Protein pI shifts due to posttranslational modifications in the separation and characterization of proteins

Proteins from breast cancer cell lines are characterized using a 2-D liquid separation technique in which protein pI is used as the first-dimension separation parameter. To effect this protein pI separation, chromatofocusing(CF) is employed whereby a pH gradient is generated on-column using a weak anion exchange medium with the intact proteins fractionated and collected every 0.2 pH unit. It is demonstrated that the pI for expressed intact proteins as generated by CF is an important parameter for identification and characterization of the actual protein modifications occurring in the cancer cell. For most proteins, the experimentally determined pI is very close to that predicted by the databases. In other cases, however, where the pI is observed to be shifted from the expected value, it is shown that this shift is often correlated to protein modifications. The modifications that cause such shifts include truncations and deletions often observed in cancer cells or phosphorylations that can shift the pI by several pH units. It is also shown that the effects of phosphorylation on the observed shift can vary depending upon the protein and the amount of phosphorylation. Moreover, large changes in the pI are often observed for proteins with a pI above 7.0 upon phosphorylation, whereas little change is observed for proteins with a pI of approximately 5.0. The expressed protein's pI value thus becomes an important parameter together with the intact MW value, peptide map, and MS/MS results for identification of the presence and type of posttranslational modifications occurring in the cancer cell.     

Genomic and proteomic identification of a DNA-binding protein used in the "fingerprinting" of campylobacter species and strains by MALDI-TOF-MS protein biomarker analysis

We have identified a prominent approximately 10-kDa protein biomarker observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five thermophilic species of Campylobacter: jejuni, coli, lari, upsaliensis, and helveticus. The biomarker was unambiguously identified by genomic and proteomic sequencing as a DNA-binding protein HU. We report the amino acid sequence of HU as determined by sequencing the hup gene of four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari(2). Confirmation of the amino acid sequence was obtained by nanoflow high-performance liquid chromatography-tandem mass spectrometry of the tryptic peptides of the extracted/digested HU protein. Protein identification was also confirmed by comparison of the molecular weight (MW) predicted from the hup gene and the MW of HU as measured by high-resolution mass spectrometry. We found the HU protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its MW varies between species and among strains within a species. Intra- and interspecies variation of the HU MW is due to changes in the amino acid sequence of the HU protein and not due to co- or posttranslational modifications. The strong ionization efficiency of HU by MALDI is likely due, in part, to four lysine residues clustered at the carboxyl end of the protein. We also report identification of the HU protein biomarker for a C. helveticus strain, whose hup gene was not sequenced, but whose HU amino acid sequence was partially conserved in C. upsaliensis strains. We have also tentatively assigned a approximately 10.5-kDa protein biomarker of a C. concisus strain as an HU protein.     

Some characteristics of kidney and liver lipids from the American antelope

Summary The percentages of total lipids, phospholipids, and cholesterol in the kidney and liver of pregnant antelope are reported. The iodine values of the total lipids in these two organs are also listed. By the use of three collections of the antelope made about one month apart during the latter part of the gestation period, it was shown that there were no significant changes in the above lipid values during this period. Published with approval of the Director, Wyoming Agricultural Experiment Station, as Journal Paper No. 123.     

Mobilities of polymer liquids at constant temperature and at constant volume

Previously developed relationships between isomobility states and equilibrium p-v-T properties of vinyl polymers are extended to predict mobilities, μ, at constant temperature and at constant volume, with poly(vinyl acetate) as an example. At constant volume, μ changes by several orders of magnitude while the ‘internal pressure’ remains constant, suggesting that kinetic energy (temperature) dominates in governing μ. From μ at constant temperature the Vogel parameters, B and T₀, are found to increase with pressure, the former increasing linearly. A new Vogel type equation is developed in which one of the parameters, B_v, depends only on the chemical composition of the polymer. Both μ and its ‘activation energy’ at constant pressure, E_p, are shown to be constant at the glass transition.     

Moving-average output interface for digital stochastic computers

A new logic circuit suitable for use as an output interface for digital stochastic computers is presented. The logic element is based on the theory of moving averages, and is demonstrated to provide a significant improvement in transient-response characteristics, without loss in accuracy.     

ELF Earth Return Coupling Into Power Systems

With the advent of the U.S. Navy's proposed ELF Communications System (Sanguine System), which would use an earth-return circuit to excite the earth-ionosphere cavity, earth-return current coupling into power systems has become important. The theoretical basis for coupling into power distribution systems is developed. A comparison is made between measured and predicted values of the induced voltage. Techniques which are useful in eliminating the effects of the induced voltage are described. The effectiveness of these interference mitigation techniques is demonstrated by comparing measurements on actual systems. Finally, data on power system interference are developed for use in communication system design tradeoff studies.     

Isothermal crystallization measurements on poly(ethylene terephthalate) by differential enthalpic analysis

The process of solid-phase crystallization in poly(ethylene terephthalate) has been followed by means of differential enthalpic analysis. Kinetic data for the process has been obtained for isothermal annealing from 100 to 115°C., showing induction times and first-order rate dependence. For the material studied, undrawn polyester filaments, an activation energy of 44 kcal./mole was determined. The technique used provides more direct monitoring of the crystallization phenomenon than that achieved with other methods.