high-throughput analysis of oligonucleotides using
TRANSCRIPT
WOD340 ASMS 2003, Hail et al.
HighHigh--Throughput Analysis of Throughput Analysis of Oligonucleotides usingOligonucleotides using
Automated Electrospray IonizationAutomated Electrospray IonizationMass SpectrometryMass Spectrometry
Mark E. HailMark E. Hail11, Brian Elliott, Brian Elliott22, Kerry Nugent, Kerry Nugent33, Jeffrey L. Whitney, Jeffrey L. Whitney11, and , and David J. DetlefsenDavid J. Detlefsen11
11Novatia, LLC, Princeton, NJ 08540Novatia, LLC, Princeton, NJ 0854022Integrated DNA Technologies, Coralville, IA 52241Integrated DNA Technologies, Coralville, IA 52241
33Michrom BioResources, Auburn, CA 95603Michrom BioResources, Auburn, CA 95603
WOD340 ASMS 2003, Hail et al.
IntroductionIntroduction
Tens of thousands of oligos produced daily in HT oligo synthesisTens of thousands of oligos produced daily in HT oligo synthesislabslabsEfficient and reliable confirmation of all synthesized products Efficient and reliable confirmation of all synthesized products desireddesiredTo date, MALDITo date, MALDI--ToF has been QC method of choiceToF has been QC method of choiceDemand for long (e.g., 70Demand for long (e.g., 70--mer) oligos for use in oligo arrays is mer) oligos for use in oligo arrays is increasingincreasingLong and/or fragile oligos have been problematic by MALDILong and/or fragile oligos have been problematic by MALDI--ToFToFWe have developed a totally automated system using LC/MS with:We have developed a totally automated system using LC/MS with:
>1000 sample per 24 hr throughput>1000 sample per 24 hr throughputcapability to analyze long (>100 mer) or fragile oligoscapability to analyze long (>100 mer) or fragile oligos< 100 ppm mass accuracy & precision across entire mass range< 100 ppm mass accuracy & precision across entire mass rangecapability for detailed LC/MS profiling on the same systemcapability for detailed LC/MS profiling on the same system
WOD340 ASMS 2003, Hail et al.
Experimental SystemsExperimental Systems
MALDIMALDI--ToFToFABI Voyager DE, linear +ve mode, external calibrationABI Voyager DE, linear +ve mode, external calibration33--HPA HPA –– ammonium citrate matrixammonium citrate matrix
LC/MS (Oligo HTCS)LC/MS (Oligo HTCS)ThermoFinnigan TSQ7000, ThermoFinnigan TSQ7000, --ve ion modeve ion modeXcalibur data systemXcalibur data systemMichrom Paradigm MS4 HPLCMichrom Paradigm MS4 HPLC
HFIP / TEA “lite” mobile phase w/trace EDTAHFIP / TEA “lite” mobile phase w/trace EDTALEAP CTCLEAP CTC--PAL autosamplerPAL autosamplerProMassProMass auto biomolecule deconvolution processing auto biomolecule deconvolution processing softwaresoftware
WOD340 ASMS 2003, Hail et al.
ProMassProMass Auto ESI Deconvolution ApproachAuto ESI Deconvolution Approach
Uses a simple scoring algorithm to determine the charge Uses a simple scoring algorithm to determine the charge of every peak in the raw mass spectrum (Zhang & of every peak in the raw mass spectrum (Zhang & Marshall, JASMS 1998)Marshall, JASMS 1998)No deconvolution artifactsNo deconvolution artifactsIncorporates signal processing and score normalization Incorporates signal processing and score normalization to improve reliability of deconvolution even on very noisy to improve reliability of deconvolution even on very noisy datadataCan confirm presence of target massesCan confirm presence of target masses
WOD340 ASMS 2003, Hail et al.
4168 4968 5768 6568 7368 8168Mass (m/z)
0
1.1E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
6168.4 MALDI-ToF6167.6
ESI ProMass Deconvolution
-A +HFIP4168 4968 5768 6568 7368 8168
Mass (m/z)
0
1.1E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
MALDI-ToF, M = 6167.4ESI, M = 6167.6
MALDI and ESI MS Data of a 20MALDI and ESI MS Data of a 20--mer Oligomer OligoM = 6168.4M = 6168.4
WOD340 ASMS 2003, Hail et al.
12415 14815 17215 19615 22015 24415Mass (m/z)
0
1579.1
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
18415.6
18541.0
MALDI-ToF
MALDI and ESI MS Data of a 60MALDI and ESI MS Data of a 60--mer Oligomer OligoM = 18410.0M = 18410.0
ESI ProMass Deconvolution18410.1
depurination
-THFIP adduct
12415 14815 17215 19615 22015 24415Mass (m/z)
0
1579.1
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
MALDI-ToF, M = 18414.6
ESI, M = 18410.1
WOD340 ASMS 2003, Hail et al.
