Electronic Supplementary Material for:
Characterization of the Saccharomyces cerevisiae ATP-Interactome using
the iTRAQ-SPROX Technique
M. Ariel Geer and Michael C. Fitzgerald
Department of Chemistry, Duke University, Durham, NC 27708
CONTENTS:
The Supplementary Material includes Supplementary Text, one figure
(Figure S1) and three tables (Tables S1-S3), two of which (Tables S1 and S2)
are provided as Excel files.
The Supplementary Text includes detailed
information about the iTRAQ-SPROX data analysis. Figure S1 shows the
distribution of the normalized iTRAQ reporter ion differences observed between
the minus and plus AMP-PNP samples for all of the methionine-containing
peptides. Table S1 summarizes the chemical denaturation data obtained for the
methionine-containing peptides assayed in iTRAQ-SPROX Experiments 1 and 2.
Table S2 lists the AMP-PNP-sensitive protein hits identified in this work and in
the SILAC-SPROX experiment described in Tran, D. et al., Mol. Cell. Proteomics,
13, (2014) 1800-1813. Table S3 summarizes the N2-normalization factors used
in the quantitative bottom-up proteomics analyses performed in iTRAQ-SPROX
Experiments 1 and 2.
1
Supplementary Text
iTRAQ-SPROX Data Analysis.
The iTRAQ reporter ion intensities
measured in the LC-MS/MS analyses were normalized as described in Strickland,
E. C. et al., Nat. Protoc., 8 (2013) 148-161. Briefly, the 8 iTRAQ reporter ion
intensities in each product ion mass spectra were subjected to two
normalizations. In the first and so-called N1 normalization, the 8 iTRAQ reporter
ion intensities in each product ion mass spectra were averaged and the raw
intensity of each reporter ion in the product ion mass spectra was divided by the
average value. In the second and so-called N2 normalization, the N1-normalized
values for all the non-methionine-containing peptides were averaged within each
reporter ion, to generate so-called N2-normalization factors for the methioninecontaining peptides in each experiment. Summarized in Table S3 are the N2normalization factors generated for each reporter ion (and corresponding
denaturant concentration) in iTRAQ-SPROX Experiments 1 and 2. Ultimately,
N1-normalized values for each methionine-containing peptide were divided by
the corresponding N2-normalization factors to obtain the final normalized reporter
ion intensities reported in Table S1. If multiple product ion spectra were obtained
for a given peptide (e.g., it was identified multiple times in the LC-MS/MS runs for
that sample) the normalized reporter ion intensity values were averaged using
Runcompare (an AWK script developed in-house).
2
The distribution of N2-normalized reporter ion intensity values for the tag
corresponding to the high and low Urea concentrations (6 and 0 M, respectively)
were plotted for the methionine-containing peptides in each experiment. The N2normalized reporter intensity value at which the two distributions crossed was
used to separate the pre- and post-transition baselines of the denaturation
curves. The GdmCl concentration corresponding to this N2-normalized reporter
intensity value, which was 1.0 in both Experiments 1 and 2, was taken as the
transition midpoint (i.e., C1/2 value) of a chemical denaturation curve.
Typically,
the GdmCl concentration values flanking the transition were averaged, and the
average value was taken as the C1/2 value. If there was a normalized reporter ion
intensity of 1.0 ± 0.1 at the transition, then that GdmCl concentration was
assigned as the midpoint. If multiple normalized reporter ion intensity values of
1.0 ± 0.1 existed at the transition, the average of all of these GdmCl
concentration values was assigned as the midpoint.
For hit selection, the chemical denaturation data sets obtained for a
specific methionine-containing peptide probe in the presence and in the absence
of AMP-PNP were visually inspected to ascertain which C1/2 values were shifted
in the presence of ligand. Significant C1/2 value shifts were taken as those that
resulted from N2 normalized reporter ion values being different at two or more
denaturant concentrations.
Hit peptides were generally those with C1/2
values >1.0 M, but the minimum detectable C1/2 varied slightly as it was
dependent on the denaturant spacing at the transition midpoint.
Chemical
denaturation data sets in which more than one normalized reporter ion intensity
3
was not >1.0 or <1.0 for methionine-containing peptides (or not <1.0 or >1.0 for
oxidized Met-containing peptides) in the pre- and post-transition baselines,
respectively, were classified as “poor quality” and not used for hit selection.
When only a single outlying value existed, the value was removed from the data
set and the remaining 7 values were used for denaturation curve construction
and transition midpoint assignment.
4
Figure S1. Distribution of the N2-normalized iTRAQ reporter ion value
differences observed between the minus and plus AMP-PNP samples generated
in iTRAQ-SPROX Experiments 1 and 2. The solid and dotted arrows point to the
iTRAQ reporter ion value differences at the 22nd and 78th percentiles, respectively.
1400
22nd Percentile
78th Percentile
Frequency
1200
1000
800
600
400
200
0
-1.2
-1
-0.8 -0.6 -0.4 -0.2
0
0.2
0.4
0.6
0.8
1
Normalized iTRAQ Reporter Ion Value Difference
5
Table S3: Summary of the N2 normalization factors used in iTRAQ-SPROX
Experiments 1 and 2. The values represent the average and standard deviation
(shown in parentheses) of the N1 values determined for each reporter ion using
the non-methionine-containing peptides at the denaturant concentrations
corresponding to each iTRAQ reporter ion.
Experiment
113
114
115
116
117
118
119
121
(-) Exp. 1
0.91
(0.18)
1.01
(0.17)
1.08
(0.26)
1.24
(0.28)
0.75
(0.17)
0.96
(0.18)
0.62
(0.28)
1.31
(0.25)
0.76
(0.14)
0.48
(0.09)
1.38
(0.22)
0.52
(0.21)
1.22
(0.21)
1.11
(0.18)
1.50
(0.27)
1.03
(0.16)
1.27
(0.15)
1.18
(0.16)
1.03
(0.23)
0.45
(0.24)
0.80
(0.13)
0.82
(0.15)
0.64
(0.13)
1.32
(0.22)
1.28
(0.21)
1.30
(0.17)
1.04
(0.24)
1.23
(0.21)
1.01
(0.15)
1.14
(0.17)
0.72
(0.21)
0.91
(0.16)
(+) Exp.1
(-) Exp. 2
(+) Exp. 2
6