Ubiquitin bovine

Proteasome inhibitors have been instrumental in identifying numerous protein substrates and in elucidating the importance of the proteasome/ubiquitin pathway in many biological processes. Initially, non-specific cell-penetrating peptide aldehydes were used for this purpose. More recently, it became possible to synthesize compounds with increased potency and selectivity (Adams et al. 1998 Elofsson et al. 1999). Furthermore, based on the crystal structure of the yeast and bovine liver CP (Groll et al. 1997 Unno et al. 2002), molecular modeling can now be used to engineer improved inhibitors. 

FIGURE 9 (A) ESI-MS of native bovine ubiquitin obtained after 20 min in a nondenaturing solution. (B) ESI-MS of native bovine ubiquitin obtained after 23 min in a denaturing solution. (Reproduced with permission from Katta and Chait.31 Copyright John Wiley Sons Limited.)... 

Identifying altered canine and bovine proteins has proved to be particularly challenging since these species are poorly represented in current genomic databases. As a result of this, new bioinformatic tools (Multildent, http //expasy.org) have had to be developed to facilitate cross-species protein identification (Wilkins et al., 1998). The most significant change observed for bovine DCM was a sevenfold increase in the enzyme ubiquitin carboxyl-terminal hydrolase (UCH) (Weekes... 

More recently we have investigated whether the ubiquitin-proteosome system is perturbed in the heart of human DCM patients (Weekes et al, 2003). As in bovine DCM, expression of the enzyme UCH was elevated more than 8-fold at the protein level and elevated more than 5-fold at the mRNA level in human DCM. Moreover, this increased expression of UCH was shown by immuno-cytochemistry to be associated with the myocytes, which do not exhibit detectable staining in control hearts. Overall protein ubiquitination was increased 5-fold in DCM relative to control hearts. Using a selective affinity purification method we were able to demonstrate enhanced ubiquitination of a number of distinct proteins in DCM hearts. We have identified a number of these proteins by mass spectrometry. Interestingly many of these proteins were the same proteins previously found to be present at reduced abundance in DCM hearts (Corbett et al, 1998). This new evidence strengthens our hypothesis that inappropriate ubiquitin conjugation leads to proteolysis and depletion of certain proteins in the DCM heart and may contribute to loss of normal cellular function in the diseased heart. 

Denisov V, Halle B (1995) Protein hydration dynamics in aqueous solution. A comparison of bovine pancreatic trypsin inhibitor and ubiquitin by oxygen-17 spin relaxation dispersion. J Mol Biol 245 682-697... 

Fig. 10.8 ESI-FT-ICR mass spectrum of bovine tions of the 6- and 12-fold protonated ions ubiquitin. A sample solution of ca. 0.01 mg ml (monoisotopic molecular weight, determined in 3 % aqueous acetic acid methanol (4 1) was 8559,5912 calculated 8559,6152 A m 3 ppm), employed. The inserts show isotopic separa-...

FIGURE 1.13 Collision cross sections for conformers of protonated bovine ubiquitin ions (z = 6-13) produced using ESI, measured in He (a) and N2 (b) gases. (From Sbvartsburg, A. A., Li, F., Tang, K., Smith, R.D., Anal. Chem., 78, 3304, ibid 8575, 2006.)... 

Purves, R.W., Barnett, D.A., Ells, B., Guevremont, R., Elongated conformers of charge states +11 to +15 of bovine ubiquitin studied using ESI-FAMS-MS. J. Am. Soc. Mass Spectrom. 2001, 12, 894. 

Figure 4,2 The repeatability and intermediate measurement precision IMP) of a robotic HX-MS apparatus. The study is based on replicant measurements of bovine ubiquitin deuterium uptake after 30s immersion in D O. (a) The peptide map of ubiquitin, comprising 29 peptides (100% protein coverage) observed in 39 analyzed peptide ions, used for the HX-MS analyses (see footnotes 2, 3 and Ref [27]). (b) The average fractional D uptake observed for each of the 39 analyzed peptide ions. As shown in the key, each symbol presents the average of 20 measurements conducted on Day 1, 2, and 37. Bars on each symbol indicate the standard deviation (SD) when it exceeds the symbol size, (c) Repeatability and IMP for each peptide set, plotted as the SD of the D fraction measurements obtained for each peptide. The horizontal solid line in each group indicates the mean SD of the peptide set (780 measurements)...

The graphs in Figure 4.2 illustrate the terms repeatability and IMP. These data were obtained by the authors during an evaluation of their robotic HX-MS system. - The experiments measured the deuterium uptake by bovine ubiquitin after immersion in D O for 30 s. This short exchange period... 

R. W. Purves, D. A. Bamnett, B. Ells, and R. Guevermont, Investigation of bovine ubiquitin conformers separated by high-field asymmetric waveform ion mobility spectrometry cross section measurements using energy-loss experiments with a triple quadrupole mass spectrometer, 7. Am. Soc. Mass Spectrom. 11, 738-745 (2000). 

Freitas, M.A., Hendrickson, C.L., Emmett, M.R., Marshall, A.G. (1999) Gas-phase Bovine Ubiquitin Cation Conformations Resolved by Gas-phase Hydrogen/ Deuterium Exchange Rate and Extent. Int. J. Mass Spectrom. 185/186/187 565-575. 

In one of the pioneering studies, conformational changes were induced in a model protein (bovine ubiquitin) by adding methanol to aqueous acidic solutions of the analyte [61], Using HDX in combination with ESI-MS, it was possible to follow conformational changes in bovine ubiquitin and in chicken egg lysozyme (due to the reduction of the disulfide... 

Protein folding studies generally concern liquid-phase phenomena however, HDX reactions can also be conducted in the gas phase (e.g., [84-90] for an overview see [91]). For instance, multiply charged ions of several model proteins reacted with DjO in a vacuum following pseudo-first-order kinetics [85]. It was also observed that removing solvent significantly increased conformational rigidity. In other work, ions of bovine ubiquitin were... 

Figure 3 Schematic diagram of an expression plasmid containing the kanamycin resistance gene, origin of bacterial replication, cytomegalovirus immediate-early promoter enhancer (CMV), ubiquitin B promoter and intron, chloramphenicol acetyltransferase reporter gene (CAT), and bovine growth hormone (BGH) polyadenylation signal.

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