)Mobile Phase: A) 25 mM Tris pH=8.0;
B) 25 mM Tris pH=8.0 with 1.0 M NaCl
Quantification of siRNA in HuPl Using Fluorescence-labeled PNA probe
(1.00-1000 ng/mL)
3’
5
’
Sense
Anti-sense
Atto-425
+3’
3
’5’ 5’
5’3’
1) 95ºC
2) 20ºCHPLC
(SAX)
Analyte: 21-mer siRNAProbe: 18-mer Atto dye labeled PNA oligo
Sample Prep: Protease K digestionColumn: DNA Pac-PA-200, 4x250 mm
Qingguo Tian, IBC’s 18th Annual TIDES May 9, 2016
Method Performance of Quantitative Determination of
a siRNA in Rat Plasma using PNA Assay
R² = 0.9989
0.E+00
2.E+06
4.E+06
6.E+06
8.E+06
1.E+07
1.E+07
0 500 1000 1500
Pe
ak A
rea
Conc. (ng/mL)
LLOQ 1 ng/mLBlank
ISR Samples %Difference from original ISR samples
< ±10% 43
±10% ~ ±15% 2
Quality Control Samples
Incurred Sample Reanalysis
45 45 (100% pass)
QC (10 Runs) Accuracy (%Bias)
2.9%~11.2% -0.7%~2.5%
Precesion (CV%)
20%Difference from Orginal
Qingguo Tian, IBC’s 18th Annual TIDES May 9, 2016
Pros and Cons of Hybridization-based HPLC-Fluorescence Assays
20
• High sensitivity (~ 1 ng/mL LLOQ )
• Good specificity (Hybridization and LC separation)
• Large dynamic range (1000x)
• Simple sample preparation
• Internal standard is not necessary
• Good reproducibility (± 15% accuracy/precision)
• Tolerance to structure modifications
Base, sugar and phosphate backbone modifications
• Long run time (15-30 minutes/sample)
• One analyte/fluorophore at a time
21
Thermo Vanquish Focused UHPLC System
With Vanquish™ FluorescenceDetector F (4 Excitation/Emission)
Chromeleon 7.2 Chromatography Data System
Fast and Mutiple Analyte Hybridizaionbased LC-Fluorescence Assays
DNApac PA200: 8 μm, (PEEK) 4.6 x 250 mm
DNApac PA200 RS: 4 μm, (Peek -lined 4.6 x 150 mmStainless Steel) 4.6 x 50 m
Data to be shared soon…
LC-MS/MS & LC-HRAM Assays
22
• Highest specificity Molecular weight identification
Product ion characterization
LC Separation
• High throughput (~5 minutes/sample)
• Wide dynamic range (1000x)
• Good sensitivity (< 5 ng/mL LLOQ)
• No enzymes or special reagents needed
• Good reproducibility (±15/20% acceptance criteria)
• Need high performance mass spectrometer
• Potentially challenging assay development
Challenges & Solutions of Developing LC-MS/MS Assays
23
• Non-specific adsorption (stickiness)– Add disruption reagent(s) in extraction buffer
– Avoid extensive drying down of samples
– Minimize use of polypropylene container
– Keep stock solution at high concentrations.
• Stability in matrix– Use K2EDTA as anticoagulant
– Add CHAPS or BSA to urine samples.
• Short column life time and carryover– Clean-up sample “appropriately”
– Re-generate the column after each injection (back flush the column).
• Charge distribution and metal ion adducts in mass spectrum– HFIP and TEA/DIPEA buffered water and MeOH/MeCN as mobile phase.
Q1 MS Scan of a 18-mer PS-ODN on a Triple Quad
24
Calculated m/z
M 5731.6
M-6H 954.3
M-7H 817.8
M-8H 715.5
M-9H 636.8
-Q1: 20 MCA scans from Sample 1 (TuneSampleID) of MT20090528154857.wiff (Turbo Spray) Max. 6.6e6 cps.
