Analysis of proteins

Methods for the analysis of proteins in body fluids include  

Specific quantitative assays of particular proteins by immunochemical methods (see Chapter 9) using specific antisera and measurement of the antigen antibody complexes by nephelometry, turbidimetry, RID, or electroim-muno assay or, if present in very low concentrations, by RIA, enzyme immunoassay (EIA), fluorescence immunoassay, or chemiluminescence. 

Analysis by mass spectrometry, which provides structural and quantitative information (see Chapter 7). 

Most immunochemical methods are applicable to the measurement of any of the proteins in this chapter (see Chapter 9). Because of their speed and ease, nephelometric and tur-bidimetric methods are most widely used for most serum proteins. These techniques are performed either by measuring the amount of Ag-Ab complex formation (equhibrium methods) or by measuring the rate of complex formation (kinetic methods). The kinetic methods are slightly faster, with measurements completed within 20 s however, kinetic assays are somewhat less sensitive because low-affinity antibodies do not have time to react. In addition, many kinetic methods obtain the baseline reading after addition of antiserum, which can reduce the measured signal with high-affinity antibodies. A compromise is often used, with timed measurement before true equilibrium. 

Important assay characteristics for immunochemical methods include (1) limit of detection, (2) precision, and (3) turnaround time. 

The analysis for proteins present in plasma or a cell extract is a challenging task due to their complexity and the great difference between protein concentrations present in the sample. Simple mixtures of intact proteins can be analyzed by infusion with electrospray ionization and more complex ones by matrix assisted laser desorption ionization. MALDI is more suited for complex mixtures because for each protein an [M+H]+ signal is observed while for ESI multiply charged ions are observed. Surface enhanced laser desorption (SEEDI) is a technique for the screening of protein biomarkers based on the mass spectrometric analysis of intact proteins [49]. However in most cases for sensitivity reasons mass spec- 

Two-dimensional electrophoresis [86] is a well established technique for the separation of intact proteins. In the first dimension the proteins are separated based on their isolectric point while the second dimension separates them based on their size. The presence on the gel of the proteins is revealed by Coomassie blue or silver staining. Under favorable conditions several thousand spots can be differentiated. The gel is digitized and computer-assisted analysis of the protein spot is performed. The spots of interest are excised either manually or automatically and then digested with trypsin. Trypsin cleaves proteins at the C-terminal side of lysine and arginine. In general one spot represents one protein and the peptides are analyzed by MALDI-TOF to obtain a peptide mass fingerprint. A peptide mass fingerprint involves the determination of the masses of all pep- 

As electrospray ionization is concentration-sensitive the last LC dimension uses a nano LC column with an internal diameter of 75 pm to achieve maximum sen- 

Two-dimensional-liquid chromatography (2D-LC) approaches are much easier to automate than 2D-electrophoresis. However 2D electrophoresis has the advantage that separation is performed at the protein and not at the peptide level and 

The calibration of total protein, albumin, and specific protein methods remains a problem when analyzing samples from laboratory animals. Many of the available protein calibration materials are bovine or human in origin, and there are international reference proteins for some human proteins (Whicher 1984 Price and Newman 1997 Tiffany 1999). However, because calibration standards for laboratory animals are not widely available, the values between methods may show wide variations due to differences in calibrators. When some protein fractions are available for laboratory animals, these materials are often less than 95% pure. The investigator therefore 

Electrophoretic methods use various support media, buffers, voltage, amperage, and times. The media include agarose gel, cellulose acetate, starch gel, and polyacrylamide gel. Techniques include single dimensional electrophoresis, crossed immunoelectrophoresis, and immunoelectrophoretic fixation (lEF). 

The application of qualitative and quantitative immunochemical protein measurements has been limited by the lack of available antisera suitable for laboratory animals, although this situation is improving. Antisera against human proteins are often useful for closely related species, and some phylogenetically distant species have similar epitopes on their corresponding proteins, which then allows protein measurements in several species (Brun, Lingaas, and Larsen 1989 Hau et al. 1990 Pineiro, Alava, and Lampreave 2003). Some proteins require species-specific antisera (e.g., CRP Balz et al. 1982 Eckersall, Conner, and Harvie 1991). When a suitable antiserum is available, it is still necessary to test that the concentrations of the antiserum 

Solid phase immunoassays—as enzyme-linked immunosorbent assays (ELISA) (Schreiber et al. 1992 Salauze, Serre, and Perrin 1994 Jones, Offutt, and Longmore 

2000) or as latex agglutination inhibition assays (Eckersall et al. 1999)—are being used more widely as species-specific antibodies become available. These methods are quicker than immunodiffusion agarose gel assays, which require 24 or 48 h for diffusion to be complete. 

