Sphingopyxis (formerly Sphingomonas) alaskensis is a model bacterium for studying adaptation to oligotrophy (nutrient-limitation). It has a unique physiology which is fundamentally different to that of the well studied bacteria such as Escherichia coli. To begin to identify the genes involved in its physiological responses to nutrient-limited growth and starvation, we developed high resolution two-dimensional electrophoresis (2-DE) methods and determined the identity of 12 proteins from a total of 21 spots using mass spectrometric approaches and cross-species matching. The best matches were to Novosphingobium aromaticivorans; a terrestrial, hydrocarbon degrading bacterium which was previously classified in the genus Sphingomonas. The proteins identified are involved in fundamental cellular processes including protein synthesis, protein folding, energy generation and electron transport. We also compared radiolabelled and silver-stained 2-DE gels generated with the same protein samples and found significant differences in the protein profiles. The use of both methods increased the total number of proteins with differential spot intensities which could be identified from a single protein sample. The ability to effectively utilise cross-species matching from radiolabelled and silver-stained gels provides new approaches for determining the genetic basis of microbial oligotrophy.