Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Reexamination Certificate
1998-05-07
2004-04-20
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
C436S043000, C436S086000, C436S091000, C436S093000, C436S173000, C436S174000, C436S181000
Reexamination Certificate
active
06723564
ABSTRACT:
BACKGROUND OF THE INVENTION
Mass spectrometry provides a means of “weighing” individual molecules by ionizing the molecules in vacuo and making them “fly” by volatilization. Under the influence of combinations of electric and magnetic fields, the ions follow trajectories depending on their individual mass (m) and charge (z). For molecules of low molecular weight, mass spectrometry has long been part of the routine physical-organic repertoire for analysis and characterization of organic molecules by the determination of the mass of the parent molecular ion. In addition, by arranging collisions of this parent molecular ion with other particles (e.g., argon atoms), the molecular ion is fragmented, forming secondary ions by the so-called collision induced dissociation (CID). The fragmentation pattern/pathway very often allows the derivation of detailed structural information.
During the last decade, mass spectrometry (MS) has become an important analytical tool in the analysis of biological macromolecules. This is due at least in part to introduction of the so-called “soft ionization” methods, namely Matrix-Assisted Laser Desorption/Ionization (MALDI) and ElectroSpray Ionization (ESI), which allow intact ionization, detection and exact mass determination of large molecules, i.e. well exceeding 300 kDa in mass (Fenn, J. B., et al., (1989)
Science
246, 64-71; Karas M. & Hillenkamp F. (1988)
Anal. Chem
. 60, 2299-3001).
MALDI-MS (reviewed in (Nordhoff E., et al., (1997)
Mass Spectrom. Rev
. 15: 67-138) and ESI-MS have been used to analyze nucleic acids. However, since nucleic acids are very polar biomolecules, that are difficult to volatize, there has been an upper mass limit for clear and accurate resolution.
ESI would seem to be superior to MALDI for the intact desorption of large nucleic acids even in the MDa mass range (Fuerstenau S. D. & Benner W. H. (1995).
Rapid Commun. Mass Spectrom
. 9, 1528-38; Chen R., Cheng X., Mitchell et al., (1995).
Anal. Chem
. 67, 1159-1163). However, mass assignment is very poor and only possible with an uncertainty of around 10%. The largest nucleic acids that have been accurately mass determined by ESI-MS, so far, are a 114 base pair double stranded PCR product (Muddiman D. C., Wunschel D. S., Lis C., Pasâ-Tolic L., Fox K. F., Fox A., Anderson G. A. & Smith R. D. (1996)
Anal. Chem
. 68, 3705-3712) of about 65 kDa in mass and a 120 nucleotide
E. coli
5S rRNA of about 39 kDa in mass (Limbach, P. A. Crain, P. F., McCloskey, J. A., (1995)
J. Am. Soc. Mass Spectrom
. 6:27-39). ESI furthermore requires extensive sample purification.
A few reports on the MALDI-MS of large DNA molecules with lasers emitting in the ultraviolet (UV) have been reported (Ross P. L. & P. Belgrader (1997)
Anal. Chem
. 69: 3966-3972; Tang K., et al., (1994)
Rapid Commun. Mass Spectrum
. 8: 727-730; Bai J., et al., (1995)
Rapid Commun. Mass Spectrum
. 9: 1172-1176; Liu Y-H-, et al., (1995)
Anal. Chem
. 67: 3482-3490 and Siegert C. W., et al., (1997)
Anal. Biochem
. 243, 55-65. However, based on these reports it is clear that analysis of nucleic acids exceeding 30 kDa in mass (i.e. ca. a 100 mer) by UV-MALDI-MS gets increasingly difficult with a current upper mass limit of about 90 kDa (Ross P. L. & P. Belgrader (1997)
Anal. Chem
. 69: 3966-3972). The inferior quality of the DNA UV-MALDI-spectra has been attributed to a combination of ion fragmentation and multiple salt formation of the phosphate backbone. Since RNA is considerably more stable than DNA under UV-MALDI conditions, the accessible mass range for RNA is up to about 150 kDa (Kirpekar F., et al., (1994).
Nucleic Acids Res
. 22, 3866-3870).
