gene regulation ͉ hydroxylation ͉ signal transduction R educed oxygen levels (hypoxia) lead to a set of cellular adaptations, including increased angiogenesis and erythropoiesis and a switch to glycolytic metabolism. The cellular machinery that senses hypoxia is composed of several proteins. A critical component is the transcription factor hypoxiainducible factor 1␣ (HIF-1␣) (1). The level and activity of HIF-1␣ are controlled by oxygen-dependent prolyl (PHD) and asparaginyl factor-inhibiting HIF-1␣ (FIH-1)] hydroxylases. PHDs hydroxylate two proline residues in the degradation domain of HIF-1␣ in normoxia, which makes HIF-1␣ a substrate for the von Hippel-Lindau E3 ubiquitin ligase and proteasomal degradation. After stabilization in hypoxia, HIF-1␣ interacts with aryl hydrocarbon receptor nuclear translocator (ARNT) to bind to hypoxia response elements (HREs)

The determination of disulfide bonds is an important aspect of gaining a comprehensive understanding of the chemical structure of a protein. The basic strategy for obtaining this information involves the identification of disulfide-linked peptides in digests of proteins and the characterization of their half-cystinyl peptide constituents. Tools for disulfide bond analysis have improved dramatically in the past two decades, especially in terms of speed and sensitivity. This improvement is largely due to the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), and complementary analyzers with high resolution and accuracy. The process of pairing half-cystinyl peptides is now generally achieved by comparing masses of non-reduced and reduced aliquots of a digest of a protein that was proteolyzed with intact disulfide bonds. Pepsin has favorable properties for generating disulfide-linked peptides, including its acidic pH optimum, at which disulfide bond rearrangement is precluded and protein conformations are likely to be unfolded and accessible to cleavage, and broad substrate specificity. These properties potentiate cleavage between all half-cystine residues of the substrate protein. However, pepsin produces complex digests that contain overlapping peptides due to ragged cleavage. This complexity can produce very complex spectra and/or hamper the ionization of some constituent peptides. It may also be more difficult to compute which half-cystinyl sequences of the protein of interest are disulfide-linked in non-reduced peptic digests. This ambiguity is offset to some extent by sequence tags that may arise from ragged cleavages and aid sequence assignments. Problems associated with pepsin cleavage can be minimized by digestion in solvents that contain 50% H(2) (18)O. Resultant disulfide-linked peptides have distinct isotope profiles (combinations of isotope ratios and average mass increases) compared to the same peptides with only (16)O in their terminal carboxylates. Thus, it is possible to identify disulfide-linked peptides in digests and chromatographic fractions, using these mass-specific markers, and to rationalize mass changes upon reduction in terms of half-cystinyl sequences of the protein of interest. Some peptides may require additional cleavages due to their multiple disulfide bond contents and/or tandem mass spectrometry (MS/MS) to determine linkages. Interpretation of the MS/MS spectra of peptides with multiple disulfides in supplementary digests is also facilitated by the presence of (18)O in their terminal carboxylates.

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of posttranslational modification). Venom proteins and their posttranslational modifications were characterized using specific antibodies, phosphoprotein-and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A 2 and ␣-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A 2 inhibitors, protein-disulfide isomerase, 5-nucleotidases, cysteinerich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.