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1.
J Mol Med (Berl) ; 75(10): 745-50, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9382998

ABSTRACT

Mutations located in the RET proto-oncogene at codon 634 associated with multiple endocrine neoplasia type 2A and medullary thyroid carcinoma are detected by low-resolution and high-resolution mass spectrometry schemes not requiring labeling or electrophoretic separation of diagnostic products. The former requires measurement by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of 21- to 27-mer oligonucleotides generated by a primer oligo base extension reaction. The latter is based upon direct measurement of artificial products which include the mutation site using matrix-assisted laser desorption ionization Fourier transform mass spectrometry. In this feasibility study a synthetic 25-mer representing the wildtype allele (7660.3 Da) was easily distinguished from G to A (7644.3 Da) and G to T (7635.3 Da) mutant alleles; the mutant alleles, which differed in mass by only 9.0 Da, were easily resolved when analyzed as a mixture. The results of both detection schemes were highly accurate and reliable, indicating mass spectrometry to be a high-quality alternative for future DNA diagnostics performed in clinical laboratories and genetic profiling studies.


Subject(s)
Drosophila Proteins , Multiple Endocrine Neoplasia Type 2a/genetics , Point Mutation , Proto-Oncogene Proteins/genetics , Proto-Oncogenes , Receptor Protein-Tyrosine Kinases/genetics , Thyroid Neoplasms/genetics , Base Sequence , Carcinoma, Medullary/genetics , Genetic Carrier Screening , Homozygote , Humans , Oligonucleotide Probes , Proto-Oncogene Mas , Proto-Oncogene Proteins c-ret
2.
Anal Chem ; 68(13): 2090-6, 1996 Jul 01.
Article in English | MEDLINE | ID: mdl-9027224

ABSTRACT

The matrix-assisted laser desorption/ionization (MALDI) method has been used with an external ion source Fourier transform mass spectrometer (FIMS) to analyze single-stranded, mixed-base oligomers of DNA. It is demonstrated that ultrahigh mass resolution (830 000 fwhm) can be achieved for small oligomers, and high resolution (136 000 fwhm) can be achieved for a 25-mer at m/z 7634. MALDI-FTMS can clearly separate the molecular ion peaks from analyte-matrix adduct peaks and alkali metal-containing species that result from replacement of hydrogen ions with sodium or potassium ions at multiple sites along the phosphate backbone. Previous MALDI-FTMS studies of oligonucleotides had two limitations: (1) low sensitivity due to difficulty in trapping the high kinetic energy ions made by the laser and (2) fragmentation of the ions due to the long delay (tens to hundreds of milliseconds) between their formation and detection. Both of these problems are alleviated in the present study. With the external ion source FTMS instrument, ions made by MALDI are injected at low energy into the analyzer cell by a rf-only quadrupole ion guide, captured by gating the voltage on the trapping plates, and cooled by a 0.5-s pulse of argon gas. Under these conditions, fragmentation is minimized, and DNA ions can be trapped in the FTMS analyzer cell for greater than 50 s. Sensitivity is also improved, as demonstrated by detection of 1 pmol of a single-stranded, mixed-base 20-mer of DNA, with a signal-to-noise ratio greater than 20:1.


Subject(s)
Oligonucleotides/analysis , Sequence Analysis, DNA/methods , Base Sequence , Fourier Analysis , Molecular Sequence Data , Sequence Analysis, DNA/instrumentation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
3.
Rapid Commun Mass Spectrom ; 8(9): 743-9, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7524807

ABSTRACT

Sensitivity in the low-femtomole range with mass resolution greater than 20,000 is demonstrated for several polypeptides analyzed by a mass spectrometer that pairs matrix-assisted laser desorption/ionization (MALDI) and Fourier-transform mass spectrometry (FTMS). The compounds investigated were substance P, renin substrate, melittin, the B-chain of bovine insulin, and bovine insulin. Standard solutions of the polypeptides were prepared with 30% acetonitrile+water, and micropipettes were used to transfer small amounts (1-20 fmol) to a sample probe. The samples were embedded in a large excess of matrix material (2,5-dihydroxybenzoic acid) and ionized by a pulse from an excimer laser. The FTMS instrument used for these experiments has the MALDI source in a separate chamber outside the magnetic field. Ions are extracted from the source and transported by an RF-only quadrupole ion guide to an FTMS analyzer cell mounted in the homogeneous region of a 6.5 T superconducting magnet. The high sensitivity of MALDI-FTMS is due, in part, to the high transfer efficiency of the ion guide, even for ions with a wide range of kinetic energies. The ion guide is easy to use because there are only two adjustments (RF amplitude and DC offset voltage), and unlike electrostatic ion transport means, alignment of it with the axis of the magnetic field is not critical. The mass resolution and sensitivity of MALDI-FTMS is compared with that of MALDI done with time-of-flight, magnetic sector, and quadrupole ion-trap mass spectrometers.


Subject(s)
Peptides/chemistry , Amino Acid Sequence , Angiotensinogen/chemistry , Animals , Cattle , Fourier Analysis , Humans , Insulin/chemistry , Lasers , Mass Spectrometry , Melitten/chemistry , Molecular Sequence Data , Substance P/chemistry
4.
Nature ; 370(6488): 393-5, 1994 Aug 04.
Article in English | MEDLINE | ID: mdl-8047148

ABSTRACT

Matrix-assisted laser desorption/ionization is an accurate and sensitive method for measuring the molecular weights of peptides and proteins. Usually time-of-flight mass spectrometry is used to detect the laser-produced ions, but a new method called Fourier transform mass spectrometry offers greater sensitivity and much higher mass measurement accuracy.


