Alfred L. Gaertner

Impact of deglycosylation methods on two‐dimensional gel electrophoresis and matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry for proteomic analysis

Glycosylation is a common post‐translational modification that can add complexity to the proteome of many cell types. We used enzymatic and chemical methods of deglycosylation to treat a heavily glycosylated exoproteome sample from the filamentous fungus Trichoderma reesei. Deglycosylated samples were resolved on one‐dimensional (1‐D) and two‐dimensional (2‐D) gels in order to determine the effect of deglycosylation on the electrophoresis patterns and on the ability to identify proteins by peptide mass matching using matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) analysis of in‐gel tryptic digests. We found that deglycosylation of the protein sample resulted in different protein patterns on 1‐D and 2‐D gels, reduced the complexity of gel patterns, and enhanced the protein identification of some proteins via MALDI‐TOF‐MS. Deglycosylation with trifluoromethanesulfonic acid (TFMS) was found to be more effective than enzymatic treatments. These deglycosylation techniques may be employed in whole proteome analysis to locate glycosylated proteins and assist in their identification by MS.

Failure mechanism determination for industrial granules using a repeated compression test

This work describes the development of a particle compression test that allows direct and repeated application of the stress. The test is designed to quickly reduce the load on a granule during its incipient failure. By so doing, the breakage can be arrested and thus the process can be studied in detail. Experimental tests have been made on samples of industrial enzyme granules, which have a complex layered structure, and reproducible results have been obtained. The contribution of the various layers to the strength of the granule has been investigated, showing that the use of coating materials results in improved granule strength. The microstructure of the granule determines the failure mode of the granule. It is concluded that the failure mechanisms can be defined from tests on only a few granules as can assessment of the relative contribution of the layers and of the granule core to its strength. A measurement of the distribution of strength requires a larger, statistically representative, sample.

The effects of plasticizers and titanium dioxide on the properties of poly(vinyl alcohol) coatings.

Poly(vinyl alcohol) has not previously been examined in much detail as a controlled release polymer for use in pharmaceutical formulations. However, this food grade polymer has barrier and tensile properties which make it attractive for such applications. The effects of several diluents and fillers on Poly(vinyl alcohol) (PVAL) coatings have been determined using both mechanical property and water vapor permeability measurements. It has been found that the alcohol ethoxylate Neodol 23-6.5 (CH3(CH2)11–O(CH2–CH2–O)6–H) acts as a plasticizer for PVAL only up to 15–20 wt% in contrast to 600 molecular weight Polyethylene Glycol (PEG 600), which continuously plasticizes PVAL. The effects of Neodol on PVAL mechanical properties and water vapor permeability at higher concentrations can be explained in terms of Neodol phase separation and has been confirmed with DSC. The inert filler and whitener titanium dioxide (TiO2) monotonically degrades film mechanical properties and increases water vapor permeability of the coating. Attempts to correlate coating dust generated during particle attrition tests with mechanical property measurements were unsuccessful. A correlation between accelerated granule stability and water vapor permeability of the PVAL coating was established.

Increasing Throughput and Data Quality for Proteomics

With the availability of microbial and mammalian genomes combined with dramatic improvements in bioanalytical methods, high-throughput analysis of transcriptomes and proteomes has become a reality for academic and industrial laboratories alike. New technologies have resulted in the discovery of a multitude of novel cellular pathways and interconnective regulatory mechanisms. For instance, the number of drug targets has grown from approximately 500 to the thousands in a short amount of time. While the information from post-genomics techniques is useful in its own right, it does not necessarily accelerate discovery, this is partly because of data management constraints. Bottlenecks also include quality of sample preparation, identification of low abundance compounds, uninterrupted unattended operations, and successful matches of the results with information available in databases.