molecule. This distribution of charge states, each corresponding to
a particular mass/charge ratio, is then deconvoluted to give a
molecular mass (Fig. 5b). In contrast, MALDI is an ionization
process from a solid phase. The protein of interest is dispersed in
a saturated solution of a small aromatic molecule called the matrix
and the whole is co-crystallized by evaporation of the solvent. The
solid phase obtained is then irradiated by a laser beam. The role of
the matrix is to absorb the energy of the laser beam, which leads to
transfer of the sample into the gas phase and subsequent desolvation. In MALDI, peptides, and even proteins, often result in only a
singly charged ion species, or at least a considerably lower number
of ion species with fewer charges as compared to ESI (Fig. 5c). One
of the main advantages of MALDI is that the sample droplets can be
prepared rapidly, allowing the analysis of a large number of soluble
or membrane proteins in a very short time, making it particularly
suited for QC screening (see Subheading 2.2.4).
A mass spectrometer consists of three main parts: (1) the ion
source, where analytes are ionized and transferred into the gas
phase and subsequently into the vacuum inside the instrument,
(2) a mass analyzer that separates the analytes according to their
m/z ratio (m being the mass of the analyte; z being the charge the
ionized analyte carries; separation in space, frequency space, or
speed), and (3) the detector registering the ions. It is worth noting
that mass spectrometers do not measure mass per se but the m/z
ratio.
For protein QC, an intact mass MS analysis (top-down strategy) is highly recommended to check the identity and integrity of
the protein of interest. This QC parameter is crucial to ensure that
the target protein is actually the desired protein (i.e., that there has
not been some mistake during cloning) and is highly pure and
without any degradation at the N- or C-terminus (which may result
from contaminating peptidase enzymes not being removed by the
purification process). The precision of intact mass MS of big proteins (e.g., bovine serum albumin, 66.4 kDa) is better with an ESI
than with a MALDI source. However, buffer components such as
detergents or glycerol can easily interfere with the ESI measurement and make the analysis of membrane proteins or proteins in
glycerol more challenging than with a MALDI source. Other
potential chemical modifications of a protein that may degrade its
functionality, such as deamination and oxidation, aspartic acid
isomerization, and nonreducible crosslinking, can also be identified
using MS [17]. Ideally, to rule out degradation later in the process,
the intact mass should be checked at different timepoints, such as
immediately postpurification and at the conclusion of downstream
experiments.
Alternatively, a peptide mass fingerprint (bottom-up strategy)
can identify and characterize the amino acid sequence of the target
12
Bertrand Raynal et al.
a particular mass/charge ratio, is then deconvoluted to give a
molecular mass (Fig. 5b). In contrast, MALDI is an ionization
process from a solid phase. The protein of interest is dispersed in
a saturated solution of a small aromatic molecule called the matrix
and the whole is co-crystallized by evaporation of the solvent. The
solid phase obtained is then irradiated by a laser beam. The role of
the matrix is to absorb the energy of the laser beam, which leads to
transfer of the sample into the gas phase and subsequent desolvation. In MALDI, peptides, and even proteins, often result in only a
singly charged ion species, or at least a considerably lower number
of ion species with fewer charges as compared to ESI (Fig. 5c). One
of the main advantages of MALDI is that the sample droplets can be
prepared rapidly, allowing the analysis of a large number of soluble
or membrane proteins in a very short time, making it particularly
suited for QC screening (see Subheading 2.2.4).
A mass spectrometer consists of three main parts: (1) the ion
source, where analytes are ionized and transferred into the gas
phase and subsequently into the vacuum inside the instrument,
(2) a mass analyzer that separates the analytes according to their
m/z ratio (m being the mass of the analyte; z being the charge the
ionized analyte carries; separation in space, frequency space, or
speed), and (3) the detector registering the ions. It is worth noting
that mass spectrometers do not measure mass per se but the m/z
ratio.
For protein QC, an intact mass MS analysis (top-down strategy) is highly recommended to check the identity and integrity of
the protein of interest. This QC parameter is crucial to ensure that
the target protein is actually the desired protein (i.e., that there has
not been some mistake during cloning) and is highly pure and
without any degradation at the N- or C-terminus (which may result
from contaminating peptidase enzymes not being removed by the
purification process). The precision of intact mass MS of big proteins (e.g., bovine serum albumin, 66.4 kDa) is better with an ESI
than with a MALDI source. However, buffer components such as
detergents or glycerol can easily interfere with the ESI measurement and make the analysis of membrane proteins or proteins in
glycerol more challenging than with a MALDI source. Other
potential chemical modifications of a protein that may degrade its
functionality, such as deamination and oxidation, aspartic acid
isomerization, and nonreducible crosslinking, can also be identified
using MS [17]. Ideally, to rule out degradation later in the process,
the intact mass should be checked at different timepoints, such as
immediately postpurification and at the conclusion of downstream
experiments.
Alternatively, a peptide mass fingerprint (bottom-up strategy)
can identify and characterize the amino acid sequence of the target
12
Bertrand Raynal et al.