Supporting Information
Studies of keratins in tongue coating samples of hepatitis B patients by
mass spectrometry
Fang Fang1, Ping Liu2*, Haoyang Wang1, Li Zhang1, Jing Zhang1, Yuping Gao1,
Liming Zeng1 and Yinlong Guo1*
1
Shanghai Mass Spectrometry Center, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China
2
Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine,
Cailun Road 1200, Shanghai 201203, China
Labeling methods of protein samples and comparative analysis with [d0]- and
[d6]-DMMSP
The protein (lysozyme) (0.1 mg) was dissolved in 1 mL of 30 mM NH4HCO3 buffer
solution (pH 7.5), to the solutions were added 5 μmol of [d0]-DMMSP, and the
mixture was incubated at 37°C for 78 h. The reaction was monitored by MALDI-TOF
MS analysis.
For quantification, the protein (lysozyme) was dissolved in 30 mM NH4HCO3
buffer solution (pH 7.5) to a concentration of 100 μg/mL, 90 μg/mL, 80 μg/mL, 60
μg/mL, 50 μg/mL, 25 μg/mL, 20 μg/mL, 14 μg/mL, 10 μg/mL, 5 μg/mL, 4 μg/mL, 2
μg/mL, respectively. Each lysozyme solution was divided into two equal parts, and
were separately treated with [d0]-DMMSP and [d6]-DMMSP, the mixtures were
incubated at 37°C for 78 h. The resultant solutions were purified by an ultrafiltration
identically with those described above. The light and the heavy labeled sample
solutions were combined with equal volumes to reach different concentration ratios:
1:50, 1:25, 1:20, 1:10, 1:8, 1:6, 1:4, 1:2, 8:10, 1:1, 10:9, 10:8, 2:1, 4:1, 6:1, 8:1, 10:1,
20:1, 25:1. For reproducibility study, lysozyme resolutions of 20 μg/mL, 14 μg/mL, 10
μg/mL, 5 μg/mL were used. The light and the heavy labeled sample solutions were
combined with equal volumes to reach different concentration ratios: 1:10, 1:1, 10:1.
All sample solutions subjected to a MS-based comparative analysis after digestion.
1
Results
The mass spectra for quantitative analyses of a Cys-containing peptide (WWCNDGR)
from lysozyme digestion are shown in Fig. S1. The combination of ‘light’ and ‘heavy’
labeled samples gave a 6 Da difference in the molecular weight, which can be visually
identified (between m/z 1074.4 and 1080.4 for WWCNDGR in Fig. S1). The intensity
ratios of the monoisotopic peaks existing in pairs were calculated and proven
consistent with the actual sample concentration ratios to a certain extent from 0.1 to 10
(Fig. S2). Peptides containing cysteine, lysine, and tyrosine residues (KVFGR,
HGLDNYR,
RHGLDNYR,
CKGTDVQAWIR,
WWCNDGRTPGSR,
KIVSDGNGMNAWVAWR) were also observed clearly in pairs (Table S1). Their
relative intensity ratios provided auxiliary and reliable information for more accurate
quantitative analysis. Here, the peptide WWCNDGR was chosen and the relative
errors of quantitative analysis were calculated (Fig. S2), which show excellent values
under 5% in certain concentration ratios from 0.1 to 10. The quantitative result could
also be obtained from any of the other labeled peptides.
Figure S1. Results from quantitative isotopic labeling at the peptide level
(WWCNDGR differentially labeled with ‘light’ and ‘heavy’ reagents). (A) The
theoretical light-to-heavy ratio for this peptide was 0.1(L/H) while the calculated ratio
was 0.097 (L/H). (B) The theoretical light-to-heavy ratio for this peptide was 4 (L/H)
while the calculated ratio was 4.186 (L/H).
2
Figure S2. Relative errors of labeling quantitative analysis of lysozyme solutions.
Table S1. Labeling peptides identified from digests of lysozyme modified by
[d0]-/[d6]-DMMSP
Observed
Matched peptides from
m/z
database
Number
No. of
No. of
Theoretic
[d0]-DM
[d6]-DM
No.
of
cysteines
tyrosines
al m/z
lysines
MSP
MSP
Position
Sequence
(before
labeling labeling labeling)
1 744.4 750.4
606.4
19-23
KVFGR
0
1
0
No. of
added
tags
1
2
1012.4 1018.4
874.4
33-39
HGLDNYR
0
0
1
1
3
1074.4 1080.4
936.4
80-86
WWCNDGR
1
0
0
1
4
1552.7 1564.7
1276.6
133-143
1
1
0
2
5
1572.5 1578.5
1434.6
80-91
1
0
0
1
0
1
0
1
CKGTDVQA
WIR
WWCNDGR
TPGSR
KIVSDGNG
6
1941.7 1947.7
1803.9
115-130
MNAWVAW
R
3