SUPPLEMENTAL TABLE LEGENDS
Table SM1 CFB calibration curve raw data given by Quasar tool.
Table SM2 Lod-Loq raw values for all transitions and labeled peptides used in the CFB
calibration curve.
Table SM3 Proteotypic Peptide Sequences and Selected SRM Transitions measured in the
human saliva. This table provides information about all peptides and the respective transitions
(fragment ion and m/z) monitored in the human saliva.
Table SM4 Peptide report result from Skyline. This table provides information about area,
retention time, dot product and ratio light-to-global standards for all peptides measured in each
sample (replicate name column).
Table SM5 MSstat input for Protein Level modeling analysis exported using Skyline. This
table contains information required for Msstats analysis: Protein Name, Peptide Sequence,
Precursor Charge, Fragment Ion, Product Charge, Isotope Label Type, Condition, BioReplicate,
Run, Intensity.
Table SM6 MSstat input for Peptide Level modeling analysis exported using Skyline.
Table SM7 MSstat results of group comparison using model-based inference at a protein
level.
Table SM8 MSstat results of group comparison using model-based inference at a peptide
level.
Table SM9 Normalized values (ratio light-to-heavy) for CFB YGLVTYATYPK and
VSEADSSNADWVTK peptides, exported using Skyline.
SUPPLEMENTAL FIGURE LEGENDS
Figure 1 Calibration curve for the Schistosoma japonicum GST peptide IEAIPQIDK. A)
A dilution curve of the unlabeled SJ GST peptide IEAIPQIDK peptide standard was spiked with
a constant amount of the heavy labeled IEAIPQIDK peptide. The unlabeled to labeled peak area
was measured by LC/SRM/MS for each standard. B) Using this linear equation the
concentration of any unknown sample (y‐value) can be obtained by inserting the light‐to‐
heavy ratio of that sample (x‐value) into a standard y = m*x + b equation (concentration =
slope * ratio + intercept). The concentration of the in vitro-synthetized protein was obtained.
Figure 2 Calibration curve for the CFB peptides. A) A dilution curve of the digested CFB
peptides was spiked in known concentration range from 0.1 to 100 fmol/µl with a constant
amount of the heavy labeled YGLVTYATYPK and B) labeled VSEADSSNADWVTCK
peptide. The unlabeled to labeled peak area was measured by LC/SRM/MS for each standard in
triplicate and the light to heavy ratio used to calculated LOD and LOQ. C) YGLVTYATYPK
was normalized by the peptides from global standards and linear regression was performed in
the standard curve with the ratio light to global standards values. D) VSEADSSNADWVTCK
was normalized by the peptides from global standards and linear regression was performed in
the standard curve with the ratio light to global standards values.
Figure 3 Quality Control plots for all proteins analyzed using MSstats at a protein level.
X-axis: run. Y-axis: log-intensities of transitions. Reference (global standards) signals are in the
left/right panel. The plots visualize potential artifacts in mass spectrometry runs.
Figure 4 Profile plot for all proteins analyzed using MSstats at a protein level. X-axis: run.
Y-axis: log-intensities of transitions. Line colors indicate peptides and line types indicate
transitions. The plots help identify potential sources of interesting and nuisance variation for
each protein.
Figure 5 Condition plots for all proteins analyzed using MSstats at a protein level. X-axis:
condition. Y-axis: log ratio of endogenous over reference intensities of each transition in a run.
Dots indicate the mean of log ratio for each condition. Error bars are confidence intervals with
0.95 significant level for each condition. The plots visualize the differences between conditions,
which are of the main biological interest.
Figure 6 Volcano plot of the comparison Control vs No Lesion condition. X-axis: practical
significance, model-based estimate of log-fold change. Y-axis: statistical significance, FDRadjusted p-values on the negative log2 scale. The dashed line represents the FDR cutoff (default
sig=0.05) and fold-change FCcutoff=1.5. A) Results at a protein level. B) Results at a peptide
level.
Figure 7 Volcano plot of the comparison No lesion vs Lesion condition. X-axis: practical
significance, model-based estimate of log-fold change. Y-axis: statistical significance, FDRadjusted p-values on the negative log2 scale. The dashed line represents the FDR cutoff (default
sig=0.05) and fold-change FCcutoff=1.5. A) Results at a protein level. B) Results at a peptide
level.
Figure 8 Relative quantification using isotope reference peptides and global standards
peptides. A) Scatter plot using ratio light to heavy for YGLVTYATYPK CFB peptide
(Student’s t-test, * p<0.05). B) Scatter plot using ratio light to global standards for
YGLVTYATYPK CFB peptide (Student’s t-test, * p<0.05). C) Scatter plot using ratio light to
heavy for VSEADSSNADWVTK CFB peptide (Student’s t-test, * p<0.05). D) Scatter plot
using ratio light-to-global standards for VSEADSSNADWVTK CFB peptide (Student’s t-test, *
p<0.05).
SUPPLEMENTAL FIGURES
SUPPLEMENTARY FIGURE 1
A)
Light-toheavy Ra o
20
15
10
5
0
0
10
-5
B)
MM
(kDa)
260
160
110
80
60
50
40
30
20
Protein
C1R
TINAG
SLPI
SERPINE1
LGR1
LNC2
TAGLN1
ANX1
FAM49B
y = 0,4674x - 0,0194
R² = 0,99983
20
30
40
50
nM IEAIPQIDK
1
2
3 4
5 6
Order
7 8 9
1
2
3
4
5
6
7
8
9
Average (nM)
2.5
2.8
4.7
5.2
6.0
12.1
14.7
22.9
40.1
Protein
C1R
TINAG
SLPI
SERPINE1
LRG1
LCN2
TAGLN2
ANXA1
FAM49B
MW
MW + GST
80
55
15
45
38
23
22
39
37
105
80
40
70
63
48
47
64
62
SUPPLEMENTAL FIGURE 2
A)
VSEADSSNADWVTK
B)
YGLVTYATYPK
6
45
40
35
30
25
20
15
10
5
0
-20 -5 0
40
60
fmol/µl
80
Ra o Light to Heavy
Ra o Light to Heavy
20
y = 0,0545x - 0,0011
R² = 0,99979
5
y = 0,3989x - 0,6123
R² = 0,99797
100
3
2
1
0
120
C)
4
-20
-1
0
20
0,5
0
0
20
40
60
fmol/µl
100
120
VSEADSSNADWVTK
80
100
120
Ra o Light to Global Standards
Ra o Light to Global Standards
y = 0,0152x - 0,0453
R² = 0,99298
1
-0,5
80
0,4
2
-20
60
fmol/µl
D)
YGLVTYATYPK
1,5
40
y = 0,0034x - 0,0016
R² = 0,99933
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
-20-0,05 0
20
40
60
fmol/µl
80
100
120
SUPPLEMENTAL FIGURE 3 (.pdf file)
SUPPLEMENTAL FIGURE 4 (.pdf file)
SUPPLEMENTAL FIGURE 5 (.pdf file)
SUPPLEMENTAL FIGURE 6
SUPPLEMENTAL FIGURE 7
SUPPLEMENTAL FIGURE 8