620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960m/z, amu
5.0e5
1.0e6
1.5e6
2.0e6
2.5e6
3.0e6
3.5e6
4.0e6
4.5e6
5.0e6
5.5e6
6.0e6
6.5e6
Inte
ns
ity
, c
ps
708.9
715.6
818.0
636.0
954.5
625.2
736.9654.6
731.1
786.3673.2
663.0631.2641.0 746.2 771.2 920.9
627.3 717.0 936.9809.1705.4 857.3 915.1875.4820.7 836.8657.1 798.6643.1 758.2 899.0683.0661.2 772.8722.9 745.2 943.0921.7
Operator: Laixin Wang
Printing Date: Thursday, May 28, 2009
Results Name: N/A
M-9H
M-8H
M-7H
M-6H
Laixin Wang, et. al., AAPS Annual Meeting and Exposition, November 2009, Los Angeles, CA,
Product Ion Scan of a 18-mer PS-ODN on a Triple Quad
25
-MS2 (715.40): 20 MCA scans from Sample 1 (TuneSampleID) of MT20090608140619.wiff (Turbo Spray) Max. 4.9e5 cps.
300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500m/z, amu
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
4.2e5
4.4e5
4.6e5
4.8e5
4.9e5
Inte
ns
ity
, c
ps
319.0
304.2
328.1
344.1
415.0346.0320.2400.1
321.9
329.2 424.0337.0305.1
384.0345.0 440.2357.1 494.8321.0 416.8 421.8362.1306.2 444.1399.0 408.3480.1382.1 445.1322.9 460.0332.3 485.1397.0348.2 437.8 464.3425.0369.6 410.1312.0 492.0
Operator: Laixin Wang
Printing Date: Monday, June 08, 2009
Results Name: N/A
SRM Transitions evaluated for TL0901
715.5 → 319.0
715.5 → 304.2
818.0 → 319.0
818.0 → 304.2
A
G
C
T
LC Separation of a 18-mer PS-ODN and its Metabolites (Neat Mixture)
26
XIC of -MRM (3 pairs): 715.4/319.0 amu from Sample 9 (25_09001_3 neat combo) of LCSET1.wiff (Turbo Spray) Max. 2.3e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Time, min
0.0
1.0e4
2.0e4
In
te
ns
it
y,
c
ps
1.51
XIC of -MRM (3 pairs): 758.4/319.0 amu from Sample 9 (25_09001_3 neat combo) of LCSET1.wiff (Turbo Spray) Max. 2.2e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Time, min
0.0
1.0e4
2.0e4
In
te
ns
it
y,
c
ps
1.28
XIC of -MRM (3 pairs): 744.5/319.0 amu from Sample 9 (25_09001_3 neat combo) of LCSET1.wiff (Turbo Spray) Max. 1.6e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Time, min
0.0
5000.0
1.0e4
1.5e4
In
te
ns
it
y,
c
ps
1.41
XIC of -MRM (3 pairs): 715.4/319.0 amu from Sample 9 (25_09001_3 neat combo) of LCSET1.wiff (Turbo Spray) Max. 2.3e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Time, min
0.0
1.0e4
2.0e4
In
te
ns
it
y,
c
ps
1.51
TL0901
TL0901 (n-4)
TL0901 (n-6)
n-4n-6
TL0901
HRAM Improves Selectivity by Using Higher Resolution (Q Exactive)
27
1906 1907 1908 1909 1910 1911 1912 1913
m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1909.18085
1909.85565
1908.52656
1910.17955
1907.78988
1907.430191910.51593
1910.83802
1911.18566
1909.51255
1909.18126
1908.84770
1909.84505
1908.52003 1910.17945
1910.51218
1908.191001910.83662
1907.793371911.18108
NL: 4.32E3
TL-Std5_1#377-390
RT: 1.69-1.75 AV:
14 T: FTMS - p ESI
SIM ms
[1907.30-1911.30]
NL: 7.32E3
TL-Std5_1#264-273
RT: 2.30-2.38 AV:
10 T: FTMS - p ESI
SIM ms
[1907.30-1911.30]
R=70,000
R=140,000
Isotope envelopes of the [M-3H]3- ion of an 18-mer PS-ODN on a high resolution Q-Exactive Orbitrap® (Thermo Finnigan, LLC)
Different Charge Distribution of a18-mer PS-ODN on Different Platforms
28
-Q1: 20 MCA scans from Sample 1 (TuneSampleID) of MT20090528154857.wiff (Turbo Spray) Max. 6.6e6 cps.
