Focus
Can MS Really Compete in the DNA World?
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n recent years, mass spectrometrists Trailing have made some big predictions of MS could eventually do for electrophoresis by anDNAwhat sequencing. In doing so, they may promised too much, too soon. The order of magnitude have time has come to ask, how realistic are those claims? in sequencing, Before the advent of electrospray and MALDI, the concept of DNA sequencing by MS may be best MS never entered people's minds. "It until electrospray and MALDI that suited for genotyping wasn't you could make reasonably sized ions from DNA There was no point in developing a applications technology for it and no point in learning the chemistry of the ions because they didn't exist," says Scott McLuckey of Oak Ridge National Laboratory. Marvin Caruthers, a professor of biochemistry at the University of ColoradoBoulder who works with DNA and DNA analogues, says, "Five years ago, the concept of using MS to sequence DNA was something that people would say was not possible. If people can now sequence small pieces of DNA, that's a giant step forward. The next giant step forward is to
learn to sequence several hundreds of bases. It's not useful in the real world to sequence short pieces of DNA because you really want to sequence genomes." Scientists don't all agree, of course, but most mass spectrometrists interviewed by Analytical Chemistry expressed a guarded optimism about the possibilities for DNA sequencing with MS. Some, however, while still optimistic, believe that those earlier promises need to be called in and that the MS community, as a whole, needs to face the user community with either more realistic expectations or an instrument that fulfills the earlier promises. Kermit K. Murray of Emory University says "We in the [MS] community oversold the abilities of MS for DNA sequencing" However, just because MS might not yet (or ever) be suited to genomic sequencing, should we count it out of sequencing and DNA analysis entirely? No, because with the end of the Human Genome Project (HGP) in sight (within the next five years, according to some estimates), the day is just dawning on DNA
Focus sequencing of other genomes and for diagnostic purposes. The competition
While mass spectrometrists have worked on DNA sequencing techniques, the DNA community has continued to push the limits of DNA sequencing by conventional methods. Automated sequencers run as many as 96 lanes in parallel and, if fhe user is willing to run the instrument long enough, can attain read lengths greater than a thousand bases an order of magnitude better than the best MS techniques. That doesn't even include the research developments in array CE instruments. According to James McCloskey of the Department of Medicinal Chemistry at the University of Utah "While the MS neoole are trans: to push this large-scale seauencins: the neoole who are doing; it bv other methods are Dushine: theirs and more of them are doing it They are not a stagnant group of people waiting to be saved " DNA sequencing is most commonly done by the electrophoretic separation of fragments generated by the Sanger dideoxy chain termination method. DNA complementary to the chain of interest is synthesized with a labeled primer with addition occurring at the 3' end of each nucleotide. Included in the reaction mixture are 2',3'-dideoxynucleoside triphosphates (ddNTPs) When a ddNTP is added to the chain in the place of its deoxy analogue the lack of a free hydroxyl group at the 3' end of the molecule terminates the chain This reaction is usually carried out in four vessels each with a different ddNTP in the mixture By carefully controlling the ratio of dideoxy terminator trt DNA a mivhire can hp creatpH in which chains of varying length are formed based on the positions of the nucleotides
The four reaction product mixtures are loaded into adjacent lanes (or a single lane if four distinct fluorescent dyes are used) and separated by electrophoresis according to their electrophoretic mobility, an extrinsic property that corresponds roughly to the length of the DNA fragment. Depending on the length of the gel, the separation can take several hours. However, the extended period is compensated for by the parallel nature of the analysis in which many lanes can be run simultaneously. Also the DNA base se244 A
quence can be read directly from the electrophoretic gel. However, the real advantage of electrophoretic sequencing is its long read length. Some of the longer gels allow the reading of sequences as long as 1200 nucleotides. Peter Oefner of the Department of Genetics at Stanford University doesn't see MS making the transition into sequencing applications. "You want to run traces mat have at least 500 to 600 bases, because your M13 clones, on average, have inserts of 800 to 1200 bases. You really want to exploit the clones as much as possible." This is the technology that MS is up against. Although MS may potentially be a more powerful way of determining the DNA sequence, as Lloyd M. Smith of the University of Wisconsin-Madison and Brian T. Chait of Rockefeller University both say, the gels work now and are very
MS provides speed and accurate mass measurements but only short read lengths. robust, making it more difficult for MS to compete. Smith, however, does say, "The potential long-term advantages of MS sequencing are quite substantial, and, even if it takes 10 or more years to acheive, MS would eventually replace electrophoretic sequencing." MS potential
