Enzymatically digestible matrix

Blood, urine, liver, other tissues, hair, saliva, injection marks Drugs Selective IA TLC, GC-MS Precipitation, acidic or enzymatic digestion, matrix dissolution LLE or SPE, Derivatization GC-MS LC-MS... 

A new approach in recovering the retained cells, now in development, is based on the principle of an enzymatically digestible matrix (Schlossman and Hudson, 1973 Phillips and Roitt, 1973). The cells isolated from these digestible columns are fully functional on adoptive transfer (Hudson and Hamilton, 1973, in prep.). 

Either acid or alkaline hydrolysis can be applied, converting nicotinamide to nicotinic acid. Alkaline hydrolysis releases also the unavailable vitamers providing the estimation of the total niacin content. Acid hydrolysis, instead, is slower than alkaline hydrolysis therefore the former is usually coupled with enzymatic digestion by using takadiastase, papain, and clarase. Extraction with water and dilute sulfuric or hydrochloride acid has been applied to release the vitamers from the matrix without degrading nicotinamide [598]. 

While the determination of total lysine is rather straightforward, the determination of free or available lysine is more problematic. In this situation, the term free is meant to imply that the e-amine of the lysine side chain has not reacted with various components of the sample matrix (most commonly carbohydrates via Maillard browning). This is important because reaction of the e-amine can render lysine nutritionally unavailable and the nutritive value for that protein is then diminished if lysine is the limiting amino acid (which is often the situation with soy proteins). While enzymatic digestion in the human gut may not release the modified lysine in a nutritionally available state, often these lysine adducts are labile to the standard acid hydrolysis in 6N HC1 at 110°C. This results in total lysine values that overestimate the amount of nutritionally available lysine. 

KL O Connell, JT Stults. Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests. Electrophoresis 18 349-359, 1997. 

Matrix-assisted laser desorption ionization is another ionization mode used for MS analysis. Enzymatically digested peptides have been studied using a 90-well microchip constmcted in a MALDI plate format (see Figure 7.41). Peptide digestion was initiated in the MALDI interface where the peptide hormone, adreno-corticotropin (ACTH) was mixed with the enzyme carboxypeptidase Y. The mixing process was self-activated in the vacuum conditions. Subsequent TOF MS analysis produced kinetic information of the peptide digestion reaction [820]. 

For MALDI-TOF MS several sample preparations are available with different matrices (26). The choice of the matrix and of the sample preparation should be adapted to the molecular mass of the compounds and to the complexity of the samples to analyze. a-Cyano-4-hydroxycinnamic acid (4HCCA) is preferred for peptides between 1 and 15 kDa, and the sandwich sample preparation can be universally used for molecular mass determination of pure peptides and enzymatic fragments or complex mixtures (e.g., crude hemolymph, enzymatic digests). The procedures reported below are the ones used for the discovery of the Drosophila immune-induced peptides (19,27,30). 

For molecular mass fingerprint of an enzymatic digestion, the acidity of the matrix and of the 1% TFA droplet is sufficient to quench any further digestion. 

Figure 4.4. Protein identification by MALDI-TOF. Steps in protein identification by MALDI-TOF are shown. Prior separation by gel or liquid chromatography to a single protein is needed prior to enzymatic digestion with trypsin. Digested peptides are spotted onto a MALDI target with an appropriate matrix that assists desorption of peptides upon activaton by laser. Peaks from the resulting mass spectrum represent peptide ions that can be searched in a database to match a theoretical tryptic digest from known proteins. The more proteins that are matched, the greater the statistical confidence in assignment of a protein identification.

Enzymatic digestion has some advantages over conventional sample pretreatment procedures based on acid or alkaline digestion thus, the former uses mild conditions as regards temperature and pH, which avoids potential analyte losses by volatilization or ohemical transformation to other species, and reduces the risk of contamination. In addition, the selectivity of enzyme catalysis is a powerful tool for distinguishing between fractions of elements associated with different components of the matrix as enzymes act on oertain ohemical bonds only. 

Probes of Cellular Structure. The membrane matrix of animal cells contains, among other compounds, enzymes, proteins, phospholipids, and triglycerides. The rate and extent of release of these compounds from the membrane during selective enzymatic digestion can be used to probe the degree of exposure of these compounds to the surface environment. 

The nuclear matrix. The lipid bilayers, the histones and other soluble proteins, and the DNA can all be removed from nuclei by exfracfion and enzymatic digestion. An insoluble residue, the nuclear matrix, is left. Largely protein in nature, this matrix is spread throughout the nucleus. Remnants of the membranes remain in the form of profeins that were in or along the bilayer. The nucleolus is clearly defined. The DNA appears to be bound to the nuclear matrix proteins. A specific 320-kb piece of a Drosophila chromosome has been mapped and used fo locate nontran-scribed scaffold (or mafrix) affachmenf regions of DNA bound to matrix proteins. These were found af intervals of 26-112 kb, fhe infervening loops confain-ing up to five or more A 120-kDa... 

Various sample preparation approaches, including microwave-assisted extraction and enzymatic digestion procedures, were examined to extract selenium from the defatted Brazil nut matrix among these approaches, enzymatic treatment with Proteinase K proved most effective [49]. SeMet was demonstrated to be the most abundant of these seleno-amino acids. In their study, another selenium species with m/z = 361 was also detected. By application of collision-induced dissociation (CID), there was evidence for this compound to be a dipeptide consisting of tyrosine and methionine with Se [49] as shown in Figure 9.1. It was proposed by the authors that this was a dipeptide with the structure p-HO(QH4)CH2CH(NH2)CONHCH(COOH)CH2CH2SeCH3. 

---