Protein, analysis

The presence of a soluble protein is indicated by the Biuret reaction. A 0.5% CUSO4 solution will give a purple colour when added dropwise to a dilute protein (or biuret, H2N CO NH CO-NH2) solution made alkaline by the addition of NaOH. In the Ninhydrin reaction, a blue or purple colour develops when the reagent is added dropwise to a protein solution, then boiled. 

Another more sensitive detector for amino acid protein components is orthophthaldehyde (14.27) which reacts to form products which fluoresce intensely at 455 nm if irradiated at 360 nm. 

Soluble proteins are precipitated by the addition of silver or lead salt solutions, by adding strong mineral acids or by the application of heat (coagulation). High molecular weight proteins form colloidal solutions which are unable to pass through manbranes such as parchment or collodion. 

The phosphorus content of proteins varies considerably. In egg-white ovalbumin, for example, there are only two phosphate groups covalently bound to a chain of 385 amino acids, whereas in egg-yolk phosvitin at least half the amino adds have covalently attached phosphate groups (Chapter 11.3). Most enzymes appear to have a variable P content, depending upon the stage of their interaction with other components in the system (Chapter 12.2). 

Historically, the quantitative determination of protein, particularly in the presence of large amounts of non-protein, was based on the estimation of the nitrogen content by the classical Kjeldahl method. Any other non-protein organic nitrogen was either assumed to be absent or was removed prior to analysis. The crude protein content was taken to be 6.25 times the % N found in the sample (this was based on the average N content of 16% in most proteins). 

This section deals with the standard repertoire of techniques needed for working with proteins, that can be used to get information about concentration and purity, molar mass, amino add composition and sequence, and tertiary conformation and stability. 

Information about the composition of protein mixtures and the purity of protein samples is usually obtained using chromatographic or electrophoretic methods, which were discussed in Ch. 4. 

The molar mass of a protein is such a fundamental characteristic that it is often used as a basis for nomenclature. The current literature of molecular and cell biol- 

Gel filtration is frequently used to determine the molar masses of native proteins (Ch. 4.1.2.2). To obtain accurate results, it is important that there should be no nonspecific interactions between the protein and the gel matrix, and this can usually be assured by the right choice of buffers. The method is not very accurate, and it needs calibration with protein standards of known molar masses and of similar shape. If the protein has a characteristic activity that can be measured, e.g., an enzyme activity, then its molar mass can be determined using crude protein mixtures by assaying the eluate from a gel filtration column for activity. 

Mass spectrometry has assumed great importance in determinations of the molar masses of biological macromolecules, even quite large ones. This is due to developments such as electrospray ionisation (ESI) and matrix assisted laser desorption/ ionisation (MALDI), which have made it possible to determine the molar masses of biopolymers up to several 100 kDa (Pitt 1996 Kellner et al. 1999 Snyder 2000). The combination of MALDI techniques with time-of-flight mass spectrometers (MALDI-TOF) is of particular significance for determination of the molar masses of proteins with high sensitivity (typically pmol quantities, although exceptionally fmol) and precision (proteins up to 100 kDa with precision of about 0.01 %). Mass spectrometry can provide very accurate measurements of protein molar mass that can yield information about even minor structural modifications not readily accessible by other means. 

---

R. A. Copeland, Methods for Protein Analysis A Practical Guide to Eaboratoy Protocols, Chapman Had, New York, 1994. 

BVB Reddy TL Blundell. Packing of secondary structural elements m proteins. Analysis and prediction of mter-helix distances. J Mol Biol 233 464-479, 1993. 

For proteins, the most useful columns are those with pores of 100-500 A, as seen in Fig. 10.2, because most proteins elute on the linear portions of the calibration curves. Figure 10.5 illustrates an analysis of a protein mixture on SynChropak GPC100. Small peptides can be analyzed on the 50-A SynChro-pak GPC Peptide column with appropriate mobile-phase modifications. Many peptides have poor solubility in mobile phases standardly used for protein analysis, as discussed later in this chapter. 