27122 31122 35122 39122 43122 47122Mass (m/z)
592.3
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
37122.6
37237.4
MALDI-ToFESI ProMass Deconvolution37030.0
27122 31122 35122 39122 43122 47122Mass (m/z)
592.3
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
ESI, M = 37030.0MALDI-ToF, M =37122.6
MALDI and ESI MS Data of a 120MALDI and ESI MS Data of a 120--mer Oligomer OligoM = 37031.0M = 37031.0
WOD340 ASMS 2003, Hail et al.
7009.8
6899.3
ESI ProMass Deconvolution7003.7
6711.50
7065.396773.44
5011 5811 6611 7411 8211 9011Mass (m/z)
0
6175.9
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
6711.5
MALDI-ToF
dissociationproduct
5011 5811 6611 7411 8211 9011Mass (m/z)
0
6175.9
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
MALDI-ToF, M =7003.7
ESI, M = 7009.8
MALDI and ESI MS Data on Fragile DualMALDI and ESI MS Data on Fragile Dual--labeled Probelabeled ProbeBlack Hole QuencherBlack Hole QuencherTMTM M = 7011.8M = 7011.8
WOD340 ASMS 2003, Hail et al.
--16.216.230950.630950.6878.8878.830978.330978.330951.130951.1100100--mermer
--27.027.037030.037030.02446.62446.637121.637121.637031.037031.0120120--mermer
--4.14.124661.024661.0146.0146.024664.724664.724661.124661.18080--mermer
5.35.318410.118410.1249.9249.918414.618414.618410.018410.06060--mermer
--24.424.412299.712299.7--113.8113.812298.612298.612300.012300.04040--mermer
--81.181.16167.66167.6--113.5113.56167.46167.46168.16168.12020--mermer
33.433.42993.12993.1--300.7300.72992.12992.12993.02993.01010--mermer
ErrorError(ppm)(ppm)
ESIESIErrorError(ppm)(ppm)
MALDIMALDIMWMWOligoOligo
Mass Accuracy of 10 to 120Mass Accuracy of 10 to 120--mersmers
WOD340 ASMS 2003, Hail et al.
Oligo HTCS Plumbing SchemeOligo HTCS Plumbing SchemeShown in HighShown in High--throughput QC Modethroughput QC Mode
Dual trap column configuration
Eluent pumps A/B, 200 uL/min
Load/wash pumps C/D, 1 mL/min
V1 toggled for each run
V2 bypass
One trap is equilibrated while other is eluting
1.4 min/sample = 10 x 96 well plates/day
1.7 min/sample = 8 x 96 well plates/day with pre-dilution & mixing steps
Michrom Paradigm MS4 HPLC
waste
to MS
2x100 mmJupiter C18
LEAP/CTCHTS-PAL
autosamplerloop
V2
V11 2
eluentpumps
loadwash
pumps
oligotraps
WOD340 ASMS 2003, Hail et al.
ProMass ProMass Automated Data ProcessingAutomated Data ProcessingXcalibur Sample Sequencedefines samples to be analyzed
Xcalibur Processing Methoddefines how peaks are selected
Parameter Setupdefines parameters for
deconvolution
Optional Amino acid or nucleotide sequence
HTML Results Reportincluding plate view,
spectra, chromatograms, and tabular results
Peak picking,auto decon-volution, and report generation
WOD340 ASMS 2003, Hail et al.
Automated Oligo ESI Mass Spectral ProcessingAutomated Oligo ESI Mass Spectral ProcessingRT: 0.00 - 0.53
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
0.17
0.42
NL:3.97E7TIC MS 7589610
7589610 #3-10 RT: 0.12-0.47 AV: 8 NL: 1.27E6T: - p ESI Q3MS [ 550.00-1500.00]
600 700 800 900 1000 1100 1200 1300 1400 1500m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
847.3
819.1
792.4 877.6
767.7 910.1
744.4 945.0 983.01068.5
1117.3722.6 1170.4702.0 1228.6 1293.3663.9606.4 1319.4 1365.8 1436.8 1474.0
Raw ESI/MS spectrumRaw ESI/MS spectrum8080--mermer
LC/MS elution profileLC/MS elution profileTotal Ion CurrentTotal Ion Current
30 sec30 sec
Expected Mass = 24600 DaExpected Mass = 24600 DaMass Error = 0.6 DaMass Error = 0.6 Da
24599.424599.4
-29
-28
-27-26
-22-24 -20
-30
-31
-32
-34
ProMass deconvolutionProMass deconvolution
ΣΣ
WOD340 ASMS 2003, Hail et al.
