Protein Analytes

Twenty-five sets of benzyloxycarbonyl-glycine-X-Y-OMe epimers were chosen to study the racemization process that occurs during peptide synthesis [470]. These tripeptides were resolved on a 40°C Cjg column (A = 245 nm) using a number of isocratic methanol/water mobile phases. The inserted amino acid residues, X and Y, included combinations of alanine, valine, leucine, isoleucine, and phenylalanine. Mobile phase compositions ranged from 45% to 65% methanol (analyte dependent), capacity factors from 3 to 14, and a values fix)m 1.11 to 1.39. 

In a similar fashion, the retention behavior of 23 tripeptide epimers (with the basic structure Z-Ala-X-Val-OMe, where X is either the d or l form of the inserted amino acid) was studied on a C g column (A = 220 nm) using a series of isocratic methanol/water mobile phases [471]. The k values for all the lll and ldl forms and the resulting a values are tabulated for each epimer. Reasonable elution times were achieved, with mobile phases ranging fixim 60% to 80% methanol. The LDL-epimers were consistently more retained than the LLL-epimers. The authors attribute this to a larger surface area presented to the packing material in the ldl conformation. 

Four cyclic heptapeptide toxins (extracted fixim Microceptis auruginosd) consisting of D-alanine, eryfAro-yJ-methylaspartic acid, glutamic acid, L-leucine, L-alanine, A -methyldehydroadenosine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dioenoic acid were baseline resolved in 20 min using a C g column (A = 238 nm) and a 58/42 methanol/water (50 mM phosphate buffer at pH 3) mobile phase [472]. Excellent peak shapes were obtained. 

Alcohols were studied as denaturants for various proteins by Herskovits et al. [473], The amount of organic solvent in water needed to denature cytochrome c, myoglobin, or chymotrypsinogen decreased in the order ethylene glycol methanol ethanol IPA -propyl alcohol 2-butanol I-butanol. Interestingly, ethylene glycol at the 60% level did not denature any of these proteins, whereas 50% methanol denatured them all. These results were supported by Bull [474], who used methanol, ethanol, n-propyl alcohol, and 1-butanol, and seven proteins. In this study 30% methanol denatured all proteins, whereas less than 10% n-propyl alcohol caused the same degree of denaturation. 

A number of human serum proteins were analyzed using a wide range of bonded phases (Cjg, phenyl, Cg, diphenyl) and either a 10/90- 70/30 or a 0/100 - 100/0 gradient of n-propyl alcohol/water (triethylamine acetate buffer at pH 4.0). Serum characterization was complete in 60 min [478]. C g bonded phases gave (he lowest absolute recovery of proteins with Cg, phenyl, and diphenyl yielding recoveries 80% at the 10 pg total mass injected level. Recovery increased to 95% at the 100 pg level. 

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Because the protein analyte is endogenous to the plant, it can be difficult to demonstrate the efficiency of the extraction procedure. Ideally, an alternative detection method (e.g., Western blotting) is used for comparison with the immunoassay results. Another approach to addressing extraction efficiency is to demonstrate the recovery of each type of protein analyte from each type of food fraction by exhaustive extraction, i.e., repeatedly extracting the sample until no more of the protein is detected. " ... 

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

While these preliminary data are encouraging, before it is possible to accept this bar code design as a standard reference material, it must be tested extensively and validated under different laboratory conditions for a range of different protein analytes commonly examined by IFIC, following principles discussed in previous chapters (Chapters 1 and 5). 

However, IHC as a practical method continues to evolve with increasing demands for standardization, and for true quantification of protein analytes by weight, in the context of their cellular microenvironment. Further studies combining proteomics by mass spectrometry and IHC are likely to lead to the refinement of both methods in the analysis of FFPE tissues. The end result may be the creation of a broader field that defines and quantifies protein expression at a cellular level, incorporating the advantages of the wide spectrum of proteins demonstrable by mass spectrometry and the precise localization offered by IHC. 

