Matrix-assisted laser desorption ionization time of flight mass spectrometry was used to sequence exons 5 to 8 of the human p53 gene. A single tube procedure was established for target amplification and mass spectrometric (MS) sequencing. The MS sequencing scheme is designed for high throughput and parallel sample processing, and is amenable to full automation. Reliable sequencing data were obtained using fmol sample amounts. The high resolution and accuracy of MS sequencing was demonstrated by direct sequencing of a heterozygous template.

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.

The only way to obtain a clear picture of the structure of these proteins is the generation of 2-dimensional or 3-dimensional crystals or the structural analysis of purified protein by NMR. For most membrane proteins, however, already the first steps towards any of these techniques are hindered by the low expression levels of these proteins, by their extreme hydrophobicity and the concomitant difficulties during purification.The attachment of hydrophilic tags to such proteins has become an important tool to overcome at least part of these problems [9 12]. The present paper describes the advantages of a bacterial biotin acceptor domain for the one-step purification of the recombinant P major sucrose carrier PmSUC2 [13] from transgenic yeast.

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