protein and detect posttranslational modifications. This approach is
possible with both a MALDI and an ESI ionization source and is
based on the analysis of peptide fragments generated by proteolytic
digestion of the target protein. Trypsin is often used for this purpose as it has good specificity, produces homogeneous peptide
fragments, and can be applied to proteins that have been analyzed
by gel electrophoresis and stained [17] (see also Subheading 2.2.1).
Before cleavage of the protein with trypsin, disulfide bonds are
reduced and free cysteines are alkylated. After digestion, the peptides are extracted and desalted. Overall, a peptide mass fingerprint
takes approximately 24 h from sample preparation to data acquisition. Note, however, that in most cases terminal truncations cannot
be detected.
1.3 Assessing
Structural Integrity
Typically, proteins adopt a defined 3D structure in their physiological/native state that is essential for their function. It is critical to
assess the structural integrity, i.e., that each batch of a protein
adopts the same (average) structure before using it in downstream
applications. In general, the 3D structure of a protein may depend
on solution conditions, such as buffer, pH, salt concentration,
reducing agents, or detergents, and it is important to examine
this possibility. The most common methods to assess the structural
state of a protein are circular dichroism (CD) spectroscopy and
nuclear magnetic resonance (NMR) spectroscopy. Both methods
can clearly distinguish folded compact structures from unfolded
structures and provide a characteristic spectrum of a protein,
which can be used to detect structural variation by comparison.
The characteristic spectrum of a protein allows the assessment of
the folding state of a protein, comparison of the structure of
a protein from different sources, production processes or batches
(see Subheading 1.5), and potential differences between protein
variants, as well as studying protein stability by thermal or chemical
denaturation, characterizing conformational changes, and analyzing protein:ligand interactions [18–21]. Introduction to protein
NMR spectroscopy is beyond the scope of this chapter and can be
found elsewhere [22, 23]. However, we note that, if NMR spectroscopy is available, it is highly recommended to include it in the
QC workflow for small-to-medium-sized proteins (<30 kDa). The
simplest NMR experiment, a one-dimensional proton NMR spectrum, may be recorded in a few minutes and not only allows
assessment of folding but also detection of the presence of lowmolecular-weight impurities or aggregation. Two-dimensional
spectra, which are readily obtainable for proteins <30 kDa by
15
N
isotope labeling, can provide information on the local chemical
environment of all individual residues. The residue chemical shifts
(peak positions) are very sensitive to the local chemical environment so that even slight structural changes, e.g., during thermal or
chemical denaturation, or due to changes in buffer composition or
Protein Quality Control
13
possible with both a MALDI and an ESI ionization source and is
based on the analysis of peptide fragments generated by proteolytic
digestion of the target protein. Trypsin is often used for this purpose as it has good specificity, produces homogeneous peptide
fragments, and can be applied to proteins that have been analyzed
by gel electrophoresis and stained [17] (see also Subheading 2.2.1).
Before cleavage of the protein with trypsin, disulfide bonds are
reduced and free cysteines are alkylated. After digestion, the peptides are extracted and desalted. Overall, a peptide mass fingerprint
takes approximately 24 h from sample preparation to data acquisition. Note, however, that in most cases terminal truncations cannot
be detected.
1.3 Assessing
Structural Integrity
Typically, proteins adopt a defined 3D structure in their physiological/native state that is essential for their function. It is critical to
assess the structural integrity, i.e., that each batch of a protein
adopts the same (average) structure before using it in downstream
applications. In general, the 3D structure of a protein may depend
on solution conditions, such as buffer, pH, salt concentration,
reducing agents, or detergents, and it is important to examine
this possibility. The most common methods to assess the structural
state of a protein are circular dichroism (CD) spectroscopy and
nuclear magnetic resonance (NMR) spectroscopy. Both methods
can clearly distinguish folded compact structures from unfolded
structures and provide a characteristic spectrum of a protein,
which can be used to detect structural variation by comparison.
The characteristic spectrum of a protein allows the assessment of
the folding state of a protein, comparison of the structure of
a protein from different sources, production processes or batches
(see Subheading 1.5), and potential differences between protein
variants, as well as studying protein stability by thermal or chemical
denaturation, characterizing conformational changes, and analyzing protein:ligand interactions [18–21]. Introduction to protein
NMR spectroscopy is beyond the scope of this chapter and can be
found elsewhere [22, 23]. However, we note that, if NMR spectroscopy is available, it is highly recommended to include it in the
QC workflow for small-to-medium-sized proteins (<30 kDa). The
simplest NMR experiment, a one-dimensional proton NMR spectrum, may be recorded in a few minutes and not only allows
assessment of folding but also detection of the presence of lowmolecular-weight impurities or aggregation. Two-dimensional
spectra, which are readily obtainable for proteins <30 kDa by
15
N
isotope labeling, can provide information on the local chemical
environment of all individual residues. The residue chemical shifts
(peak positions) are very sensitive to the local chemical environment so that even slight structural changes, e.g., during thermal or
chemical denaturation, or due to changes in buffer composition or
Protein Quality Control
13