With electrophoresis able to achieve such long read lengths and to analyze so many samples simultaneously, why do mass spectrometrists continue to hold fast to the dream of DNA sequencing? In other words, what can MS offer to DNA sequencing? MS might be applied to DNA in sequencing in numerous ways: as a replacement for the gel in sequencing reactions (either the Sanger method or the enzy-
Analytical Chemistry News & Features, April 1, 1997
matic cleavage method) or as a completely self-contained tool with gas-phase fragmentation. The most straightforward of these applications is as a replacement for the electrophoretic gel in the Sangersequencing reaction. Rather than run four lanes, four mass spectra are obtained, which can be overlaid. The sequence is then read directly from the spectra in much the same way as the sequence is read from a gel. Dick Smith of Pacific Northwest National Laboratory this method as a potential approach to realworld sequencing because it requires lower resolution than methods that would involve effectively mixing; all four lanes The best approach will ultimately depend on the ionization method as well as the ar*tiievable rearl leno+ti and flie lilzplir win
ner is far from ohvious'at this time cording- to Smith
MS offers the ability to separate the fragments from an entire sequencing reaction in less than a second. For example, ,he data acquisition for a single scan takes only a millisecond. However, that value can lead to some unrealistic expectations for the ultimate speed of DNA sequencing by MALDI-MS, says Lloyd Smith, because ii doesn't include thetimenecessary for ancillary activities such as placing the sample in the spectrometer, finding the best spot on the sample averaging the signal or transferring and analyzing the data McCloskey worries that people are misled by such unrealistic claims: "People say, 'You have a way to make ions in the gaa phase, don't you? You can shoot it in microseconds, can't you? You can do hundreds in a shorttime,can't you?' You add up those attributes, and out of it comes a claim that you ought to be able to produce this marvelous method. It isn't quite so simple." Because m/z is an intrinsic property of the species (in contrast with the electrophoretic mobility), the electrophoretic artifact of compression disappears in MS sequencing. Compression results from secondary structure formation and causes the location of the fragment in the gel to inaccurately represent a particular position in the sequence. Conventional sequencing has advanced through the use of the terminator method which reduces the anomalies caused by gels. Ideally, MS would provide results tlist were more reliable and easily interpreted MS P3T1
also distinguish a "false termination" in the Sanger reaction (a fragment that is not actually terminated with a ddNTP) because of the mass difference between a deoxy and a dideoxy nucleotide. The greatest advantage of MS for sequencing is also its most obvious: It provides an accurate mass measurement. "Just as in all MS measurements, knowing the mass gives you a lot of information that you can use intelligently to learn other things," says Lloyd Smith. According to McCloskey, who has focused much of his attention on RNA, the ability to determine modifications which can be identified by mass shifts, is the most important contribution MS can make. When analyzing RNA by conventional methods, the RNA is converted to DNA with reverse transcriptase and the DNA is sequenced by conventional means However the conventional methods no longer work if the RNA has hpen mnriified "The people who are makin g mnHifiprl
RNA have a particularly difficult time because thev are cut off from conventional methods " says Closke "That's one reason why we've concentrated on RNA, because the need for those folks is greater. The DNA folks i
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are less needy. Clearing the hurdles A consensus exists on the current barriers to DNA sequencing by MS, the most formidable of which involves the related issues of mass range and resolution. For MS Sanger sequencing to be feasible, the ionization of DNA fragments several hundred-bases long (on the order of 90 kDa) must be achieved with a mass resolution of 300 or more. With even larger proteins being routinely analyzed by MS, 90 kDa might not sound like much of a problem, but fragmentation and metal adduct formation make it much difficult with DNA Two hurdles to measuring high masses with MALDI are contamination and fragmentation. Metal adducts can be reduced or eliminated by using an ammonium salt, such as ammonium citrate, to exchange with the metal cations present as sample impurities. The ammonium is eliminated as ammonia, leaving H+ behind and preventing die adduction of metals such as sodium. Fragmentation is also a problem because the DNA molecule cleaves easily along the phosphodiester
backbone. According to Chait, chemical modifications such as die inclusion of diaza analogues of the DNA nucleotides can make the molecules less susceptible to fragmentation. Murray's group will be attempting to combat fragmentation with ice matrices, an extension of Peter Williams' work at Arizona State University. They hope that when the ice absorbs IR laser light, numerous water molecules will be ejected in a supersonic expansion that can generate cooling collisions with the DNA before it has an opportunity to fragment. 'You can focus on the MS [as opposed to sequencing per se] and that forces you to focus on the physical chemistry of the desorption [in MALDI] " says Murray.