Subject(s)
Mass Spectrometry/methods , Proteins/chemistry , Animals , Cattle , Fourier Analysis , Insulin/chemistry , Lasers , Mass Spectrometry/instrumentation , Vasopressins/chemistry
5.
Anal Chem ; 66(13): 2077-83, 1994 Jul 01.
Article in English | MEDLINE | ID: mdl-8067524

ABSTRACT

A new calibration method has been developed for Fourier transform mass spectrometry (FTMS) that is accurate to better than 0.001% (10 ppm) for peptides and proteins up to 5700 Da. The custom-designed FTMS instrument used for this work has a matrix-assisted laser desorption/ionization (MALDI) source located outside of the magnetic field in a differentially pumped chamber, and ions are injected through the fringing fields of the magnet into the FTMS analyzer cell by a long quadrupole ion guide. The mass spectrometer is calibrated with four model compounds ([Arg8]-vasopressin, melittin, bovine insulin B-chain, and bovine insulin) of known molecular mass. The set of measured ion resonance frequencies (f) for these compounds are fit to a three-term calibration equation of the form f = A(z/m) + B(V) + C(V2) (m/z), where m/z is the mass-to-charge ratio of a calibrant peak, V is the trapping voltage, and A, B, and C are calibration coefficients that depend on the magnetic field strength and the dimensions of the analyzer cell. The same set of calibration coefficients can be used for many weeks because the magnet and the electronics of the FTMS instrument are very stable. This method is useful because unknowns can be run separately without the need to add an internal calibration compound in with the sample.


Subject(s)
Peptides/analysis , Proteins/analysis , Fourier Analysis , Mass Spectrometry , Molecular Weight
6.
Proc Natl Acad Sci U S A ; 91(11): 4801-5, 1994 May 24.
Article in English | MEDLINE | ID: mdl-8197138

ABSTRACT

Matrix-assisted laser desorption/ionization (MALDI) has been used with an external ion source Fourier-transform mass spectrometer to obtain the highest mass resolution ever, to our knowledge, demonstrated for laser-produced ions (m/delta m = 1,100,000 for [Arg8]vasopressin, 228,000 for melittin, and 90,000 for bovine insulin). The peaks in the isotope cluster for bovine insulin are fully resolved, and the mass measurement accuracy is an order of magnitude better than can be achieved with time-of-flight mass spectrometry. With the method described here, analyte is applied to a sample probe and mixed with a solution containing a matrix material (2,5-dihydroxybenzoic acid) that strongly absorbs ultraviolet light. Upon irradiation with a pulse from an excimer laser (353 nm, 2 mJ), a large number of intact protonated molecular ions are produced. The ions are focused by a 117-cm-long quadrupole ion guide and injected into an ion cyclotron resonance analyzer cell located inside the bore of a 6.5-T superconducting magnet. A pulse of argon buffer gas cools the ions prior to detection. One of the principal advantages of an external ion source Fourier-transform mass spectrometer is that the ion formation and ion detection processes are separated and can be independently optimized.


Subject(s)
Mass Spectrometry , Peptides/chemistry , Proteins/chemistry , Animals , Cattle , Fourier Analysis , Insulin/chemistry , Ions , Lasers , Mass Spectrometry/methods
7.
Rapid Commun Mass Spectrom ; 8(3): 237-41, 1994 Mar.
Article in English | MEDLINE | ID: mdl-7513214

ABSTRACT

Mass spectra of several model oligopeptides (substance P, [Arg8]-vasopressin, tyrothricin, and the B-chain of bovine insulin) have been obtained with a matrix-assisted laser desorption/ionization (MALDI) source and a custom-designed Fourier-transform mass spectrometer (FTMS). The MALDI source is outside the magnetic field in a separately pumped external chamber, and ions are injected through the fringing fields of the magnet and into the FTMS analyzer cell by a long quadrupole mass filter that is operated in the RF-only mode. A large window on the ion source housing makes it easy to point and focus the laser beam onto the sample probe tip. A good quality mass spectrum is achieved for 0.5 pmol of substance P, and the mass resolution for the B-chain of bovine insulin is 19,000.


Subject(s)
Oligopeptides/analysis , Animals , Arginine Vasopressin/isolation & purification , Cattle , Fourier Analysis , Insulin/analysis , Lasers , Mass Spectrometry , Substance P/analysis , Tyrothricin/analysis
12.
Rev Sci Instrum ; 49(1): 111, 1978 Jan.
Article in English | MEDLINE | ID: mdl-18698950

ABSTRACT

A pulsed ion cyclotron resonance mass spectrometer utilizes the cyclotron resonance principle for mass analysis of ions trapped at low pressures by electric and magnetic fields. Both mass analysis and ion trapping are accomplished in a one-region device called a trapped ion analyzer cell. A pulsing sequence is described which allows for generation of ions by electron impact, reaction with added gases, and mass analysis of the products of ion-molecule reactions. Methods are described for measuring rate constants and equilibrium constants for ion-molecule reactions. The high ion trapping efficiency and open geometry of the analyzer cell make it well suited for studying the interaction of laser radiation with gaseous ions and may also be useful for high-accuracy isotope ration mass spectrometry.

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