6
Rapid Peptide Mapping via Automated Integration of On-line Digestion, Separation and Mass Spectrometry for the Analysis of Therapeutic Proteins
Conclusion
An automated workflow was developed for protein therapeutics peptide mapping
which combined online trypsin digestion and high resolution accurate mass MS.
A trypsin column was developed capable of operation over prolonged periods at
elevated temperatures.
Operation under denaturing conditions enabled rapid digestion without
pretreatment.
Direct coupling of this system to the Q Exactive Orbitrap hybrid quadrupole-
Orbitrap mass spectrometer provided confident amino acid sequence information
for the monoclonal antibody.
A confident peptide mapping experiment including online trypsin digestion and
Orbitrap LCMS/MS analysis was achieved within a hour.
Potential future applications include use of this set-up in fast diagnostics, process
monitoring and disulfide bond mapping.
References
1. Götze,et al. J. Am. Soc. Mass Spectrom. (2011), DOI: 10.1007/s13361-011-0261-
2.
2. Vermeer et. al. Biophysical Journal Volume 78 January 2000 394–404.
3. Kumakura et. al. Journal of Molecular Catalysis, 23 (1984) 1 – 8.
FIGURE 7: Base peak chromatogram of native HSA digestion without reduction
and alkylation.
Perfinity is a trademark of Perfinity Biosciences, Inc. Mascot is a trademark of Matrix Science Ltd. All other
trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
igests.
RT:
0.00 -30.02
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
8.21
575.80
9.08
547.83
4.95
395.65
7.15
464.70
9.17
501.28
3.49
441.13
14.92
683.01
12.17
657.02
10.53
490.56
1.96
538.26
13.62
672.57
17.23
802.27
19.36
813.31
15.38
812.42
21.32
1398.41
0.63
515.64
20.05
823.40
17.78
821.23
22.60
974.78
27.71
316.73
23.33
1353.92
26.92
316.75
28.89
799.41
NL:
1.09E8
BasePeak
MS
50ul_300ugml_
hsa_eppdig_to
pten_mar_7_3
0min_r1
12 13 14 15 16 17 18 19
11.88
634.75 12.82
693.24
16.44
1273.71 17.58
971.42
14.20
730.08
17.61
971.44
12.88
693.30 14.25
773.50
14.30
773.48
1.41
2.58
16.05
1122.28
15.67
1052.34
17.68
971.35
17.73
971.08
19.56
742.95
NL:
1.28E8
BasePeak
MS
r&ahuiggin10%
bc_10000ngml
_r1_topten_12j
uly2012
e mAb digestion without reduction
HSA digests.
alkylated mAb samples yielded
the samples of digested without
heavy chain and 97% for light chain. The
antibody digestion is shown in Figure 5.
d to native human serum albumin. The
kes it an especially challenging case for
eterminations of native HSA digests.
f native HSA digestion without reduction
er of canonical peptides suggests native
ested. Given the highly bridged nature of
s of less stable proteins and antibodies
ns.
inutes it is possible that this workflow
ose to real time monitoring would be
ause reduction and alkylation are not
ld also be successfully applied to disulfide
hain
Heavy chain
91%
79%
