Protein Characterization Fingerprinting

On occasion, nonmicrobial sediment and/or hazes may develop in bottled wines and juices. Inasmuch as some of these may, upon microscopic examination, appear bacterialike the following section is included to assist the reader in their identification. For the sake of discussion, instabilities may be classified as particulate, including crystalline (or crystallike), fibrous, and amorphous which lack regular structural properties (See Table D-1). 

Preliminary sample evaluation and review of processing records often provides useful information as to the nature of the problem. In the case of haze or precipitates present in small amounts, it may be necessary to concentrate the latter by either centrifugation or filtration. Most laboratory filtration units utilize 47-mm membranes. Alternatively, syringe filters equipped with a filter disk of appropriate pore size may also be used. For general purposes, 1-5-pm cellulose acetate filters are useful. Alternatively, the bottle may be stood upright for several hours and the precipitate pipetting directly off the bottom. 

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Usui, K., Ojima, T., Takahashi, M., Nokihara, K. and Mihara, H. (2004a). Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns. Biopolymers 76, 129-139. 

The characteristic derivative-shaped feature at g 1.94 first observed in mitochondrial membranes has long been considered as the sole EPR fingerprint of iron-sulfur centers. The EPR spectrum exhibited by [4Fe-4S] centers generally reflects a ground state with S = I and is characterized by g values and a spectral shape similar to those displayed by [2Fe-2S] centers (Fig. 6c). Proteins containing [4Fe-4S] centers, which are sometimes called HIPIP, essentially act as electron carriers in the photoinduced cyclic electron transfer of purple bacteria (106), although they have also been discovered in nonphotosynthetic bacteria (107). Their EPR spectrum exhibits an axial shape that varies little from one protein to another with g// 2.11-2.14 and gi 2.03-2.04 (106-108), plus extra features indicative of some heterogeneous characteristics (Pig. 6d). 

The ability to resolve and characterize complicated protein mixtures by the combination of 2DLC and online mass spectrometry permits the combination of sample fractionation/simplification, top-down protein mass information, and bottom-up peptide level studies. In our lab, the simplified fractions generated by 2D(IEX-RP)LC are digested and analyzed using common peptide-level analysis approaches, including peptide mass fingerprinting (Henzel et al., 1993 Mann et al., 1993), matrix-assisted laser desorption/ionization (MALDI) QTOF MS/MS (Millea et al., 2006), and various capillary LC/MS/MS methodologies (e.g., Ducret et al., 1998). 

Characterization of noncovalent bonding of the proteins can also be done using MS. For example MALDI MS has been used in measurement of the molecular mass of the noncovalendy linked tetramer of glucose isomerase, a complex consisting of identical monomers of 43.1 kDa each. MALDI-TOFF peptide mass fingerprinting combined with electrospray tandem mass spectrometry can efficiently solve many complicated peptide protein analysis problems. 

CD complements fluorescence as a technique for characterizing the folded conformation in solution and for providing, in a single spectrum, a highly specific fingerprint for the native state. It requires only modest amounts of protein and measurements are relatively quick to make. Like fluorescence, its use as a comparative method requires a control spectrum from a carefully characterized sample of the natural or recombinant protein, recorded under defined and optimal conditions, using a spectrometer in good condition. 

The capillary LC/MS-based approach for peptide mapping performed by Arnott and colleagues features miniaturized sampleloading procedures, which are routinely amenable to small quantities of peptides. The reliable characterization of protein/peptide mixtures in conjunction with the widely used 2-DGE methods offers a powerful fingerprinting approach in the pharmaceutical industry. Low femtomole detection limits (typically <50 femtomole) with a mass accuracy of +0.5Da provide unique advantages for protein identification. Liberal parameters for mass range and unmatched masses are used for the initial protein search, whereas more conservative parameters are used to reduce the number of matches and to improve the confidence in the search. 

Finally, the goal of this first phase is to analyze and to compare the resulting proteins and peptides by alignment search tools, such as BLAST (http / blast.ncbi.nlm.nih.gov/Blast.cgi CMD=Web PAGE TYPE=BlastHome), in order to identify and characterize specific peptide fingerprints, which will be used in the monitoring approach of the next phase of the pipeline. 

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