laser desorption of dna oligomers larger than one kilobase from cooled 4-nitrophenol

Laser Desorption of DNA Oligomers LargerThan One Kilobase from Cooled 4-Nitrophenol

Hua Lin*, Joanna M. Hunter and Christopher H. BeckerGeneTrace Systems Inc., 1401 Harbor Bay Parkway, Alameda, CA 94502, USA

SPONSOR REFEREE: Professor Peter Williams, Arizona State University, Tempe, AZ, USA

A unique matrix system consisting mostly of 4-nitrophenol has shown to be very effective for matrix-assistedlaser desorption/ionization time-of-flight mass spectrometric analysis of large DNA oligomers when a cooledsample stage was used to prevent the sublimation of this matrix under vacuum. Using this 4-nitrophenolmatrix with UV laser desorption, detection of picomole quantities of DNA oligomers containing up toapproximately 800 nucleotides was routinely achieved. The effectiveness of this matrix was furtherdemonstrated by the observation of a double-stranded DNA oligomer larger than 1000 base pairs, seen as adenatured single-stranded species, with a molecular ion mass exceeding 300000 Da. The potentialapplications of 4-nitrophenol as a matrix for DNA sizing are discussed. Copyright# 1999 John Wiley &Sons, Ltd.

Received 5 October 1999; Accepted 7 October 1999

Over the past five years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has received wide attention as a potential tool toreplace conventional gel electrophoresis for rapid DNAsequencing and sizing studies.1–4 In MALDI, samples areusually prepared by embedding analytes in a large molarexcess of photoabsorbing small organic molecules via a co-crystallization process. Laser desorption and ionization ofthese crystals provides a relatively gentle method togenerate large intact molecular ions in the gas phase.5,6

The ions produced in this single desorption event are thenseparated and detected effectively by simple time-of-flightmass spectrometry. Therefore MALDI-TOFMS has theadvantages of speed and accuracy for the mass determina-tion of large biomolecules such as proteins and DNAoligomers.

Soon after its invention, the MALDI technique wasdeveloped to detect subpicomole amounts of proteins with amass exceeding several hundred thousand Daltons (Da).7,8

However, because of their polyanionic nature and relativelyfragile structure, the detection of nucleic acids up to thesame mass was found to be difficult resulting fromextensive salt-adduct formation, and prompt and metastablefragmentation.1,2 The extent of the fragmentation in thiscase strongly depends on the matrix systems used and thebase composition of the analyte.2,9–12 Using matricescommonly employed for proteins, such as 2,5-dihydroxy-benzoic acid, early efforts to analyze nuclei acids werelimited to very small mixed-base oligonucleotides.13

The application of the MALDI technique in the area ofDNA analysis was significantly advanced due to thediscovery of 3-hydroxypicolinic acid (3-HPA), a very

effective matrix for oligonucleotides.14,15Using this matrix,femtomole sensitivity for modest size oligomers up to20000 Da was achieved.15,16Subsequently, a mixture of 3-HPA and picolinic acid (PA) was developed to obtain massspectra of double-stranded DNA segments as large as 500and 622 base pairs.17,18 Since then, genomic applicationsincluding DNA sequencing,16,19–23 polymorphism detec-tion24–34 and microsatellite analysis4,35–37 have emerged.However, the attainment of acceptable mass resolution andsensitivity for DNA oligomers of increased size remains achallenge. Routine detection of DNA oligomers larger than200 bases has been problematic in practice. Adductformation and fragmentation are still the major limitationsin DNA mass spectrometry.

It is therefore desired to develop new matrices andMALDI experimental strategies that can minimize thelimitations for DNA analysis, and improve sensitivity, massresolution and reproducibility. One area of current studieshas been focused on matrices having significant volatility atroom temperature.38–40 In principle, the relatively weakintermolecular interactions (matrix to matrix and matrix toanalyte) of these volatile molecules are expected to result ina lower energy threshold for desorption, thus minimizingthe analyte internal energy, decreasing the fragmentation,and increasing the sensitivity. Very recently, Hillenkamp’sgroup demonstrated the benefit of reduced fragmentation ofDNA with a liquid matrix of glycerol.41 They were able toextend the mass upper limit of detection to exceeding akilobase using an infrared laser emitting at 2.94mm.

