289
trap or triple quadrupole detection (Ferranti et al. 2007). An alternative modern
approach to 2-DE is a multi-dimensional liquid phase based separation techniques
such as MudPIT (Multidimensional Protein Identification Technology) pioneered
by Yates and colleagues (Washburn et al. 2001).
Chemical Methods
Foods being heterogenic materials are composed of different nutrients, such as carbohydrates, lipids and other micronutrients. There components and interactions
between them may result in inaccuracy in protein content measurements. Moreover,
results of these measurements are used in other calculations like measurement of
enzyme activity, proteins loading of SDS-PAGE gels, such measurements are thus
critical. Any error in such measurement results in errors in calculation. Thus methods that provide quicker and accurate assessment of protein concentrations are
acceptable. Many chemical methods determining the amount of protein either
directly or indirectly in a sample are based on different analytical principles, such as
nitrogen content determination, presence of peptide bond and aromatic amino acids,
dye binding capacity, light scattering and ultraviolet absorptivity properties of protein molecules.
Direct methods are based on analysis of amino acid residues in the sample while
indirect protein determination relies on nitrogen content determination with subsequent conversation to protein using nitrogen-to-protein conversion factor that varies
with sample type, usually 6.25 or interference from other chemical substances
(Maehre et al. 2018).
Most of these are colorimetric methods, where a portion of the protein solution
is reacted with a reagent that produces a coloured product which is then measured
spectrophotometrically. The amount of colour relates to the amount of protein present by appropriate calibration. However, none of these methods is absolute.
Direct Protein Determination
Amino Acid Analysis
The direct method for analysis of amino acid is considered the gold standard for
protein quantitation as it measures the quantity of each individual amino acid in a
protein. The method is composed of three basic steps hydrolysis of peptide bonds,
which link amino acids with each other and chromatographic separation of individual amino acids and detection and quantification of separated amino acids.
Protein content is calculated after subtraction of molecular mass of water from sum
total of individual amino acid residues. Hydrolysis of peptide bond is usually carried out under vacuum using 6 M HCl and heating at 100–165 °C for upto 72 h.
Recent Advances in Analysis of Food Proteins
trap or triple quadrupole detection (Ferranti et al. 2007). An alternative modern
approach to 2-DE is a multi-dimensional liquid phase based separation techniques
such as MudPIT (Multidimensional Protein Identification Technology) pioneered
by Yates and colleagues (Washburn et al. 2001).
Chemical Methods
Foods being heterogenic materials are composed of different nutrients, such as carbohydrates, lipids and other micronutrients. There components and interactions
between them may result in inaccuracy in protein content measurements. Moreover,
results of these measurements are used in other calculations like measurement of
enzyme activity, proteins loading of SDS-PAGE gels, such measurements are thus
critical. Any error in such measurement results in errors in calculation. Thus methods that provide quicker and accurate assessment of protein concentrations are
acceptable. Many chemical methods determining the amount of protein either
directly or indirectly in a sample are based on different analytical principles, such as
nitrogen content determination, presence of peptide bond and aromatic amino acids,
dye binding capacity, light scattering and ultraviolet absorptivity properties of protein molecules.
Direct methods are based on analysis of amino acid residues in the sample while
indirect protein determination relies on nitrogen content determination with subsequent conversation to protein using nitrogen-to-protein conversion factor that varies
with sample type, usually 6.25 or interference from other chemical substances
(Maehre et al. 2018).
Most of these are colorimetric methods, where a portion of the protein solution
is reacted with a reagent that produces a coloured product which is then measured
spectrophotometrically. The amount of colour relates to the amount of protein present by appropriate calibration. However, none of these methods is absolute.
Direct Protein Determination
Amino Acid Analysis
The direct method for analysis of amino acid is considered the gold standard for
protein quantitation as it measures the quantity of each individual amino acid in a
protein. The method is composed of three basic steps hydrolysis of peptide bonds,
which link amino acids with each other and chromatographic separation of individual amino acids and detection and quantification of separated amino acids.
Protein content is calculated after subtraction of molecular mass of water from sum
total of individual amino acid residues. Hydrolysis of peptide bond is usually carried out under vacuum using 6 M HCl and heating at 100–165 °C for upto 72 h.
Recent Advances in Analysis of Food Proteins