Digest proteins

FIGURE 2.16 pH versus enzymatic activity. The activity of enzymes is very sensitive to pH. The pH optimum of an enzyme is one of its most important characteristics. Pepsin is a protein-digesting enzyme active in the gastric fluid. Trypsin is also a proteolytic enzyme, but it acts in the more alkaline milieu of the small intestine. Lysozyme digests the cell walls of bacteria it is found in tears. 

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. 

Pancreatic secretion for many, if not most, species is regulated in order to insure adequate protein digestion. Correspondingly, protease inhibitors have a greater impact on pancreatic secretion than do inhibitors of amylase and lipase (Toskes, 1986). The secretory response of the exocrine pancreas to protease inhibitors can be rapid (< 10 min), does not involve parallel increases in the secretion of all enzymes (Holm et al., 1992), and is probably mediated by a signaling pathway (see below). 

FIGURE 98-1. Schematic representation of carbohydrate, fat, and protein digestion. (From KumpfVJ, Chessman KH. Enteral nutrition. In DiPiro JT, Talbert RL, Yee GC, et al, (eds.) Pharmacotherapy A Pathophysiologic Approach. 6th ed. New York McGraw-Hill 2005 2616.)... 

M Yoshioka, RH Erikson, JF Woodly, R Gulb, D Guam, YS Kim. Role of rat intestinal brush-border membrane angiotensin-converting enzyme in dietary protein digestion. Am J Physiol 253 G-781-G-786, 1987. 

Cholecystokinin Endocrine cells in mucosa of duodenum Breakdown products of lipid and, to a small extent, protein digestion in duodenum Inhibits gastric emptying and gastric secretion stimulates contraction of gallbladder stimulates secretion of digestive enzymes from pancreas... 

Pepsinogen is produced by the chief cells. Within the lumen of the stomach, this precursor molecule is split by HCl to form the active enzyme pepsin. Optimally active at an acidic pH (pH = 2), pepsin begins protein digestion by fragmenting proteins into smaller peptide chains. 

Dipeptides and tripeptides are also presented to the brush border of the absorptive cells. As the nutrient molecules are absorbed, aminopeptidases split them into their constituent amino acids. The activity of aminopeptidases accounts for approximately 60% of protein digestion. The amino acid molecules then exit the absorptive cells by way of facilitated diffusion and enter the blood capillaries. 

Chylomicrons leave the absorptive cell by way of exocytosis. Because they are unable to cross the basement membrane of the blood capillaries, the chylomicrons enter the lacteals, which are part of the lymphatic system. The vessels of the lymphatic system converge to form the thoracic duct that drains into the venous system near the heart. Therefore, unlike products of carbohydrate and protein digestion that are transported directly to the liver by way of the hepatic portal vein, absorbed lipids are diluted in the blood... 

Ramsey, J.D., Jacobson, S.C., Culbertson, C.T., Ramsey, J.M. (2003). High efficiency, two-dimensional separations of protein digests on microfluidic devices. Anal. Chem. 75, 3758-3764. 

Table 5.1 lists several heart-cut and comprehensive techniques. Heart-cut 2DLC is very common and has great application for the increased resolution of one or several components from the first dimension (Augenstein and Stickler, 1990 Majors, 1980 Pasch et al., 1992 and Dixon et al., 2006). Heart-cut 2DLC for the analysis of polymers is often referred to as cross-fractionation (Balke and Patel, 1980). Protein digest analysis with MS/MS identification has been called multidimensional protein identification technology or MUDPIT. This is described in detail in Chapter 11. 

AEC RPLC LIF Protein digest 6-port Holland and Jorgenson (1995)... 

CEC RPLC UV Protein digest 10-port Stoll and Carr (2005)... 

Issaq, H.J., Chan, K.C., Cheng, S.L., Qingho, L. (2001). Multidimensional high performance liquid chromatography-capillary electrophoresis separation of a protein digest an update. Electrophoresis 22, 1133-1135. 

Y. Ma, Y. Lu, H. Zeng, D. Ron, W. Mo, T. A. Neubert, Characterization of phosphopeptides from protein digests using matrix assisted laser desorption/ionization time of flight mass spectra metry and nanoelectrospray quadrupole time of flight mass spectrometry, Rapid Commun. Mass Spectrom., 15, 1693 1700 (2001). 

Delcroix, M., Sajid, M., Caffrey, C.R., Lim, K.-C., Dvorak, J., Hsieh, I., Bahgat, M., Dissous, C., and McKerrow, J.H. (2006) A multienzyme network functions in intestinal protein digestion by a platy-helminth parasite./. Biol. Chem. 281, 39316-39329. 

J. Gao, J.D. Xu, L.E. Locascio, and C.S. Lee, Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification. Anal. Chem. 73, 2648-2655 (2001). 

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