Our early approach to MALDI development with a UVlaser utilizes volatile matrix systems consisting of frozenthin films of photoabsorbing substituted phenols in aqueous/methanol solutions.38 The extent of DNA fragmentationobtained with this method was indeed found to besignificantly reduced compared with that of the room-temperature crystalline matrix with the same photoabsorber.Furthermore, the greater spatial homogeneity of frozen thinfilm matrices results in a more uniform DNA signal across

*Correspondence to: H. Lin, GeneTrace Systems Inc., 1401 HarborBay Parkway, Alameda, CA 94502, USA.Contract/grant sponsor: National Institutes of Health, National HumanGenome Research Institute; Contract/grant number: R01 HG00174.

CCC 0951–4198/99/232335–06 $17.50 Copyright# 1999 John Wiley & Sons, Ltd.

RAPID COMMUNICATIONS IN MASS SPECTROMETRYRapid Commun. Mass Spectrom.13, 2335–2340 (1999)

the sample spot.With thesefrozen thin film matrices, thedetection sensitivity is still not as good as that with theroom-temperature 3-HPA matrix, however.

The explorationof frozensolution-phasematrices led usto an investigation of cooled crystalline solid matricescontaining volatile compoundsat room temperature. Thelow sublimation temperaturesof thesecompoundsresult ina rapidevaporationundervacuumat roomtemperatureandtherefore dictate the useof a cryogenically cooledsamplestage.Our searchfor potential effective ‘volatile matrices’started with substituted aromatic compoundscontaining ahydroxyl group for proton transfer ionization. Two suchmolecules, 4-nitrophenol and 8-hydroxyquinoline, werefound to be particularly attractive matrices for DNAanalysis.42

Hereinwepresentourstudiesonthe4-nitrophenolmatrixsystemfor sensitivedetection of DNA oligomersexceedingonekilobase.Our results demonstratethatthe4-nitrophenolmatrix is a powerful new systemfor mass spectrometricanalysisof largeDNA oligomers.

EXPERIME NTAL

Oligonucleotides

Three DNA oligomers were usedfor massspectrometricanalysis: (1) Thegel-purified synthetic single-stranded(ss)DNA mixed-base55-mer (calculatedmass:16911Da) waspurchasedfrom BioSource International(Menlo Park,CA,USA) anduseddirectly afteraseriesdilution with deionizedwater. (2) The ss-DNA of 468 basesin length (468-mer)with a known sequence(calculatedmass: 144755Da) wasprepared from polymerasechainreaction (PCR)amplifica-tion of thewild typep53transcript encompassingExons6–9.ThePCRamplification wasperformedin athermalcycler(MJ Research,Watertown, MA, USA) using 1 mL oftemplateDNA in a 100mL reaction containing30pmol ofboth forwardandreverseprimers,0.25mM dNTP,1.5mMMgCl2, 1X PCR buffer and 2.5U Taq polymerase(Promega,Madison,WI, USA). The cycle conditionswereasfollows: initial denaturingat95°C for 2 min; 50cyclesof95°C for 15s, 52°C for 30s, 72°C for 45s, and finalextension at 72°C for 5 min. The 468-mer ss-DNA waspurified from PCR products by a solid phaseclean-upprocedure.43 Theforward primer for this PCRreactionwaslabeled with a biotin. The biotinylated strand was thencaptured by streptavidin-coated magnetic beads. Af terdenaturing andwashing, thesingle-strandedDNA productswere released and dried in a Speed-Vac concentrator(Savant Inc. Farmingdale,NY, USA). (3) Double-stranded(ds) DNA oligomer exceeding 1000basepairs wasalso aPCR product. It was derived from a cDNA insert ofunknown sequencein a vector. The PCRprotocol wasthesame as the one for the 468-mer. By polyacrylamide gelelectrophoresis(1%)andcomparingto a100basepairDNAladder, thesizeof this ds-DNA samplewasestimatedto bebetween 1000and1100bases.Purification of this productwasdoneby ultrafiltration usingmembranefilt ration units(Microcon-30, Amicon Inc., Beverley, MA, USA). Thefilt erwaspre-washedwith 400mL of deionizedwater.Then100mL of PCR product solution and 300mL of 1 Mammonium acetatewereaddedto the filter andspundownat 10000 rpm to a volume of about 40mL. This wasfollowed by three additional washes with 400mL of

deionized water. After the final wash, the DNA wascollected off the filter andlyophilized.

