high-throughput analysis of oligonucleotides using

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


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


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


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


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


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


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


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



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


--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


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


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


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


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


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)


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


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


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


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


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


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


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


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

high-throughput analysis of oligonucleotides using

Documents