Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients

Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3–10 and narrow range pH 4–7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6–11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2‐D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well‐resolved or visible using wide‐range pH 3–10 IPG gel strips. Twenty‐seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N‐terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5–10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI ≈︁ 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6–11) IPGs has allowed the visualisation of a significantly greater percentage of the ‘functional proteome’, that portion of the total protein complement of a genome actively translated within a specific time frame, on 2‐D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.