In 1988, Karas and Hillenkamp developed a way to produce
ions from proteins with a molecular mass greater than 10 kDa [16]
by laser desorption using a matrix. As a result of this work, MALDI
(matrix-assisted laser desorption ionization) was made possible and
opened new perspectives in proteomic analysis. MALDI is a soft
ionization which is crucial to preserve intact mass of fragile and
nonvolatile compounds such as proteins. The principle of this
ionization mode is based on the irradiation by laser pulses of a
crystalline phase containing the cocrystallized PDZ and an organic
matrix. This irradiation is carried out using a laser pulsed usually at a
UV wavelength, which causes the desorption of the PDZ–matrix
pair that is charged by the transfer of protons from the matrix to the
analyte. Importantly, the sample must be able to release or capture
protons (transfer with the matrix), depending if the positive or
negative ionization mode is used to ionize the sample. For proteins,
a linear and positive mode is usually applied and the matrix must
absorb within the laser emission range to allow the sample to
vaporize. The ions are then accelerated by the application of an
electrical pulse and pass through a separation tube, called Time of
Flight (TOF), maintained under vacuum. As the difference of
potential is constant for all ions, ions with a smaller m/z value
(lighter ions) move faster until they reach the detector. Therefore,
the time of flight is different depending on the mass to charge ratio
(m/z). The signal at the detector is recorded and amplified and then
processed electronically to generate a m/z spectrum. In MALDITOF, proteins mainly result in singly charged or doubly charged
ion species.
One of the main advantages is that the sample droplets can be
prepared rapidly on the metal target, allowing the analysis of a large
number of proteins in a short time. Also, nowadays, most of the
scientific centers have access to this technology and research teams
can easily be trained to complement their SDS-PAGE
electrophoresis.
1.2.2 Top-Down PDZ
Sequencing
Top-down sequencing (TDS) with MALDI is a complementary
application of trypsin digestion. The generated trypsin digestion
peptides are analyzed and compared to queries databases to determine a protein identity from SDS gel bands or liquid samples. The
objective of the TDS strategy is to ensure that the protein sample is
the expected one by comparison of both the N- and C-termini
sequences with the generated sequencing, assuming no degradation or enzymatic cleavage. This technique allows researchers to
work directly on intact and undigested PDZs. TDS is truly suitable
for the analysis of small domains such as PDZs. The fragmentation
is done directly in the source (in-source decay or ISD) using a laser
power much higher than the one used for intact mass. In practice,
ISD fragmentation, compared to collision-induced fragmentation
(CID), triggers a much more independent fragmentation of the
PDZ Sample Quality Assessment
93
ions from proteins with a molecular mass greater than 10 kDa [16]
by laser desorption using a matrix. As a result of this work, MALDI
(matrix-assisted laser desorption ionization) was made possible and
opened new perspectives in proteomic analysis. MALDI is a soft
ionization which is crucial to preserve intact mass of fragile and
nonvolatile compounds such as proteins. The principle of this
ionization mode is based on the irradiation by laser pulses of a
crystalline phase containing the cocrystallized PDZ and an organic
matrix. This irradiation is carried out using a laser pulsed usually at a
UV wavelength, which causes the desorption of the PDZ–matrix
pair that is charged by the transfer of protons from the matrix to the
analyte. Importantly, the sample must be able to release or capture
protons (transfer with the matrix), depending if the positive or
negative ionization mode is used to ionize the sample. For proteins,
a linear and positive mode is usually applied and the matrix must
absorb within the laser emission range to allow the sample to
vaporize. The ions are then accelerated by the application of an
electrical pulse and pass through a separation tube, called Time of
Flight (TOF), maintained under vacuum. As the difference of
potential is constant for all ions, ions with a smaller m/z value
(lighter ions) move faster until they reach the detector. Therefore,
the time of flight is different depending on the mass to charge ratio
(m/z). The signal at the detector is recorded and amplified and then
processed electronically to generate a m/z spectrum. In MALDITOF, proteins mainly result in singly charged or doubly charged
ion species.
One of the main advantages is that the sample droplets can be
prepared rapidly on the metal target, allowing the analysis of a large
number of proteins in a short time. Also, nowadays, most of the
scientific centers have access to this technology and research teams
can easily be trained to complement their SDS-PAGE
electrophoresis.
1.2.2 Top-Down PDZ
Sequencing
Top-down sequencing (TDS) with MALDI is a complementary
application of trypsin digestion. The generated trypsin digestion
peptides are analyzed and compared to queries databases to determine a protein identity from SDS gel bands or liquid samples. The
objective of the TDS strategy is to ensure that the protein sample is
the expected one by comparison of both the N- and C-termini
sequences with the generated sequencing, assuming no degradation or enzymatic cleavage. This technique allows researchers to
work directly on intact and undigested PDZs. TDS is truly suitable
for the analysis of small domains such as PDZs. The fragmentation
is done directly in the source (in-source decay or ISD) using a laser
power much higher than the one used for intact mass. In practice,
ISD fragmentation, compared to collision-induced fragmentation
(CID), triggers a much more independent fragmentation of the
PDZ Sample Quality Assessment
93