Dibujo 12: Situación de los tanques de combustible y agua dulce
3.2 Espesor de los paneles del casco
The principles of mass spectrometry (MS) are well known. Only a brief outline is provided here. A food sam- ple is bombarded with high energy electrons in an evacu- ated chamber. Molecular ions produced by bombardment
are accelerated via an electric field, past a set of electro- magnets, towards a detector. The detection time for molecular ions is proportional to their mass-to-charge ratio (mi/zi)½. Techniques for generating protein ions for
MS analysis were only developed in the mid-1980s. The best known of these desorption techniques are fast atom bombardment (FAB), matrix assisted laser desorption ion- ization (MALDI), and electrospray ionization (ESI). The theory, principles, and instrumentation for protein MS analysis have been reviewed (92). Table 5.6 summarizes each desorption method and types of information avail- able from MS analysis of proteins. Process-induced changes in food proteins appear to be readily detectable. For example, ESI-MS analysis of beta-lactoglobulin from 109 cows showed it to be covalently modified by a 324 Da species, probably lactose (93). The mechanism of heme protein denaturation was also examined by ESI-MS (94). Proteolysis of caseins during cheese manufacture and ripening was readily followed by MALDI-MS (95). Virtually all the major food protein groups have been ana- lyzed by MS including milk, egg, meat and cereal proteins as reviewed in References 96–98.
IV. PROTEOMICS
The term proteome was introduced by Wilkins et al. in 1995 to describe the “entire PROTEin complement expressed by a genOME, or by a cell or tissue type” (99). Proteomics is the wholesale identification of proteins comprising a proteome using large-scale, high-throughput technologies, primarily 2D gel electrophoresis and MS. In
TABLE 5.6
Mass Spectrometric Analysis of Food Proteins
Technique Comments
FAB ● Protein + glycerol are bombarded by Xe or
Cs ions at 8–40 keV. Sample is introduced into MS port.
ESI ● Protein solution disintegrates from the tip
of capillary polarized at ⫾3000–5000 V. Electrospray is fed to MS port.
MALDI ● Protein + large excess of crystalline matrix
is irradiated by a laser. The matrix absorbs energy, vaporizes, and ionizes protein. Application areas*
Aggregation Molecular mass analysis
Cheese ripening Polymorphism
and maturation
Denaturation Post-translational modification
Glycation Process effects
Heat effects Proteolysis
Irradiation Purity
Meat postmortem Sequence determination Sulfur/disulfide exchange * Compiled from References 92–98.
the so-called post-genomics era, interest has extended to the subsidiary topics of transcriptomics, proteomics, and metabolomics (Figure 5.2). The transcriptome and metabolome refer to the total profile of RNA and metabo- lites within a cell, respectively (100). Protein functions other than metabolism – the entirety of enzymatic reac- tions within a cell – are also of interest. Proteomics is con- sidered a protein-based method for gene expression analysis.
The rationale for proteomics can be seen from the central dogma of molecular biology, postulated by Francis Crick in 1958. The direction of information flow within cells is DNA RNA protein function (101). Transcription of DNA leads to time-dependent expression of cell function. However, the relation between genome and cell function is complicated due to the presence of regulatory mechanism, redundancy, or amplification processes during information transfer. The study of pro- teomics is clearly essential because (i) proteins are the machinery that perform day-to-day functions within a cell, (ii) there is a low correlation between the number of genes and the number of proteins within a cell, (iii) dif- ferential gene expression occurs at different times and in different parts of the organism, and (iv) single genes can encode for more than one protein due to post-translational modification.
There are five essential steps for proteomics research: (i) sample preparation – cells, tissues, or organelles are homogenized to produce a protein extract. Care is needed to avoid protein modification by endogenous proteases, chemical modification or denaturation; (ii) protein separa- tion by 2D electrophoresis. Typically, IEF (1D) and SDS- PAGE (2D) analysis is followed by protein visualization and densitometry leading to a 2D digital representation of the separated proteins. With the new generation of densit- ometers it is possible to perform a comparative image analysis of hundreds of protein spots resolved by 2D elec- trophoresis. The key is to discover differences between specific protein (spots) for the control and treatment sam- ple; (iii) protein identification – protein spots of interest
are excised from the 2D gel, digested with trypsin, then subjected to HPLC or CE analysis; (iv) peptide sequenc- ing – the products of proteolysis are analyzed by MALDI- MS or ESI-MS as described in Section IIIB. Sophisticated MS instrumentation can now provide protein molecular mass as well as sequence information, (v) Bioinformatics – the application of computerized informatics tools to bio- logical data. Peptide sequences are compared with the DNA- sequence database in order to identify the protein of interest. Digitized 2D gel patterns can also be compared directly with computerized library data for protein identi- fication (99).
The impact of proteomics on food science and tech- nology could be considerable (100,102). It may be possi- ble to correlate changes in protein expression or post-translational modification with specific treatments, be they developmental, environmental, nutritional, or hor- monal. Potential areas for proteomics research in food related areas include the study of protein structure func- tion relations, functional ingredients, food-borne pathogens (103), allergens (104), food adulteration, novel ingredients, starter cultures (105), muscle or meat science (106), and the identification of protein markers for grain quality (107).
V. CONCLUSION
Aspects of food protein analysis are reviewed in this chapter. Food proteins were defined as those proteins which are of interest in food science. Food protein analy- sis is a vast topic but still emerging as an integrated dis- cipline. Novel techniques for protein quantitation and characterization are being developed. The food system and food science expertise areas (Tables 5.1–5.2) were suggested as schema for defining the perspective of this rapidly evolving subject. The first part of the chapter emphasized “approved” methods for protein quantitation which are used within the food industry (Figure 5.1). Further research on Kjeldahl and Dumas analysis is needed to establish national and international protocols for a wide range of foods. Further developments in NIR analysis are needed in the areas of instrumentation and pattern recognition software. On-line NIR analysis for products on conveyor belts will probably increase (108). An example of a commercially available NIR on-line instrument is the MM170 analyzer from NDC Infrared Engineering (109). The trend towards diode array NIR instrumentation will lead to more rapid acquisition of spectra, increased portability, and more affordable instru- mentation. Section III dealt with selected methods for protein fractionation, and the chapter culminated with a discussion of proteomics (Section IV). Though less than 10-years old, high-throughput protein analysis within a proteomics framework is now firmly on the food science agenda.
Food Protein Analysis: Determination of Proteins in the Food and Agriculture System 5-7
(A)
• DNA→ • mRNA→ • Proteins→ • Function
(B) Metabolomics Proteomics Proteomics Genomics Proteome Transcriptoms Genome Metabolome Transcriptomics
FIGURE 5.2 Information flow in cells is from DNA (genome) to mRNA (transcriptome) to proteins (proteome) to a variety of protein functions (phenome, physiome, and metabolome).
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