pCAP-based peptide substrates: the new tool in the box of tyrosine phosphatase assays.

Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls.

Posttranscriptional modification of transfer RNA in the submarine hyperthermophile Pyrolobus fumarii.

In the RNA of hyperthermophiles, which grow optimally between 80 degrees C and 106 degrees C, posttranscriptional modification has been identified as a leading mechanism of structural stabilization. Particularly in the Archaeal evolutionary domain these modifications are expressed as a structurally diverse array of modification motifs, many of which include ribose methylation. Using mass spectrometric techniques we have examined the posttranscriptional modifications in unfractionated tRNA from the remarkable organism Pyrolobus fumarii, which grows optimally at 106 degrees C, but up to 113 degrees C (Blöchl et al.

Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii.

Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T(1), accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence.

Posttranscriptional modifications in 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfataricus.

Posttranscriptional modification is common to many types of RNA, but the majority of information concerning structure and function of modification is derived principally from tRNA. By contrast, less is known about modification in rRNA in spite of accumulating evidence for its direct participation in translation. The structural identities and approximate molar levels of modifications have been established for 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfactaricus by using combined chromatography-mass spectrometry-based methods.

Structural characterization of U*-1915 in domain IV from Escherichia coli 23S ribosomal RNA as 3-methylpseudouridine.

Mass spectrometry-based methods have been used to study post-transcriptional modification in the 1900-1974 nt segment of domain IV in 23S rRNA of Escherichia coli, a region which interacts with domain V in forming the three- dimensional structure of the peptidyl transferase center within the ribosome. A nucleoside constituent of M r 258 (U*)which occurs at position 1915, within the highly modified oligonucleotide sequence 1911-psiAACU*Apsi-1917, was characterized as 3-methylpseudouridine (m3psi). The assignment was confirmed by chemical synthesis of m3psi and comparison with the natural nucleoside by liquid chromatography-mass spectrometry.