Protein/peptide analysis determination

Modern methods of amino-acid and peptide analysis, have enabled the complete amino-acid sequence of a number of proteins to be worked out. The grosser structure can be determined by X-ray diffraction procedures. Proteins have molecular weights ranging from about 6 000 000 to 5 000 (although the dividing line between a protein and a peptide is ill defined). Edible proteins can be produced from petroleum and nutrients under fermentation. 

Raman spectroscopy is a related vibrational spectroscopic method. It has a different mechanism and therefore can provide complementary information to infrared absorption for the peptide protein conformational structure determination and some multicomponent qualitative and/or quantitative analysis (Alix et al. 1985). 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

The most widely used mass spectrometric identification procedure is MALDI-Tof analysis of the entire peptide mixture. Gas-phase matrix interaction with peptide ions in MALDI-Tof results in singly charged ions, giving a mass profile that is highly characteristic of the protein from which the peptides are derived. These peptide masses (actually protonated peptide molecular ions, MH+) can be used to search databases (either protein or nucleic acid databases) to identify the proteins. The two most important factors in successfully identifying proteins by this approach are the number of matching peptide masses and the accuracy of the peptide mass determination. 

About 0.5-200 picomoles per digestion is sufficient for MS/MS analysis. After a reversed-phase HPLC fractionation, the molecular mass of each peptide is determined. Peptides with masses lower than 3000 Da that yield important signals are sequenced using MS/MS. Sequenced peptides then are assembled by matching the peptides in both series while considering the molecular masses of the large unsequenced peptides that contain several smaller sequenced peptides or the known sequence of a homologous protein. 

The basic goal of the mass spectrometry measurement in the context of peptide analysis in proteomics and phosphoproteomics is to determine specific attributes that are then used in subsequent database searches to provide 1. the identity of the proteins present in the sample 2. location of the site(s) of phosphorylation in these proteins. Both pieces of information are derived from the mass of the peptide and, most importantly, from the gas-phase dissociation patterns that are diagnostic of the peptide s amino acid sequence and phosphosite location. The gas-phase dissociation patterns are obtained via tandem mass spectrometry (MS/MS). On a phosphoproteome-wide scale, the analysis includes measurement of the attributes for many thousands of individual peptides. 

The structure of the protein has been determined as part of a protein complex or with a peptide ligand bound. Repeats/ Blades indicates the number of sequence repeats identified by sequence analysis, followed by the number of blades (and therefore, additional poorly-conserved sequence repeats) identified after structural determination. See text for information on closure mechanisms. Aipip is organized into two 7-bladed propellers the N-terminal strand closes the C-terminal propeller and then proceeds to blade 1 of the first propeller. There is no similar closure of the first propeller. ... 

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