Two adjacent signals in the ESI mass spectrum of a pure protein have mlz values of 1428.6 and 1666.7, respectively. Since adjacent signals differ in the value of their respective charge states by 1, the following expressions can be solved for the value of one of the charge states (z2 in this case) and then for the molecular mass of the protein analyte, m. 

After device construction, structural and functional analysis are critical. One might argue that only the second issue matters, but structural data often give insights into why devices perform suboptimally, and provide important clues about how to improve device function. We routinely use protein analytics (matrix-assisted laser desorption-ionization mass spectroscopy, amino acid composition analysis, gel electrophoresis, Western blotting, circular dichroism, vari-... 

The column packings used for SEC must be compatible with aqueous mobile phases and therefore must be hydrophilic in nature. The support surface must be inert to minimize interactions with protein analytes. The packing must be available in pore sizes suitable for permeation of a wide range of proteins, and the pores should be uniform in diameter. Because the separation only takes place within the pore system, the porosity of the packing should be as large as possible. The support material should be chemically compatible with SEC mobile phases and mechanically stable under high flow rates and pressures. 

The selection considerations for appropriate p7 markers for cIEF with proteins/anti bodies included purity and stability of the p7 markers, p7 values of the protein analytes, and potential protein—p7 marker interactions. High purity, stable p7 markers that give reliable p7 values with no protein—p7 marker interaction are desirable. Table 6 lists sets of p7 markers used for optimization. The antibody of interest had a p7 range of approximately 6.3 to 7.0. In this case, six different vendor sources were evaluated. These p7 markers vary in nature, from proteins and peptides to small molecules. The e-grams obtained using these markers with the antibody of interest are shown in Figure 22. Although the nature of the p7 markers and exact p7 marker values were different, the cIFF profiles of the antibody were the same. 

Moore, R. E., Licklider, L., Schumann, D., and Lee, T. D., A microscale electrospray interface incorporating a monolithic, poly(styrene-divinylbenzene) support for on-line liquid chromatography tandem mass spectrometry analysis of peptides and proteins. Analytical Chemistry 70(23), 4879-4884, 1998. 

CNCbl, HOCbl, Milk dairy Extraction and protein Analytical A acetonitrile. Detection of vitamers in External standardization. 177... 

Tolosa L, Gryczynski I, Eichhom LR, Dattelbaum JD, Castellano FN, Rao G, Lakowicz JR. Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein. Analytical Biochemistry 1999, 267, 114—120. 

Hamaguchi N, Ellington A, Stanton M. Aptamer beacons for the direct detection of proteins. Analytical Biochemistry 2001, 294, 126-131. 

Kussmann M, Nordhoff E, Rahbek-Nielsen H, Haebel S, Rossel-Larsen M, Jakobsen L, Gobom J, Mirgorodskaya E, Kroll-Kristensen A, Palm L, Roepstorff P (1997) Matrix-assisted laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptides and protein analytes. J Mass Spectrom 32 593-601... 

Because protein microarrays (antibody microarrays) have been successfully used for a variety of applications, such as antibody response profiling, identification and detection of bacterial and protein analytes, as well as disease proteomics with a clear focus toward oncoproteomics, it can be assumed that they will also prove to be a useful tool in studies on allergens (Wingren and Borrebaeck, 2007). 

While the combination of 2DGE with protein analytic techniques has been established for toxicoproteomics, the integration of bioinformatic and appropriate software has yet to be implemented. 

Rowe CA, Tender LM, Feldstein MJ et al (1999) Array biosensor for simultaneous identifl-cation of bacterial, viral, and protein analytes. Anal Chem 71 3846-3852... 

Homolka, Jiri, Polarography of Proteins, Analytical Principles and... 

T. J. Richmond, /. Mol. Biol., 178, 63 (1984). Solvent Accessible Surface Area and Excluded Volume in Proteins—Analytical Equations for Overlapping Spheres and Implications for the... 

H20. Homolka, J., Polarography of proteins, analytical principles, and applications in biological and clinical chemistry. Methods Biochem. Anal. 19, 435-555 (1971). 

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