MALDI samplepreparation

4-Nitrophenol (99�% purity) waspurchasedfrom AldrichChemical Co. (Milwaukee, WI, USA) and used withoutfurther purification. A 0.5 M matrix solution waspreparedby dissolving 4-nitrophenol in a 1:1 (v/v) methanol/watermixture containing diammonium citrate at 50mM finalconcentration. The addition of diammonium citrate, whichbuffers the sample solution to approximately pH 5, isimportantfor this matrix system becauseit reducesalkali-metal adduct formation and enhances the molecular ionsignalof theanalyte.44,450.5mL of this matrix solutionwasaddedto the dried DNA sample, andthe resultingmixturewaspipettedonto a silicon substrate mounted on a coppersamplestage.A gold-coated samplestagewas also testedand found to work aswell as the silicon substrate. Beforepipettingthesample, thesurfaceof thesiliconsubstratewasgentlyscratchedwith fine sandpaperto providenucleationsites that facilitate fast crystallization. After drying thesolventin air and resultantcrystallization of the matrix atambienttemperature,the samplestagewas inserted into acryogenically cooled sample stage holder in the massspectrometer.

Time-of flight massspectrometer

Massspectrawere recorded in positive ion modeusing acustom 1-meter linear time-of-flight mass spectrometerequippedwith a cooled stage.The detailsof the apparatushave beendescribed previously.15,38 The volatility of thematrix crystalsrequires that the samplestage of the massspectrometer be cooled substantially below room tempera-ture.This wasachievedby flowing liquid nitrogen throughthecoppersampleholder.Thetemperatureof thesampleonthesilicon surfacewasmaintainedat approximately 180K,as determined by a thermocoupleattachedto the surface.The cooled sample-matrix crystals were desorbedandionizedat 355nm by a third harmonic generation Nd:YAGlaser(Spectra PhysicsModel DCR-1,Mountain View CA,USA). It is significant that the massspectraobtained fromthe 355nm laser show less fragmentation of DNAoligomers than those from the 266nm laser.The typicalbackgroundpressurein the ion sourcechamberof themassspectrometer was 2� 10ÿ7 Torr. Several groups haveshowndramaticimprovementsin mass resolution andmassaccuracy using delayed extraction.46–49 Therefore, thistechnique was also used in the presentstudy. The pulsevoltage for delayed extraction was 5 kV and the totalaccelerating voltage was 25kV. The ions were detectedusing a dual microchannel plate detector,and the signaloutput from the detector was amplified and digitized.Spectrareported in the presentwork were collected using5 ns time resolution.

RESULTS AND DISCUSSIONS

Volatilit y of 4-nitrophenol at room temperature

Previously,using a 4-nitrophenol matrix at room tempera-ture ss-DNA synthetic oligonucleotides were analyzedinour laboratory by the MALDI technique.15 Gooddetectionsensitivity was reportedthen, even though 4-nitrophenolgivesrise to broader peaksthan3-HPA. Furtherinvestiga-

Rapid Commun.MassSpectrom.13, 2335–2340(1999) Copyright# 1999JohnWiley & Sons,Ltd.

2336 LD OF DNA OLIGOMERS LARGER THAN ONE KILOBA SE

tion of this matrix reveals that 4-nitrophenol sublimesquickly undervacuumat room temperature.To determinethe rate of sublimation, 250nmol of 4-nitrophenol persample(dried from 0.5mL of the matrix solution) weredeposited on the probe surface and placed into the massspectrometer. Figure 1 presentsthe UV absorption spectraof this matrix re-dissolved from the probe surface usingdeionizedwaterbeforeandafterbeingplacedundervacuumfor 10 and 30min, respectively. Indeed, the decreaseofabsorption intensity indicatesthatmost of the4-nitrophenolsublimed in less than half hour at room temperature.Correspondingly, we also observed that MALDI massspectral quality deteriorated with time as the matrixsublimed. The bestquality and most reproducible spectrawereobtained only whenthecooledsamplestagewasused.