ProMassProMass Sample BrowserSample Browser9696--well analysis of 50well analysis of 50--130 mer oligos130 mer oligos
Color codes indicate Color codes indicate presence or absence of presence or absence of target massestarget masses
ColorColor--coded wells are coded wells are hyperlinked to detailed reporthyperlinked to detailed report
Target mass found as major Target mass found as major componentcomponent
Target mass found with Target mass found with other significant componentsother significant components
Target mass found but not Target mass found but not as major peakas major peak
Target mass not foundTarget mass not found
WOD340 ASMS 2003, Hail et al.
9696--well Platewell Plate--Level Mass AccuracyLevel Mass Accuracy
Average and Standard Deviation of Green Average and Standard Deviation of Green and Purple wells from 3 plates run on different daysand Purple wells from 3 plates run on different days
Plate 1 (70Plate 1 (70--mers):mers):Average Mass Error: 0.0021% (21 ppm)Average Mass Error: 0.0021% (21 ppm)Standard Deviation: 0.0045% (45 ppm)Standard Deviation: 0.0045% (45 ppm)
Plate 2 (70Plate 2 (70--mers):mers):Average Mass Error: 0.0000% (0.4 ppm)Average Mass Error: 0.0000% (0.4 ppm)Standard Deviation: 0.0042% (42 ppm)Standard Deviation: 0.0042% (42 ppm)
Plate 3 (50Plate 3 (50--130130--mers):mers):Average Mass Error: Average Mass Error: --0.0046% (0.0046% (--46 ppm)46 ppm)Standard Deviation: 0.0046% (46 ppm)Standard Deviation: 0.0046% (46 ppm)
WOD340 ASMS 2003, Hail et al.
SM: 5G
700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400m/z
1.2
0.0
0.2
0.4
0.6
0.8
1.0
3.0
0.0
0.5
1.0
1.5
2.0
2.5
28
0
5
10
15
20
Rel
ativ
e Ab
unda
nce
100
20
40
60
80
912.4 947.5
985.5879.7
1026.6849.3
1071.21120.0821.1 942.2 1173.3980.0794.4 988.6 1065.3 1231.8769.7 1078.3 1296.6746.5 1191.9
912.4947.6
879.7
849.4 985.4
1026.61071.2821.1 942.2794.7 980.0 1120.0 1173.4988.4 1065.3769.5751.6 1296.51199.6 1231.3 1369.1
912.3
879.7947.4
849.5 985.31026.2969.0 1120.0807.2 1006.9
1070.9 1173.3703.5 733.0 773.0 1388.51190.1 1227.4 1342.01289.1
913.3
985.6947.5
897.4879.3850.4
1009.9951.3 1026.6973.2 1139.5 1161.9838.9 1086.5 1337.1821.9737.6 1163.5 1221.7779.7 1282.2 1346.7 1371.91324.4
-26-27
-29-30 -23 -22 -21
-25
-24
-28 100 pmoles100 pmoles
10 pmoles10 pmoles
1 pmole1 pmole
500 fmoles500 fmoles
ESI Mass SpectraESI Mass Spectra
700 800 900 1000 1100 1200 1300
1.2
3.0
28
100
M/z
% R
elat
ive
Abun
danc
e
24660.4
24661.0
24661.0
24661.8
100 pmoles100 pmoles
10 pmoles10 pmoles
1 pmole1 pmole
500 fmoles500 fmoles
Deconvoluted Mass SpectraDeconvoluted Mass Spectra
Oligo HTCS Sensitivity Study Oligo HTCS Sensitivity Study –– 80 mer oligo80 mer oligoM = 24661.1M = 24661.1
WOD340 ASMS 2003, Hail et al.
ESI Mass Spectra of Long OligosESI Mass Spectra of Long Oligoslow purity makes analysis of long oligos challenginglow purity makes analysis of long oligos challenging
ESI mass spectrum120-merbefore baseline removal
ESI mass spectrum120-mer after baseline removal
Baseline removal is Baseline removal is key for reliable key for reliable deconvolutiondeconvolution
HT QC of crude synthesis HT QC of crude synthesis products is highly desirableproducts is highly desirable
Low purity due to presence Low purity due to presence of multiple failure sequence of multiple failure sequence productsproducts
Causes baseline “hump” Causes baseline “hump” under ESI mass spectrumunder ESI mass spectrum
WOD340 ASMS 2003, Hail et al.