620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960m/z, amu
5.0e5
1.0e6
1.5e6
2.0e6
2.5e6
3.0e6
3.5e6
4.0e6
4.5e6
5.0e6
5.5e6
6.0e6
6.5e6
In
te
ns
ity
, c
ps
708.9
715.6
818.0
636.0
954.5
625.2
736.9654.6
731.1
786.3673.2
663.0631.2641.0 746.2 771.2 920.9
627.3 717.0 936.9809.1705.4 857.3 915.1875.4820.7 836.8657.1 798.6643.1 758.2 899.0683.0661.2 772.8722.9 745.2 943.0921.7
Operator: Laixin Wang
Printing Date: Thursday, May 28, 2009
Results Name: N/A
M-9H
M-8H
M-7H
M-6H
Oligo_st_07 #495 RT: 2.22 AV: 1 NL: 5.28E6T: FTMS - p ESI Full ms [500.00-2500.00]
600 800 1000 1200 1400 1600 1800 2000 2200 2400
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
1909.1829
778.9326
1158.8998551.0519
968.91601200.8896
1431.6390
1348.88681948.2201
646.9078 817.6472 1052.89281864.16601532.8566 1989.77931686.7766
904.9360
2165.3530 2391.9988
M-3H
M-4H
Q-ExactiveAPI-5000
Weiwei Yuan, et. al. “Quantitative Determination of TL0901 Oligonucleotide in Human Plasma Using LC-HR/AM,“ ASMS, 2012
-Q1: 1.407 to 1.525 min from Sample 15 (NBA1520_neat_50ugml_10ul_MS) of 20160203_381_257_HBV_TUNE.wiff (Turbo Spray) Max. 6.1e6 cps.
450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250m/z, Da
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.7e6
Inte
ns
ity
, c
ps
797.8
911.6
709.1
638.0
1063.9
541.4
746.3471.3 503.1547.2 609.8
781.0648.7 805.4757.4693.6525.2 563.1 574.3439.3 865.7654.4 1041.1
10-9-
8-7-
6-
API5000 Triple Quads
Different Charge Distribution of a20-mer PS-ODN on Different Platforms
Q Exactive Plus
NBA1520_FullMS_5ul #183-218 RT: 3.22-3.40 AV: 36 NL: 4.90E7T: FTMS - p ESI Full ms [600.0000-2500.0000]
600 800 1000 1200 1400 1600 1800 2000 2200 2400
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
2127.8546z=3
797.3193z=8
1595.6443z=4
1063.4284z=6
1276.3142z=5
2020.8549z=3
2276.9763z=3
3-
4-5-6-7-8-
9-
Full Scan Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (1/3)
Extraction: SPE from 200 µL HuPl
LC Column:Thermo DNApac RP4 µm, 2x50 mm
Column Temp: 60 °CMobile Phase:
HFIP/TEA buffered water/MeOH Gradient
Run Time: 8 minutesLC System: Themo Vanquish UPLC
MS: Q-Exactive PlusIonization Mode: NegativeResolution: 70KScan Range 600-2500 m/z-
NBA1520: 5’--TAG TCA ATC TGC TTA TGT CA--3’ C196H249N68O102P19S19
NBA1519: 5’--TAG TCA ATC TGC TTA TGT C--3’ C186H237N63O98P18S18
NBA1514: 5’--TAG TCA ATC TGC TT--3’ C137H175N46O72P13S13
31
Full Scan Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (2/3)
NBA1520: 5’--TAG TCA ATC TGC TTA TGT CA--3’ C196H249N68O102P19S19
NBA1519: 5’--TAG TCA ATC TGC TTA TGT C--3’ C186H237N63O98P18S18
NBA1514: 5’--TAG TCA ATC TGC TT--3’ C137H175N46O72P13S13
(A)
(B)(C)
(A): NBA1520
(B): NBA1519
(C): NBA1514
Full-MS_STD210 #192-207 RT: 3.25-3.33 AV: 16 NL: 2.09E6T: FTMS - p ESI Full ms [600.0000-2500.0000]
600 800 1000 1200 1400 1600 1800 2000 2200
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
1053.5355z=1
2127.8553z=3
962.9642z=2736.9414
z=1
926.9245z=1
2166.8953z=3
1595.6417z=4
1282.5730z=2
1116.9090z=1
1926.9323z=1
2083.1716z=3
1769.8316z=1
8-7-
4-
3-X
XX
X
Full Scan Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (3/3)
NBA 1520 (Analyte)
LLOQ(0.5 ng/mL)
NBA 1514 (IS)
(200 ng/mL)
Extraction: SPE from 200 µL HuPlLC Column: Thermo DNApac RP
4 µm, 2x50 mmMobile Phase:
HFIP/TEA buffered water/MeOHLC System: Themo Vanquish Flux UPLC
MS: Q-Exactive PlusIonization Mode: NegativeResolution: 70Kt-SIM:
Analyte: 2123.8610-2131.8610IS: 1473.8065-1481.8065
Target-SIM Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (1/3)
Target-SIM Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (2/3)
NBA 1520 (Analyte)
ULOQ(500 ng/mL)
NBA 1514 (IS)
(200 ng/mL)
Target-SIM Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (3/3)
Nominal C.