Detection of synthetic oligonucleotides

Figure 2 shows a typical mass spectrum of the single-stranded 55-mer(MW 16911Da) with 4-nitrophenolas acryogenic matrix. The total loading of the analyte was 40fmol, andtheanalyte to matrix molar ratio was1.6� 10ÿ7.Thespectrum wasobtainedby summingover100shots.Theraw datawas smoothed twice, eachtime with a 21-pointSavitzky-Golay smoothing, and then the baseline wassubtracted.Themajorpeaksin thespectrum areprotonatedmolecularions[M � H]� anddoubly charged[M � 2H]2�.The less intense peaksobserved are base-loss fragment[M ÿ 135]� and matrix adduct[M � 139]� correspondingto thecombination of anintactDNA andamatrix molecule.The mass resolution, M/DM(FWHM), for the singlyprotonatedmolecular ion signalat 16915 is 154.

Detection of PCR products

An example of the detection of higher massDNA with the4-nitrophenolmatrix is given in Fig. 3, which exhibitshighsignal-to-noiseratio for molecular ionsof thess-DNA468-mer (MW 144755Da). The amount of sample loadedwasapproximately 5 pmol andtheanalyte to matrix molar ratiowas2� 10ÿ5. Thespectrum wasobtainedby summingover400shots.Therawdatawassmoothed twice,eachtimewitha21-pointSavitzky-Golaysmoothing,andthenthebaselinewas substracted. The massresolution for the parentpeak,M�, at 146000 is about30.

Figure 4 illustrates a spectrum of the largest DNAoligomerdetected so far, seenassingly chargedmolecularions. Theanalyteis ads-DNA exceeding onekilobasepairs.(The massanalysis of single-strandedDNA extractedfromthe same PCR product using a solid phase cleaningprocedure failed, possibly due to the low efficiency forcapturing DNA larger than 1000 bases.)The amount ofsample loaded was approximately 10pmol, which corre-spondsto ananalyte to matrix molar ratio of 4� 10ÿ5. Thespectrum was obtained by summingover 1000 shots.Theraw datawassmoothedtwice, eachtime with a 101-pointSavitzky-Golay smoothing, and then the baseline wassubtracted.During the MALDI analysis, the two strands

Figure 1. UV absorptionspectraof 4-nitrophenolrecoveredfrom thesampleplateby re-dissolvingin 2.5mL of deionizedH2O beforeandafterbeingplacedundervacuumfor 10and30min. Theinitial loadingof matrixwas250nmol.Thedecreaseof absorptionintensityindicatesthatmostof the4-nitrophenolsublimedin lessthanhalf hourat roomtemperature.

Figure 2. Positiveion TOF massspectrumof a mixed-basesynthetic55-mer obtained from 4-nitrophenol matrix using a cryogenicallycooledsamplestage.The parentmolecularion is protonated55-mer[M � H]�. Doubly chargedion [M � 2H]2� and triply chargedion[M � 3H]3� arealsoplotted.Thespectrumwasobtainedby summingover 100 shots.The datawasfurther processedwith Savitzky-Golaysmoothingand baselinesubtraction.Total analyteloading: 40 fmol;Accelerationstatic voltage: 20kV; Extraction pulse voltage: 5 kV;Extractiondelaytime: 400ns.

Figure 3. Positive ion massspectrumof a single-stranded468-merusinga4-nitrophenolmatrix.Themassresolutionis 30.Thisspectrumwas obtained by summing over 400 shots and using an 800nsextraction delay time. The amountof sampleloadedwas approxi-mately 5 pmol. The datawas further processedwith Savitzky-Golaysmoothingandbaselinesubtraction.

Copyright# 1999JohnWiley & Sons,Ltd. Rapid Commun.MassSpectrom.13, 2335–2340(1999)

LD OF DNA OLIGOMERS LARGER THAN ONE KILOBASE 2337

of the DNA, which arebelievedto havebeendenatured inthematrix solution,form singly anddoubly chargedsingle-stranded molecular ions, M� and M2�, respectively. Theresolution for M� of 320000 Dais about18.Thepeakwidthoriginates in part from the different mass of the twocomplementary DNA strands and partly from adductformation aswell asfragmentation.

Sensitivity

The detection sensitivity of oligonucleotideswas found tobeexcellentwhen4-nitrophenol wasusedasthematrix,anddiammoniumcitrate asthematrix additive.Low femtomolelevels of synthetic oligomersrangingin length from 4 to 65baseswereeasily detectedin bothpositive andnegative ionmassspectra. Similar signal intensity for oligomersof thesesizes could also be obtained using 3-HPA, but morevigoroussampledesalting wasnecessary in the latter case.When analyzing MALDI samples containing DNA frag-mentsexceeding 100bases,4-nitrophenol consistently gavebetterresultsthananyotherUV matrix systemtestedsofar.Less than picomole quantity of up to approximately 800-nucleotide oligomers (about240 kDa) hasbeenroutinelydetected from this matrix. Above 800 bases, the detectionsensitivity with the 4-nitrophenol matrix decreasessig-nificantly, but it is still possible to detectDNA more than1000 bases in length. In contrast, no signal of DNAoligomerslargerthan468-merwasobservedusing either3-HPA or the 3-HPA/PA mixture as the matrix underotherwise identicalexperimentalconditions.

