Supporting information S1
Combination of Online Enzyme Digestion with Stable Isotope Labeling for High
Throughput Quantitative Proteome Analysis
Fangjun Wang1, Xiaoluan Wei1, Hu Zhou2, Jing Liu1, Daniel Figeys2, Hanfa Zou1*
1. CAS Key Lab of Separation Sciences for Analytical Chemistry, National
Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, Dalian 116023, China
2. Ottawa Institute of Systems Biology, University of Ottawa
*Correspondence: Prof. Dr. Hanfa Zou, CAS Key Lab of Separation Sciences for
Analytical Chemistry ， National Chromatographic R&A Center, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
Tel: +86-411-84379610
Fax: +86-411-84379620
E-mail: hanfazou@dicp.ac.cn
Supporting Figures:
Figure 1
Base peak chromatogram of 1D nanoLC-MS/MS analysis of 2 μg mouse liver protein tryptic
digests (1 mg protein sample was in-solution digested overnight and 2 μg of the digest
was analyzed).
Figure 2: Log2(Ratio) distribution of quantified proteins by the online chemical isotope
labeling system (Analytical Chemistry 2010, 82 (7), 3007-3015). 15 μg tryptic digest of
normal human liver protein was labeled by light or heavy isotope labeling reagents,
respectively, by the automated online labeling system, and then analyzed by online
multidimensional nanoLC-MS/MS. The data from our previous work (Analytical
Chemistry 2010, 82 (7), 3007-3015) were re-analyzed by Maxquant as described in
experimental section. Black: one time quantification analysis; Red: replicate
quantification analyses (CVs<50%).
Figure 3: Ratio distribution of quantified proteins by online enzyme digestion for protein sample
extracted from 5×104 Hela cells (light and heavy isotope labeled cells mixed with the ratio 5:1).
Two replicate quantification analyses were performed and the CVs were controlled less than 50%.
One dimensional nanoLC-MS/MS analysis with 150 min separation gradient (0-35% ACN) was
applied, and LTQ-OrbiTrap XL was used for detection.
Table 1 The level of missed cleavage obtained from previous reports.
Ref.
J. Proteome Res. 2005, 4 (2),
481-490
Anal. Chem. 2008, 80 (8),
2949-2956
Proteomics 2007, 7 (14),
2330-2339
Mol. Cell. Proteomics 2011,
10 (2), M110 000679.
Number of
peptides
Number of
peptides with
missed cleavage
Percentage of
missed cleavage
46
29
63.0%
15
9
60.0%
13
9
69.2%
4736
3634
76.7%
Table 2 The rates of miss-cleavage in proteome analysis of 5 µg BSA by 60 or 600 min’s
online enzyme digestion. Peptides were analysis by 40 min gradient nanoLC-MS/MS
analysis on LTQ. And database search was performed by Mascot, the data filtering
criteria were as follows: bold red, rank 1, and ion score >30.
Online
digestion time
Number of
peptides
Number of
peptides with
miss-cleavage
Percentage of
missed
cleavage
Protein
coverage
60 min-1
1015
685
67%
93%
60 min-2
937
656
70%
95%
60 min-3
1016
656
65%
95%
600 min-1
1268
683
54%
95%
600 min-2
1357
693
51%
94%
600 min-3
1217
642
53%
94%