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Amadei et al. 1993] Amadei, A., Linssen, A.B.M., Berendsen, H.J.C. Essential Dynamics of Proteins. Proteins 17 (1993) 412-425 [Balsera et al. 1997] Balsera, M., Stepaniants, S., Izrailev, S., Oono, Y., Schiilten, K. Reconstructing Potential Energy Functions from Simulated Force-Induced Unbinding Processes. Biophys. J. 73 (1997) 1281-1287 [Case 1996] Case, D.A. Normal mode analysis of protein dynamics. Curr. Op. Struct. Biol. 4 (1994) 285-290... 

M. Oobatake and G.M. Crippen, Residue-residue potential function for conformational analysis of proteins, J.Phys. Chem. 85 (1981), 1187-1197. 

D. A. Case. Normal mode analysis of protein dynamics. Curr. Opin. Struc. Biol., 4 385-290, 1994. 

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Oxford University Press, ISBN 0199636788 (paperback) T.L.Blundell and L.N.Johnson Protein Crystallisation, Academic Press, NY, 1976 A,McPherson Preparation and Analysis of Protein Crystals, J.Wiley Sons, NY, 1982 A.McPherson, Crystallisation of Biological Macromolecules, Cold Spring Harbour Laboratory Press, 2001 ISBN 0879696176.]... 

Analysis and prediction of side-chain conformation have long been predicated on statistical analysis of data from protein structures. Early rotamer libraries [91-93] ignored backbone conformation and instead gave the proportions of side-chain rotamers for each of the 18 amino acids with side-chain dihedral degrees of freedom. In recent years, it has become possible to take account of the effect of the backbone conformation on the distribution of side-chain rotamers [28,94-96]. McGregor et al. [94] and Schrauber et al. [97] produced rotamer libraries based on secondary structure. Dunbrack and Karplus [95] instead examined the variation in rotamer distributions as a function of the backbone dihedrals ( ) and V /, later providing conformational analysis to justify this choice [96]. Dunbrack and Cohen [28] extended the analysis of protein side-chain conformation by using Bayesian statistics to derive the full backbone-dependent rotamer libraries at all... 

GW Carter Ir. Entropy, likelihood and phase determination. Structure 3 147-150, 1995. RL Dunbrack Ir, EE Cohen. Bayesian statistical analysis of protein sidecham rotamer preferences. Protein Sci 6 1661-1681, 1997. 

Koch, I., Kaden, R, Selbig, J. Analysis of protein sheet topologies by graph theoretical methods. Prot Struc. Func. Genet. 12 314-323, 1992. 

McPherson, A. The Preparation and Analysis of Protein Crystals. New York Wiley, 1982. 

The Shodex OHpak SB-800 HQ series is usually not the best choice for the analysis of proteins. However, in some cases, such as when an alkaline... 

Mann, M., and Wilm, M., 1995. Electro.spray ma.ss. spectrometry for protein characterization. Trends in Biochemical Sciences 20 219-224. A review of die ba.sic application of ma.ss. spectrometric methods to the analysis of protein. sequence and. structure. 

Qnadroni, M., et al., 1996. Analy.sis of global re.spon.ses by protein and peptide fingerprinting of protein.s i.solated by two-dimensional electrophore-.sis. Application to snlfate-starvation re.sponse of Escherichia coli. European Journal of Biochemistry 239 773-781. This paper de.scribes the n.se of tandem MS in the analysis of protein.s in cell extracts. 

R. Consden, A. H. Gordon and A. J. P. Martin, Qualitative analysis of proteins partition chromatographic method using paper , 7. Biochem. 38 224-232 (1944). 

A. W. Moore-Jr, J. P. Lamiann-Jr, A. V. Lemmo and J. W. Jorgenson, Two-dimensional liquid chromatography-capillary electrophoresis teclmiques for analysis of proteins and peptides . Methods Enzymol. 270 401-419 (1996). 

Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

Gavin AC et al (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415 141-147... 

Fig. 1. SDS gel analysis of proteins synthesised by excised maize roots incubated at continuous 40 °C. Roots of 3-day-old maize seedlings were excised and incubated at 40 °C for increasing times as indicated. Labelling with [ Sjmethionine was carried out in the final 20 min of the incubation. Proteins were visualised by fluorography. Mol wt distribution in kDa indicated at left. From Cooper Ho (1983).

Fancy, D.A. and Kodadek, T., Chemistry for the analysis of protein-protein interactions Rapid and efficient cross-linking triggered by long wavelength light, Proc. Natl. Acad. Sci. USA, 96, 6020-6024, 1999. 

Applications of Molecular Dynamics for Structural Analysis of Proteins and Peptides... 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

A new chapter on the primary structure of proteins, which provides coverage of both classic and newly emerging proteomic and genomic methods for identifying proteins. A new section on the appHcation of mass spectrometry to the analysis of protein structure has been added, including comments on the identification of covalent modifications. 

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