absorbance of 5580 M
À1 .cm
À1 and 1480 M
À1 .cm
À1 , respectively.
The quantification at 280 nm is therefore suitable in presence of at
least 1 tryptophan or 1 tyrosine in the PDZ sequence.
The molar absorption relates the measured absorbance to the
concentration of the protein through the Beer–Lambert law: A
(λ) ¼ ε.l.c, where A is the absorbance at a given wavelength λ
(280 nm for a protein), ε (in M
À1 cm
À1 ) is the molar absorption
coefficient (also called the extinction coefficient) for a given PDZ,
and c is the concentration of PDZ of interest (in M) (see Note 1).
These properties can be used in protein analysis, either to identify
protein-containing fractions or to determine the concentration of
protein in a purified sample. Quantification of PDZs in a complex
mixture (e.g., isolated fractions of cells) by UV visible absorption is
difficult since protein compositions and their absorption coefficients are not known. Absorption in the 205 nm region (peptide
bonds) can also be used for the spectrophotometric assay of proteins [13], this is of particular importance as some PDZs do not
contain tryptophan or tyrosine. For the majority of proteins,
UV-vis absorption allows to measure a mass concentration with
concentration as low as 100 μg/ml.
In addition to the measure of the concentration using the
molar absorption coefficient of the PDZ, a complete spectrum
will also inform about the general quality of the preparation. It
should be emphasized that the exact PDZ buffer should be used as
a blank to avoid misinterpretation. A strong signal at 260 nm is
usually a sign of nucleic acid contamination or small compounds
such as DTT, detergents, imidazole, ATP, etc. [13–15]. In the
presence of a 260 nm contamination, the quantification at
280 nm can easily be overestimated. In addition, a regularly increasing absorbance between 340 nm and 300 nm is generally indicative
of scattering due to aggregation, also resulting in quantification
overestimation.
1.2 Identity
1.2.1 Intact Mass
Spectrometry
Mass spectrometry is a sensitive analytical method that can be easily
implemented to determine the intact molecular mass of PDZ
domains. The combination of the small size of the PDZs and the
high accuracy in m/z of mass spectrometry devices allows, in addition to ensuring the molecular mass, to determine potential posttranslational modifications (PTMs) of the domains. Proteolytic
processing due to the loss of one or several amino acids is crucial
to monitor and nearly impossible to detect by gel electrophoresis.
In addition, the fragmentation of PDZs into small peptides allows
sequencing and thus confirms the identity of the PDZ of interest
(see in-source decay (ISD) part). Many mass spectrometer configurations have been developed, each with its strengths and limitations. In this chapter, only the MALDI-TOF configuration will be
explained.
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Ce ´ lia Caillet-Saguy et al.
À1 .cm
À1 and 1480 M
À1 .cm
À1 , respectively.
The quantification at 280 nm is therefore suitable in presence of at
least 1 tryptophan or 1 tyrosine in the PDZ sequence.
The molar absorption relates the measured absorbance to the
concentration of the protein through the Beer–Lambert law: A
(λ) ¼ ε.l.c, where A is the absorbance at a given wavelength λ
(280 nm for a protein), ε (in M
À1 cm
À1 ) is the molar absorption
coefficient (also called the extinction coefficient) for a given PDZ,
and c is the concentration of PDZ of interest (in M) (see Note 1).
These properties can be used in protein analysis, either to identify
protein-containing fractions or to determine the concentration of
protein in a purified sample. Quantification of PDZs in a complex
mixture (e.g., isolated fractions of cells) by UV visible absorption is
difficult since protein compositions and their absorption coefficients are not known. Absorption in the 205 nm region (peptide
bonds) can also be used for the spectrophotometric assay of proteins [13], this is of particular importance as some PDZs do not
contain tryptophan or tyrosine. For the majority of proteins,
UV-vis absorption allows to measure a mass concentration with
concentration as low as 100 μg/ml.
In addition to the measure of the concentration using the
molar absorption coefficient of the PDZ, a complete spectrum
will also inform about the general quality of the preparation. It
should be emphasized that the exact PDZ buffer should be used as
a blank to avoid misinterpretation. A strong signal at 260 nm is
usually a sign of nucleic acid contamination or small compounds
such as DTT, detergents, imidazole, ATP, etc. [13–15]. In the
presence of a 260 nm contamination, the quantification at
280 nm can easily be overestimated. In addition, a regularly increasing absorbance between 340 nm and 300 nm is generally indicative
of scattering due to aggregation, also resulting in quantification
overestimation.
1.2 Identity
1.2.1 Intact Mass
Spectrometry
Mass spectrometry is a sensitive analytical method that can be easily
implemented to determine the intact molecular mass of PDZ
domains. The combination of the small size of the PDZs and the
high accuracy in m/z of mass spectrometry devices allows, in addition to ensuring the molecular mass, to determine potential posttranslational modifications (PTMs) of the domains. Proteolytic
processing due to the loss of one or several amino acids is crucial
to monitor and nearly impossible to detect by gel electrophoresis.
In addition, the fragmentation of PDZs into small peptides allows
sequencing and thus confirms the identity of the PDZ of interest
(see in-source decay (ISD) part). Many mass spectrometer configurations have been developed, each with its strengths and limitations. In this chapter, only the MALDI-TOF configuration will be
explained.
92
Ce ´ lia Caillet-Saguy et al.