Mass resolution, precision and accuracy

For the4-nitrophenol matrix, thereis generally morepromptand metastable fragmentation as well as photo-adductformation than with the 3-HPA matrix, which appears toresult in a somewhat lower effective mass resolution.Typically, for the nucleotide oligomersup to 55-mer (asshown in Fig. 2), massresolution is approximately half ofthe best massresolution obtained for the samemolecule

using3-HPA asthematrix.In this low massrange, thepeakscorresponding to prompt fragmentationand photo-adductformation areusuallyresolved from themainpeakandpeakintensitiesweresignificantly higherthanthosefrom 3-HPA.For DNA massesexceeding 30 kDa (approximately 100bases),thefragmentation andphoto-adduct peakscannotberesolvedfrom the main molecular ion peakand the massresolution is limited to below 50.

The massspectraobtained with 4-nitrophenolare veryreproducible from ‘shot-to-shot’ as well as ‘spot to spot’.Theprecision of the massdetermination using centroidsofthepeaksis usually0.1–0.3%for theDNA oligomersin thelow massrangeup to 100basesandbetterthan0.5%in thehigh massrangeup to 800 bases. For mass accuracy, arelativeerror of lessthan1%canbeobtained within abroadmassrangewhen the single-stranded DNA oligomersofknownsequencesareusedasthe calibrants.

The massin the spectrum of the ds-DNA about 1000basesin length(shownin Fig. 4) wasexternally calibratedusingsingly anddoubly chargedmolecular ionsof the468-mer shown in Fig. 3. Linear extrapolation from thiscalibration resulted an average mass of approximately320000Da for M�, which is 5000Da less than thatcalculatedby doubling them/zvalueof thedoublychargedM2�. Thereasonfor thissignificantpeakshift towardlowermass is the initial kinetic energy of the ions.8,50,51 AllMALDI ions possesssimilar initial velocity after desorp-tion. As themolecular weightof theion increasesand/oritschargestatedecreases, thecontributionof theinitial kineticenergyto the total kinetic energyof the ion increases,andthusthedifferencebetween theobservedandthecalculatedm/z increases.This nonlinear effect of the initial kineticenergyof the MAL DI ions on masscalibration hasbeenobservedpreviously and was successfully correctedfor inthe analysis of humanimmunoglobulin.8 However,the useof the delayedextraction for the linear TOF instrument inthepresentexperimentmade thecorrection very difficul t.50

For DNA aslargeas1000bases,anerrorof 10 to 20 baseswasexpected from the mass calibration. Note that a moreaccuratemass measurementof 325000Da, correspondingto a DNA fragment of approximately 1060 bases, can beobtainedfrom theM2� ion thanthat from M�, becausetheformer hasa m/zvalue close to that for ss-DNA 468-merusedfor calibration.

Rationalization

Therearetwo possibleexplanationsfor 4-nitrophenol beingausefulMALDI matrix, especially for largeDNA. First,the4-nitrophenol matrix systemshows high tolerancetowardsinorganic impurities in the sample analyzed, especiallysodiumandpotassiumsalts.Analyte-adductionsof Na� orK�, thatoftendegradeMALDI massspectraof DNA using3-HPA, wereeitherof low intensity or not observedfor 4-nitrophenol. Consequently,much less effort for sampledesaltingwasrequired to detectDNA at low concentrationlevelswith the4-nitrophenol matrixsystemthanthatwith 3-HPA. This is a very important factor for large DNAfragmentsthat havemanycationbinding sitesavailable.

