Protein identification/characterization

A. Riggin, J. R. Sportsmen, and F. E. Regnier, Immunochromato-graphic analysis of proteins identification, characterization and purity determination, J. Chromatogr., 632 37-44 (1993). 

Dosztanyi, Z., Fiser, A. Simon, I. (1997). Stabilization centers in proteins identification, characterization and predictions. J Mol Biol 272,597-612. 

Proteomics Protein identification, characterization, expression, and interactions. 

Figure 9.15 Typical mass spectrometry based experiment for protein identification/characterization. (1) Proteins are fractionated by chromatography, separated by sodium dodeyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE), then excised from gel (2) the protein of interest is digested into peptide fragments, that are then (3) identified by ESI MS/MS (4) the first dimension involves molecularion analysis for peptide mass finger-printing (ql, MS only). (5) Tandem MS/MS is used when protein identification is not unambiguous, in which case parent molecular ions are activated by CID (q2) and daughter (product) ions are characterized (ToF) according to the technique of product ion scanning (illustration from Aebersold and Mann, 2003, Fig. 1).

Chemical derivatization methods provide a useful additional tool for protein structural analysis, particularly when conpled with the multistage tandem mass spectrometric capabilities of modern ion trap mass spectrometers. The objective of this chapter was to provide a brief overview of the chemical derivatization strategies that are employed currently to address the challenges associated with protein identification, characterization, and quantitative analysis as well as for the characterization of protein-protein interactions. 

LeDuc, R.D., Taylor, G.K., Kim, Y.B., Januszyk, T.E., Bynum, L.H., Sola, J.V., Garavelli, J.S., Kelleher, N.L. (2004). ProSight PTM an integrated environment for protein identification and characterization by top-down mass spectrometry. Nucleic Acids Res. 32, W340-W345. 

This multidimensional protein identification technology (MudPIT) specifically incorporates a strong cationic exchange (SCX) column in tandem with an RP column to achieve maximal resolution and exquisite sensitivity. MudPIT is effective for studying complex proteomes such as mammalian cellular samples. It has been applied to large-scale protein characterization with identification of up to 1484 proteins from yeast in a single experiment.12... 

ExPASy Proteomics tools (http //expasy.org/tools/), tools and online programs for protein identification and characterization, similarity searches, pattern and profile searches, posttranslational modification prediction, topology prediction, primary structure analysis, or secondary and tertiary structure prediction. 

Lehmann, W.D. Bohne, A. von der Lieth, C.W. The Information Encrypted in Accurate Peptide Masses-Improved Protein Identification and Assistance in Glyco-peptide Identification and Characterization. J. Mass Spectrom. 2000, 35. 1335-1341. 

A further advantage, as described by Thomas et al. [19], is the possibility of protein identification that follows the functional characterization of the enzyme. The activity of an enzyme is initially determined by following the substrate consumption and product formation in the first assay (Fig. 8.10). Since no matrix components are present in the sample spot, the immobilized enzyme is then directly... 

Wolbers R, Landrey G (1987) The use of direct reactive fluorescent dyes for the characterization of binding media in cross sectional examinations. Preprints of 15th Annual Meeting of the American Institute for Conservation and Artistic Works, Vancouver, 168-202. Heginbotham A, Millay V, Quick M (2006) The use of immunofluorescence microscopy and enzyme-linked immunosorbent assay as complementary techniques for protein identification in artist s materials. J Am Inst Conserv 45 89-105. 

The reason for this dissatisfaction in most cases is that the gene may have been identified but not necessarily the associated protein(s). Protein identification has been achieved in a few limited cases, but it is more difficult to produce a protein in sufficient quantities for testing or, more critically, for delivery to the patient. That these proteins will be found, characterized, and produced in sufficient quantities for human trial and evaluation cannot be doubted just how long this will take in individual situations is an entirely different matter. 

Over the last decade or so, new ionization techniques such as ES and MALDI have been introduced and have increased still further the use of mass spectrometry in biology. Identification of proteins and characterization of their primary structure is a rapidly growing field in the postgenomic era. ES ionization was the first method to extend the useful mass range of instruments to well over 50 kDa (Mano and Goto... 

MALDI-TOF provides limited capabilities for mixture analysis, LC/MS methods are used to provide more detailed interrogation of protein expression and peptide sequence. The use of LC/MS approaches for protein identification in conjunction with 2-DGE offers distinct advantages such as the ability to handle low picomole (miniaturized) level samples, enhanced separation, detection, the amenability to N-terminally blocked proteins, and fast analysis. The LC/MS methods for protein characterization focus on four distinct goals (1) confirmation of putative sequence, (2) identification of amino acid modifications, (3) identification of known proteins, and (4) sequence determination of unknown proteins. 

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. 

Blackburn, R. K. Moseley, M. A. Ill 1999. Quadrupole time-of-flight mass spectrometry a powerful new tool for protein identification and characterization. Am. Pharm. Rev., 2, 49-59. 

Database Interrogation (Profound, Sequest) Protein Identification Post-translational Modification Characterization... 

Lalwani, N. D., Alvares, K, Reddy, M. K, Reddy, M. N., Parikh, I., and Reddy, J. K. (1987). Peroxisome proliferator-Binding Protein Identification and Partial Characterization of Nafenopin-Clofibric Acid-, and Ciprofibrate-Binding Proteins from Rat liver. Proc. Natl. Acad. Sci. U.S.A. 84, 5242-5246. 

The comprehensive molecular biology server, ExPASy (Expert Protein Analysis System), provides Proteomic tools (http //www.expasy.ch/tools) that can be accessed directly or from the ExPASy home page by selecting Identification and Characterization under Tools and Software Packages. The Protein identification and characterization tools of the Proteomics tools (Figure 11.4) provide facilities to ... 

Figure 11,4. ExPASy Proteomic tools. ExPASy server provides various tools for proteomic analysis which can be accessed from ExPASy Proteomic tools. These tools (locals or hyperlinks) include Protein identification and characterization, Translation from DNA sequences to protein sequences. Similarity searches, Pattern and profile searches, Post-translational modification prediction, Primary structure analysis, Secondary structure prediction, Tertiary structure inference, Transmembrane region detection, and Sequence alignment.

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