(ng/mL)
Average C.
(ng/mL) % Bias CV%
High QC 400. 395 -1.25% 4.51%
Medium QC 200. 193. -3.50% 5.29%
Low-Medium 15.0 14.2 -5.33% 7.10%
Low QC 1.50 1.41 -6.00% 4.48%
Carryover blank
(NBA 1520)
~21% of LLOQ
NBA 1514
(IS)
Curve range:
0.5-500 ng/mL
Fragmentations of PS-ODN on Q-Exactivefor PRM/DIA Analysis Plus (1/2)
NBA1520_DIA2_160520143926 #421 RT: 3.26 AV: 1 NL: 3.16E7T: FTMS - p ESI Full ms2 [email protected] [301.0000-6000.0000]
1750 1800 1850 1900 1950 2000 2050 2100 2150 2200
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
-95.3694-50.3518
-45.0183
0.0000
-140.7185
-90.03541.3352
-190.7373-235.0587 -36.6804
-280.4053
2.3384
-102.7004
-153.0322
-330.7554 53.4163
-G
Precursor
2127.8611-A
-C
-AG
-GG
-AA
-AC
-GC
-AAG
-AAGG
-AGG
-AAAGG
-AAAGGG
5’--TAG TCA ATC TGC TTA TGT CA-3’
(NBA1520: A4C4G3T8)
NBA1520_DIA2_160520143926 #425 RT: 3.27 AV: 1 NL: 6.11E5T: FTMS - p ESI Full ms2 [email protected] [302.0000-2470.0000]
310 320 330 340 350 360 370 380
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
346.0387
319.0165
337.0272328.0281
304.0168 325.0277
347.0417344.0230
329.0316348.0361305.0202 359.4888 379.4913372.5412
A
G
C
T
Fragmentations of PS-ODN on Q-Exactivefor PRM/DIA Analysis Plus (2/2)
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1) Aliquot 200 μl sample a) Add 50 μl IS to each wellb) Add 500 μl loading bufferc) Vertex briefly and spin down
2) Load the samples onto the SPE plate (Pre-equilibrated with 1 mL MeOH and 1 mL loading buffer)
3) Wash the plate with 1 ml loading buffer and 1 mL of washing buffer
4) Heat sample plate for 1 hr incubation at 70°C5) Elute the sample with 250-500 μl elution buffer
(contains 10% methanol)6) Inject 10 μl for LC-MS analysis
Extraction SPE Plate: 96-Well WAX SPE plate, 30 mg,
Sample Aliquot: 200 µL Human Plasma
Extraction Procedure:
SPE Extraction of the 20-mer PS-ODN From Human Plasma
LC-MS/MS vs LC-HRAM
• Q1 mass spectra of oligos have different charge distributions on different instrument platforms
• LC-HRAM on Q-Exactive can achieve comparable sensitivity as LC-MS/MS on triple quads for some analytes.
• LC-HRAM can be performed without any prior knowledge of the compound, thus avoiding the need for compound-specific tuning (setting up the SRM transition). Therefore, it is very convenient for monitoring unknown metabolite information or estimate the known metabolite concentration(s) without standard reference material while quantifying the parent compounds.
39
Size Maters for The Assay Sensitivities
40
Length of the oligonucleotide
10 20 30 40-mer
Sen
siti
vity
• LC-MS/MS (HRAM) assays are a good choice for small oligonucleotides;• Hybridization-based assays are advantageous for large
oligonucleotides.
41
THANK YOU