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choice of matrix which is important for good MALDI-MS spectra. From aspect of
peptide analysis, following matrices can be considered most effective which
includes cinnamic acid derivatives (α-cyano-4-hydroxycinnamic) and benzoic acid
derivatives (2,5-dihydroxybenzoic and that for protein includes sinapinic acid
(trans-3,5-dimethoxy 4-hydroxycinnamic acid) (Beavis et al. 1992; Beavis and
Chait 1989).
An advance of this technique is MALDI-TOF, MALDI coupled with a time-offlight (TOF) mass spectrometer. This conjugation is used due to pulsed nature of
MALDI which makes the process much convenient. This technique allows the
simultaneous measurement of masses from 1000 to 100,000 without chromatographic purification in the low picomol range within few minutes. Thus, not only
intact proteins, but also protein hydrolyzates can be analyzed. The technique has
been proved very helpful in evaluating authenticity of milk by detecting presence of
even small amount of cow milk in either raw ewe and buffalo milk samples and the
addition of powdered milk to samples of fresh raw milk, adulteration was evaluated
from protein patterns coming from the most abundant whey proteins, alphalactalbumin and beta-lactoglobulin, used as molecular markers (Cozzolino
et al. 2001)
Chromatographic Methods of Food Protein Analysis
In the post-genomic era some of the greatest challenges faced by scientist were to
identify and quantify all expressed protein components in cells, tissues, and organisms. In rescue of this situation chromatography has found one important position
in protein analysis scenario. Proteins can be purified based on characteristics such
as size and shape, total charge, hydrophobic groups present on the surface, and
binding capacity with the stationary phase. Four separation techniques based on
molecular characteristics and interaction type use mechanism of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are
based on the stationary bed, including column, thin layer, and paper
chromatography.
Column Chromatography
Column Chromatography is one of the most frequently used methods of protein
purification (Coskun 2016). The technique is performed on chromatography columns filled with a variety of column matrices (mostly derivatives of agarose) using
different characteristics of proteins for separation such as size, charge, affinity
toward substrates or inhibitors, hydrophobicity, lectin, or antibody binding. In column chromatography stationary phase suitably moistened with mobile phase is
either coated onto discrete small particles (the matrix) and packed into the column
Recent Advances in Analysis of Food Proteins
choice of matrix which is important for good MALDI-MS spectra. From aspect of
peptide analysis, following matrices can be considered most effective which
includes cinnamic acid derivatives (α-cyano-4-hydroxycinnamic) and benzoic acid
derivatives (2,5-dihydroxybenzoic and that for protein includes sinapinic acid
(trans-3,5-dimethoxy 4-hydroxycinnamic acid) (Beavis et al. 1992; Beavis and
Chait 1989).
An advance of this technique is MALDI-TOF, MALDI coupled with a time-offlight (TOF) mass spectrometer. This conjugation is used due to pulsed nature of
MALDI which makes the process much convenient. This technique allows the
simultaneous measurement of masses from 1000 to 100,000 without chromatographic purification in the low picomol range within few minutes. Thus, not only
intact proteins, but also protein hydrolyzates can be analyzed. The technique has
been proved very helpful in evaluating authenticity of milk by detecting presence of
even small amount of cow milk in either raw ewe and buffalo milk samples and the
addition of powdered milk to samples of fresh raw milk, adulteration was evaluated
from protein patterns coming from the most abundant whey proteins, alphalactalbumin and beta-lactoglobulin, used as molecular markers (Cozzolino
et al. 2001)
Chromatographic Methods of Food Protein Analysis
In the post-genomic era some of the greatest challenges faced by scientist were to
identify and quantify all expressed protein components in cells, tissues, and organisms. In rescue of this situation chromatography has found one important position
in protein analysis scenario. Proteins can be purified based on characteristics such
as size and shape, total charge, hydrophobic groups present on the surface, and
binding capacity with the stationary phase. Four separation techniques based on
molecular characteristics and interaction type use mechanism of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are
based on the stationary bed, including column, thin layer, and paper
chromatography.
Column Chromatography
Column Chromatography is one of the most frequently used methods of protein
purification (Coskun 2016). The technique is performed on chromatography columns filled with a variety of column matrices (mostly derivatives of agarose) using
different characteristics of proteins for separation such as size, charge, affinity
toward substrates or inhibitors, hydrophobicity, lectin, or antibody binding. In column chromatography stationary phase suitably moistened with mobile phase is
either coated onto discrete small particles (the matrix) and packed into the column
Recent Advances in Analysis of Food Proteins