Use of a nitrocellulose film substrate in matrix-assisted laser desorption/ionization mass spectrometry for DNA mapping and screening

Abstract

A nitrocellulose film is used as a substrate in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) studies of DNA. PCR products and DNA fragment digests obtained from biochemical procedures can be analyzed with the use of a nitrocellulose substrate in MALDI MS whereas no signal is observed with the use of a stainless steel substrate. In this method, on-probe purification allows for effective elimination of the interfering effects of salts, buffers, and other contaminants that are usually present in DNA samples, which serve as important limiting factors in the DNA molecular ion yield in the MALDI process. The use of the nitrocellulose film substrate also appears to improve the shot-to-shot and sample-to-sample reproducibility of the ion yield due to the more homogeneous coverage of matrix/analyte over the sample surface. With the use of the nitrocellulose substrate, DNA fragments of up to 622 base pairs in complex mixtures provide mass spectra with minimal sample purification. Although only species corresponding to single-stranded DNA were detected, a mass calibration scheme was established allowing an accuracy of within one base pair for fragments of < 250 bp under the experimental conditions. Despite the low mass resolution of the spectra obtained, the method has been successfully used for rapid DNA screening for sample disease genes and PCR products.

Fragmentation pathway studies of oligonucleotides in matrix-assisted laser desorption/ionization mass spectrometry by charge tagging and H/D exchange

“…Since Matrix-Assisted Laser Desorption/ Ionization (MALDI) has been introduced, [3,4] much effort has been devoted to the development of oligonucleotide analysis by MALDI mass spectrometry. To date, Tang et al [5] and Liu et al [6] have reported the analysis of PCR products ϳ500 to 600 bases using UV-MALDI (at 337/355 nm), and Hillenkamp and co-workers extended the upper limit of DNA and RNA analyses up to ϳ2180 bases using IR-MALDI [7]. Applications such as analyzing short tandem repeats [8], gene-defect diseases [9 -11], and genotyping of single nucleotide polymorphisms [12] have been attempted.…”

Accurate characterization of the tyrosine hydroxylase forensic allele 9.3 through development of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

“…To date, MALDI-TOF mass spectrometry has been the technique associated with the majority of PCR analyses [23][24][25][26][27][28][29][30][31][32][33][34][35] which was a direct extension of ongoing work in large-scale DNA sequencing efforts. 15,[36][37][38][39][40][41][42] Initial reports from Chen and co-workers 26 and Lubman and co-workers 27 showed MALDI-TOF analysis of restriction endonuclease digestion fragments derived from PCR products.…”

The observation of chaperone‐ligand noncovalent complexes with electrospray ionization mass spectrometry

“…Mass spectrometry has evolved to contribute to biological research on many frontiers (Ganem et al, 1991;Goodlett et al, 1993;Light-Wahl et al, 1993;Gale et al, 1994;Verentchikov et al, 1994;Little et al, 1995;Liu et al, 1995;Loo, 1995;Fitzgerald et al, 1996;Muddiman et al, 1996;Smith et al, 1996). The analysis of many types of biomolecules, including DNA, RNA, proteins, and their noncovalent complexes with mass spectrometry is rapidly becoming of great utility.…”