need for high resolution, which can be achieved if Fourier transform ion cyclotron resonance (FT-ICR) is considered a viable alternative. However, the FT-ICR mass spectrometer is still a research tool that will probably not help make the transition into DNA sequencing in the short term. The combination of electrospray ionization with FT-ICR has already demonstrated that measurements of intact DNA can be made well into the megadalton size range However measurements of 1000component sequencing mixtures present significant challenges 'We're trying to strike a balance between charge state reduction and the resolution needs of the analysis " Smith says "The other essential element of this approach tfiat aDDears
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range enhancement" The ion trap in an FT-ICR mass spectrometer has a limited hi t removing the most abundant species' from the trap clears the way to analyze the
The ultimate test ofMS methods less abundant species, which are generally the higher molecular weight species. for DNA analysis will Sensitivity is an issue in every method of MS DNA sequencing. Murray comments that many examples in the literature use be their use in 100 times more material than the Sanger reaction would provide, thus making these applications. methods less than realistic. Christopher 1
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Becker, president and CEO of GeneTrace (Menlo Park, CA), says, "We have been The resolution requirements for fourusing realistic amounts and types of temspectrum Sanger sequencing are not as plates. One of our primary focuses is on stringent as those for enzymatic cleavage cDNA sequencing and the expressed seladder sequencing, in which mass accuquence tags of cDNA Typical amounts of racy and mass resolution must be suffiplasmids that we seniience fire 0.25 pmoll cient to distinguish the 9 Da difference We can routinely get to 100 bases ,ncludbetween adenine and thymine. When all ing a 20-base primer " four Sanger reactions are carried out in Fragmentation need not always be a the same reaction vessel (so that a single drawback for DNA sequencing. A method spectrum is obtained), the resolution rethat relies solely on gas-phase fragmentaquirements are identical for the Sanger tion is undoubtedly the most elegant for and enzymatic cleavage methods. MS DNA sequencing. Imagine putting Most groups working on DNA seDNA in the instrument, pressing a button, quencing by MS are using MALDI, but and getting the DNA sequence, without some are using electrospray ionization. lengthy sample preparation. However, The multiply charged ions generated by gas-phase fragmentation turns out to be electrospray ionization can be an advanthe most difficult method to interpret betage and a disadvantage. They bring the cause the mass spectrum quickly bemass spectrum into a lower m/z range, comes very complicated. where sensitivity and resolution are imMcLuckey, McCloskey, and Dick proved, according to Dick Smith, but they Smith all believe that gas-phase analysis complicate the spectrum and increase the using an enzyme to sequentially remove one nucleotide at a time would be the ideal mass spectrometric technique. Analytical Chemistry News & Features, April DNA 1, 1997 245 A
What's new
in LC-MS
Focus McLuckey says, "To the ion chemistry community, an ideal would be to have a little 'Pac-Man' molecule that could chew from one end, so you could clip off one base at a time and just measure the mass shift. We haven't found that molecule yet." Each of the MS methods has demonstrated an ability to routinely sequence oligonucleotides 15- to 20-bases long, and 50 to 100 mers would appear to be the current "state of the art", but these demonstrations are usually with known sequences. Murray says, "The problem with a lot of papers and presentations is that they're working with a known they already know the sequence. If you sit there with the sequence and the mass spectrum you can find a peak every place you want one but that's a lot different from starting off with an unknown and figuring out what it is Hardly anybody does that test" It's a test that MS will eventually have t o rtaQQ Hpfore it c a n m o v e i n t o
the r e a l w o r l d of D N A cpniipncincr a n d diaff
nostic aDDlications Becker emphasizes that thev routinely analyze unknowns at GeneTrare although they have thus far chosen not'to nnblish these results
PRODUCT Platform LC
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Research applications also seem broad. Not only can MS be used to measure the exact mass and location of covalent modifications, its role in the study of drug binding and biologically crucial interactions with proteins has been suggested by recent studies. Although questions remain, clear evidence suggests that the study of such noncovalent interactions is feasible. In the long run, such applications may constitute some of the most exciting. At least one company is betting on MS techniques for DNA analysis, in both sequencing and sizing applications. GeneTrace, led by Becker and Joseph A Monforte (vice president for research and development), is a spin-off from SRI International to commercialize MS of DNA for analytical purposes. According to Becker, research applications are where MS will really shine. "If you want to do genomic sequencing, especially of humans where have lot of rcoGcits you Wcint to use a. £fel " he S3vs "If you're looking at more repetitive types of assays including diagnostics or research applications where you're looking for an identification MS can be faster cheaper and inst as armore
What would it take for MS to become a The post-genomic world When the Human Genome Project is com- more widely used sequencing technique? Caruthers says, "You can come up with a pleted, the need for DNA sequencing will sophisticated method using MS that evbe even greater, and that is the time that erybody would be using if it had come to mass spectrometrists are anticipating the fore 10 years ago but now, because of making the biggest contribution. That's not to say there's no disappointment at not the advances in gel technology, may never compete. Timing is important in these being a larger player in the HGP. "It's things, too. You've got to give them somekind of a feeling you get of being on the thing that they can't get with the current sidelines," Murray says. "You want to go technology because they have to equate in and get your chance, but it seems that the expenses with the higher 'whatever' the star running back is racking up the More reliable data nucleotides yards. It looks like the sequencing game might be before MS has a chance to run—whatever it is they have to be able shine." to equate it MS will never compete with Dick Smith says that his group is anti- the current technology if there aren't some really serious advantages of the cipating the "postgenomic era", when mass spectrometer over the gel" PCR will be used more routinely to compare small pieces of genome to the referThe ultimate test of MS methods for ence genome. "When you start talking DNA analysis will be their use in applicaabout PCR, you may be talking about tions. McCloskey says, "All of these oligonucleotides that are often no more [methods] have every right to exist for a than 80 or 100 bases long. MS has already time in development. 'Look what I can do. demonstrated the ability for quickly deter- Here's a new way to do it' But ultimately, mining whether there are differences in it has to make it to the problem-solving base composition and offers the potential stage. If it does, it's a success. If it doesn't, of getting sequence information for PCR we judge it to be either a stepping stone products of that size " he or a dead end." Celia Henry