Second,DNA in the 4-nitrophenol matrix systemmaygain detectionsensitivity from apparently high desorption/ionization efficiency. Ultimately, the detectionsensitivityfor DNA is determined by combining the efficiency togenerate ions in the initial desorption plume and theprobability for these ions to survive during the extraction

Figure 4. Positiveion massspectrumof approximately10pmol of adouble-strandedPCRproductgreaterthan1000nucleotidesin lengthusinga 4-nitrophenolmatrix. The samplewasderivedfrom a cDNAinsert in a plasmid vector. M� and M2� correspondto singly anddoubly chargedions of the two denaturedDNA strands.The massresolutionis 20. The spectrumwasobtainedby summingover 1000lasershotsand using an 800ns extractiondelay time. The datawasfurther processedwith Savitzky-Golay smoothing and baselinesubtraction.



time. The fact that 4-nitrophenol produces significantfragmentation (as shown in Fig. 2) indicatesa decreasedprobability for intactmolecular ionsto survivecomparedtothe 3-HPA matrix. Therefore, the increased detectionsensitivity couldonly beattributedto the increasedamountof analyte ionsproducedinitially after the laserdesorption/ionization pulse by the 4-nitrophenol matrix system.Thesupporting evidence for the argument of the ionizationefficiency with the 4-nitrophenolmatrix is the observationof relatively high amounts of protonation associated withmultiply chargedspeciesin the massspectra.

Perspectives and applications

In principle, the amount of fragmentation and adductformation can be significantly reducedby introducing anadditive into thematrix systemthatgivesadditionalcoolingin the plumeexpansion. For example, we haveobservedadecreasein base-lossfragmentation in the massspectraofDNA in the 4-nitrophenol matrix by adding solventmolecules into the frozen matrix. However,the sensitivityfor DNA oligomers obtained from the frozen solutionmatrix is typically an order of magnitudeworse. Morerecently, theKarasgroupreportedanotherbeneficial effectfrom additives,suchas fucose, for slowing sublimation.40

Finetuningof thematrix conditionsaswell assearchingforother effective additivesarecurrentlyunderinvestigation.

Rapid progressin utili zing massspectrometry for DNAanalysis is driven by its potentialgenomicapplicationsforDNA sequencing and DNA sizing. With the impressiveimprovementof massresolution made by delayed,pulsedextraction for TOF-MS, single-baseresolution approaching100 bases has been achieved using a 3-HPA matrix.Sequencing readlengthfor thefour-baseSangersequencingreaction has been extended to approximately 100 basesincluding the primer.4 Even though the massresolutionofferedby the4-nitrophenolmatrix is nothighenoughyettoextend the current read length of DNA sequencing byMALDI, the 4-base resolution in the 150–200basesizerangeis sufficient for massspectroscopicdetermination ofallelesof short tandem repeat (STR) loci usedfor humanidentification,35–37aswell asscreeningof geneticdisorders.The combination of mass accuracyof better than 1% andpicomole sensitivity for DNA aslargeas800 basesin sizemakes the 4-nitrophenol matrix especially attractive forapplicationssuchasDNA sizing.With this matrix,wehavebeenableto optimize PCRandclean-up conditions for theongoingdevelopmentof STRloci of forensicinterest.35 Wehave also successfully used4-nitrophenol to detect somefragile-X samplescontaining extensive GC-rich regionswhich were especiallyproblematic for conventional gel-basedanalysis. In the areaof developing a rapid assaybydetection of DNA mutation throughthemass measurementof restriction length fragment polymorphism(RLFP), thisnewmatrix is expected to play an important role.

CONCLUSIONS

A matrixsystemcontaining cooled4-nitrophenolhasshownto be useful for TOF mass spectrometric analysis of largeDNA oligomers. The detection sensitivity permittedby thismatrix for DNA oligomerslargerthan100baseswasfoundto be significantly better than that from any UV matricespreviously reported. Using 4-nitrophenol, the accessiblemassrangefor oligonucleotides can be extendedbeyond

3000000Da, which corresponds to a DNA oligomer sizelarger than1000bases. In addition, thepicomolesensitivityandbetterthan1% massaccuracyfor the routinedetectionof DNA aslargeas800basesmake4-nitrophenolapotentialmatrix system for applications of DNA sizing by massspectrometry.

Acknowledgements

The authorsthankDrs. JoeA. Monforte,ThomasA. Shaler,JohnM.Butler, JiaLi andMs. Yuping Tan for helpful discussions.This workwas supportedby the US National Institutes of Health, NationalHumanGenomeResearchInstitute(GrantR01HG00174).

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laser desorption of dna oligomers larger than one kilobase from cooled 4-nitrophenol

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