FIGURE 10.5 Protein analysis on SynChropak GPC 100. Column 300 X 7.8 mm i.d. Mobile phase 0.1 /H potassium phosphate, pH 7. Flow rate I ml/min. (From MICRA Scientific, Inc., with permission.)... 

Gels made in this way have virtually no usable porosity and are called Jordi solid bead packings. They can be used in the production of low surface area reverse phase packings for fast protein analysis and in the manufacture of hydrodynamic volume columns as well as solid supports for solid-phase syntheses reactions. An example of a hydrodynamic volume column separation is shown in Fig. 13.2 and its calibration plot is shown in Fig. 13.3. The major advantage of this type of column is its ability to resolve very high molecular weight polymer samples successfully. 

E. Rohde, A. J. Tomlinson, D. H. Johnson and S. Naylor, Protein analysis by membrane preconcenti ation-capillaiy electi ophoresis systematic evaluation of parameters affecting preconcenti ation and separation , 7. Chromatogr. 6 713 301-311 (1998). 

Domon B, Aebersold R (2006) Mass spectrometry and protein analysis. Science 312 212-217... 

An area worthy of study is the development of systems of increasing sample throughput beyond the single column operation. Scott has introduced a prototype multicolumn system based on the centrifugal analyzer principle (53). In this set-up a series of LC colimns is rotated on a disc, with sample delivery at the center of the disc and elution and spectrophotometric analysis on the outside. He has suggested using affinity columns for rapid serum protein analysis by this approach. Of course, other principles, such as segmented flow, could be envisioned in an automated LC system as well. Undoubtedly, we can expect to see the availability of such systems in the next few years. 

Bolton, J. L. Le Blanc, J. C. Y. Siu, K. W. M. Reaction of quinone methides with proteins analysis of myoglobin adduct formation by electrospray mass spectrometry. Biol. Mass... 

Fenselau, C. MALDI MS and strategies for protein analysis. Anal. Chem. 1997, 69, 661A-665A. 

Chen, S.-H., Lin, Y.-H., Wang, L.-Y., Lin, C.-C., Lee, G.-B. (2002). Flow-through sampling for electrophoresis-based microchips and their applications for protein analysis. Anal. Chem. 74, 5146-5153. 

Murphy, R.E. (2001). Comprehensive two-dimensional liquid chromatography for comparative protein analysis. Presented at the International Symposium on the Separation of Proteins, Peptides and Polynucleotides. (ISPPP 2001) Orlando, FL. 

Peng, J., Elias, J.E., Thoreen, C.C., Licklider, L.J., Gygi, S.P. (2003). Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC—MS/MS) for large-scale protein analysis the yeast proteome. J. Proteome Res. 2, 43-50. 

The consensus among proteome researchers is that separation is an essential part of complex protein analysis methods. A simplification of complex samples using 2DLC is beneficial for state-of-the-art MS/MS analysis. While 2DLC potentially provides a higher peak capacity than 1DLC, the orthogonality of separation has to be taken into consideration. 

Von Haller, P.D., Yi, E., Donohoe, S., Vaughn, K., Keller, A., Nesvizhskii, A.I., Eng, J., Li, X.J., Goodlett, D.R., Aebersold, R., Watts, J.D. (2003). The Application of New Software Tools to Quantitative Protein Profiling Via Isotope-coded Affinity Tag (ICAT) and Tandem Mass Spectrometry II. Evaluation of Tandem Mass Spectrometry Methodologies for Large-Scale Protein Analysis, and the Application of Statistical Tools for Data Analysis and Interpretation. Mol. Cell. Proteomics 2, 428 -42. 

Instrument We reported the first two-dimensional capillary electrophoresis system for protein analysis (Michels et al., 2002, Hu et al., 2004, Michels... 

Michels, D.A., Hu, S., Schoenherr, R.M., Eggertson, M.J., Dovichi, NJ. (2002). Fully automated two-dimensional capillary electrophoresis for high sensitivity protein analysis. Mol. Cell. Proteomics 1, 69-74. 

Neukirchen, R.O., Schlosshauer, B., Baars, S., Jackie, H., Schwarz, U. (1982). Two-dimensional protein analysis at high resolution on a microscale. J. Biol. Chem. 257,15229-15234. 

---