The analysis of nucleic acids by IR-MALDI with solid matrices (mostly succinic acid and, to a lesser extent, urea and nicotinic acid) has been described (Nordhoff, E. et al., (1992)
Rapid Commun. Mass Spectrom
. 6: 771-776; Nordhoff, E. et al., (1993)
Nucleic Acids Res
. 21: 3347-3357; and Nordhoff, E. et al., (1995)
J. Mass Spec
. 30: 99-112). The 1992 Nordhoff et al., paper reports that a 20-mer of DNA and an 80-mer of RNA were about the uppermost limit for resolution. The 1993 Nordhoff et al. paper, however, provides a distinct spectra for a 26-mer of DNA and a 104-mer of tRNA. The 1995 Nordhoff et al., paper shows a substantially better spectra for the analysis of a 40-mer by UV-MALDI with the solid matrix, 3-hydroxy picolinic acid, than by IR-MALDI with succinic acid (See FIGS.
1
(
d
) and
1
(
e
)). In fact the 1995 paper reports that IR-MALDI resulted in a substantial degree of prompt fragmentation.
Nucleic acid analysis can be useful, for example, for diagnosing the existence of a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism or to provide information relating to identity, heredity or compatibility. Since mass spectrometry can be performed relatively quickly and is amenable to automation, improved methods for obtaining accurate mass spectra for larger nucleic acid molecules (e.g. larger than about 90 kDa of DNA and 150 kDa of RNA) are clearly needed.
SUMMARY OF THE INVENTION
In one aspect, the invention features processes for rapidly and accurately determining the mass of nucleic acids (e.g. DNA or RNA) using infrared matrix assisted laser desorption ionization (IR-MALDI) mass spectrometry and a liquid matrix.
In a preferred embodiment, a solution containing the nucleic acid and a liquid matrix is deposited onto a substrate to form a homogeneous, transparent thin layer of nucleic acid solution, which is then illuminated with infrared radiation, so that the nucleic acid is desorbed and ionized, thereby emitting ion particles, which are then analyzed using a mass analyzer to determine the identity of the nucleic acid. Preferably, sample preparation and deposition is performed using an automated device.
Preferred liquid matrices for use herein have a sufficient absorption at the wavelength of the laser to be used in performing desorption and ionization and are a liquid (not a solid or a gas) at room temperature (20° C.). For absorption purposes, the liquid matrix can contain at least one chromophore or functional group that strongly absorbs infrared radiation. Preferred functional groups include: nitro, sulfonyl, sulfonic acid, sulfonamide, nitrile or cyanide, carbonyl, aldehyde, carboxylic acid, amide, ester, anhydride, ketone, amine, hydroxyl, aromatic rings, dienes and other conjugated systems.
Particularly preferred liquid matrices are substituted or unsubstituted: (1) alcohols, including: glycerol, 1,2- or 1,3-propane diol, 1,2-, 1,3- or 1,4-butane diol, triethanolamine; (2) carboxylic acids including: formic acid, lactic acid, acetic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid and esters thereof; (3) primary or secondary amides including acetamide, propanamide, butanamide, pentanamide and hexanamide, whether branched or unbranched; (4) primary or secondary amines, including propylamine, butylamine, pentylamine, hexylamine, heptylamine, diethylamine and dipropylamine; (5) nitriles, hydrazine and hydrazide.
Preferably, a liquid matrix for use herein, is miscible with a nucleic acid compatible solvent. It is also preferable that the liquid matrix is vacuum stable, i.e. has a low vapor pressure, so that the sample does not evaporate quickly in the mass analyzer. Preferably the liquid should also be of an appropriate viscosity to facilitate dispensing of micro- to nano-liter volumes of matrix alone or mixed with a nucleic acid compatible solvent. Mixtures of different liquid matrices and additives to such matrices may be desirable to confer one or more of the above-described properties.
Once prepared, the nucleic acid/matrix solution is deposited as a thin layer on a substrate, which is preferably contained within a vacuum chamber. Preferred substrates for holding the nucleic acid/matrix solution are selected from the group consisting of: beads, capillaries, flat supports, pins and wafers, with or without filter plates. Preferably the temperature of the substr
Heller Ehrman White & McAuliffe LLP
Seidman Stephanie L.
Sequenom Inc.
Soderquist Arlen
LandOfFree
IR MALDI mass spectrometry of nucleic acids using liquid... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with IR MALDI mass spectrometry of nucleic acids using liquid..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and IR MALDI mass spectrometry of nucleic acids using liquid... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3250881