Oligo HTCS Plumbing SchemeOligo HTCS Plumbing SchemeShown in Detailed LC/MS Profiling ModeShown in Detailed LC/MS Profiling Mode
waste
to MS
oligotraps
loop
Switch from HT to Switch from HT to detailed mode detailed mode automaticallyautomatically
V2 activates 2 x V2 activates 2 x 100 mm C18 column 100 mm C18 column
Column wash w/ Column wash w/ 100% D100% D
Useful for detailed Useful for detailed profiling profiling
55--40% B using 20 40% B using 20 min gradientmin gradient
V2
V1 2
Michrom Paradigm MS4 HPLC
LEAP CTC- PALautosampler
2x100 mmJupiter C18
eluentpumps
loadwash
pumps
1
WOD340 ASMS 2003, Hail et al.
LC/MS Analysis of Crude 80LC/MS Analysis of Crude 80--mer Oligomer OligoJupiter C18 300A 2x100 mm, 5Jupiter C18 300A 2x100 mm, 5--40%B in 20 min40%B in 20 min
RT: 0.00 - 25.32
0 2 4 6 8 10 12 14 16 18 20 22 24Time (min)
0
5
10
15
20
mill
iVol
ts
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
13.1412.99
16.56
12.69
12.39
11.9911.44
13.75 17.1210.84
14.15 17.4710.44 16.219.43 18.128.83 18.82 23.2422.6319.98 23.897.937.225.824.921.81 2.510.60 3.11
13.12
12.77
16.67
12.4012.0311.511.83
10.879.451.68
9.165.25 5.46 8.86 14.487.950.04 2.54 4.38 23.424.10 23.1218.17 18.92 21.47
NL:6.71E6TIC MS test80mer_lcms01
NL:2.42E1A/D Card Ch. 1 A/D card test80mer_lcms01
Failure productsTarget OligoDMT-on
LC/UV 260 nmLC/UV 260 nm
LC/MS TICLC/MS TIC
WOD340 ASMS 2003, Hail et al.
ESI Mass Spectra of DMTESI Mass Spectra of DMT--on Oligoon Oligo8080--mer from LC/MS run, RT = 16.5 minmer from LC/MS run, RT = 16.5 min
ProMass DeconvolutionProMass DeconvolutionExpected Mass: 24988.4 DaObserved Mass: 24988.6 Da
ESI/MSESI/MS
WOD340 ASMS 2003, Hail et al.
Detailed LC/MS Analysis of Failure ProductsDetailed LC/MS Analysis of Failure ProductsLC/MS of 80LC/MS of 80--mermer
T61T61--G80G80MW 6125MW 6125
G60G60--G80G80MW 6453MW 6453
G58G58--G80G80MW 7112MW 7112
G59G59--G80G80MW 6783MW 6783
5’-TAATACGACTCACTATAGGGTAATACGACTCACTATAGGGTAATACGACTCACTATAGGGTAATACGACTCACTATAGGG-3’
LC/MS TICLC/MS TIC
WOD340 ASMS 2003, Hail et al.
ConclusionsConclusions
A fullyA fully--automated system for HT oligo MS analysis was describedautomated system for HT oligo MS analysis was described--ve ion detection with optimized mobile phaseve ion detection with optimized mobile phaseHighHigh--speed onspeed on--line desaltingline desaltingAutomated ESI deconvolution and webAutomated ESI deconvolution and web--based reportingbased reportingSample throughput of >1000 samples/day in HT MW confirmation modSample throughput of >1000 samples/day in HT MW confirmation mode e
MALDIMALDI--ToF ToF and ESIand ESI--MS offer comparable mass accuracy below ~40MS offer comparable mass accuracy below ~40--mersmersBetter resolution with Better resolution with quadrupolequadrupole ESIESI--MS vs. linear MALDIMS vs. linear MALDI--ToF ToF across across entire mass rangeentire mass rangeESIESI--LCMS is the technique of choice for analysis of long and/or fragLCMS is the technique of choice for analysis of long and/or fragile ile oligonucleotidesoligonucleotides
“Softer” ionization“Softer” ionizationNo degradation of mass resolution with increasing massNo degradation of mass resolution with increasing massMass accuracy (< 100 ppm) across entire mass rangeMass accuracy (< 100 ppm) across entire mass range
Detailed LC/MS profiling capability on the same system via automDetailed LC/MS profiling capability on the same system via automatic valve atic valve switchingswitching
WOD340 ASMS 2003, Hail et al.
AcknowledgementsAcknowledgements
Brian Elliott Brian Elliott –– IDTIDTCoCo--workers workers –– Jeff Whitney, David Detlefsen, Jeff Whitney, David Detlefsen, Kathleen AndersonKathleen AndersonKerry Nugent Kerry Nugent –– Michrom BioResourcesMichrom BioResources