4
Rapid Peptide Mapping via Automated Integration of On-line Digestion, Separation and Mass Spectrometry for the Analysis of Therapeutic Proteins
tion for automated peptide mapping.
Results
Historically, Kumakura et. Al (2), showed that in the best-case scenario trypsin that
was simply immobilized exhibited a 60% reduction of the activity 30 minutes at 70 C.
Shown in Figure 3, the proprietary stabilization process of the enzymes three
dimensional structure enables prolonged operation under these harsh conditions.
Table 2. Coverage deter
RT:
0.00 -19.93
0
1
2
3
4
5
6
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
3.86
390.97
6
53
3.92
391.05
3.84
391.03
5.28
367.28
2.91
415.98
5.30
550.1
1.68
446.86
1.57
377.77
2.78
349.29
FIGURE 5: Base peak chr
and alkylation.
TABLE 6. Coverage deter
As shown in Figure 4, when the digestions of native antibody samples were performed
at various times and temperatures, a dramatic increase in efficiency was observed at
70 C. These results suggest that under these conditions the samples were
simultaneously denatured and enzymatically digested. This observation validates
results obtained by Vermeer et. al. that showed that the denaturation of antibody
variable regions occurs at 60 C and constant region at 70 C (3).
FIGURE 4: Native mAb digested at various times and temperatures.
As shown in Table 2, diges
sequences coverage exce
pretreatment sequence co
base peak chromatograms
id quadrupole-Orbitrap mass
Capillary
s for
Sensitivity
Bent
Flatapole
ass Filter for
n Selection
Settings
400-2000
100
1e6, fullMS
5e4,MS/MS
2, fullMS
60,MS/MS
2.0
27
1%
30sec
-Orbitrap mass spectrometer
This online digestion experi
extensive disulfide linkages
native digestion. Figure 6 sh
Figure 7 shows the base pe
and alkylation. The identifica
human serum albumin (HSA
HSA this data suggests that
can effectively be digested u
Since the total digest time fo
would be extremely useful i
advantageous such as proc
necessary, it is possible that
bond mapping.
Sample
Reduced and Alkylate
Native
oftware revision 1.3 was used to search the
engine for all database searches. The
StavroX (1). Raw files generated by the Q
ectrometer were searched directly using a
u fragment mass tolerance.
FIGURE 3. Data suggests that a trypsin column was developed capable of
operation over prolonged periods at elevated temperatures.
