SUPPLEMENTAL MATERIAL:
False Positive Rate of Protein Target Discovery using a
Covalent Modification- and Mass Spectrometry-Based Proteomics Platform
Erin C. Strickland, M. Ariel Geer, Jiyong Hong, and Michael C. Fitzgerald*
Department of Chemistry, Duke University, Durham, North Carolina 27708
*Corresponding Author
CONTENTS:
The Supplemental Material includes Supplemental Text and Tables S-1-4. The
Supplemental Text includes detailed information about the iTRAQ® normalization
procedure and the difference analysis used in this work. Table S-1 summarizes the hit
peptides and proteins identified in the control experiment. Table S-2 summarizes the
N1 normalization values and standard deviations for the non-methionine containing
peptides identified in the control and manassantin A binding experiments. Table S-3
summarizes the false positives that arise from comparison of the (-) ligand technical
replicates analyzed in the manassantin A binding experiment. Table S-4 summarizes
the potential hit peptides and proteins identified from comparison of the (-) and (+)
ligand samples in the manassantin A binding experiment.
1
Supplemental Text
iTRAQ Normalization. The eight iTRAQ® reporter ion intensity values extracted
from each product-ion mass spectrum were averaged, and the raw intensity value of
each reporter ion in the product-ion mass spectra was divided by the average intensity
value obtained for that spectra. This generated a set of N1-normalized iTRAQ® reporter
ion values for each peptide identification in the LC-MS/MS analyses. The N1-normalized
iTRAQ® reporter ion values for all the non-methionine-containing peptides identified in
each iTRAQ® labeled (non-enhanced) sample were used to generate a normalization
factor for each reporter ion. This normalization factor was obtained by averaging the N1normalized reporter ion intensity values for the non-methionine-containing peptides
identified in a given iTRAQ® labeled sample. For example, all the N1-normalized values
obtained for the 113 reporter ion were averaged, all the N1-normalized values obtained
for the 114 reporter ion were averaged, etc. The non-methionine-containing peptides
used to generate the normalization values were those that were not missing reporter ion
intensities (or in the case of the control experiment those in which the raw iTRAQ ®
reporter ion intensities summed up to >1,000), that had a Spectrum Mill identification
score of greater than 8 (or in the case of the manassantin A binding experiment were
identified with high or medium confidence in Proteome Discoverer). Ultimately, the N1normalized reporter ion intensity values of the methionine-containing peptides were
divided by the appropriate normalization factor to generate the normalized iTRAQ ®
reporter ion intensity values reported in this work. In cases where multiple product-ion
spectra were obtained for a given peptide (e.g., multiple product ion mass spectra were
collected in a single LC-MS/MS run or in multiple LC-MS/MS runs), the normalized
2
intensities for each iTRAQ® reporter ion were averaged to obtain a single set of
chemical denaturation data for the peptide. This averaging step was performed using
an AWK script developed in-house.
Hit Peptide Selection. In the control experiment, all the chemical denaturation
data sets were visually inspected to assign a transition midpoint, and hit peptides were
identified as those that had transition midpoint shifts of > 0.5 M.
Transition midpoints
were assigned using a set of rules that assumed the data had a specific structure (e.g.,
a single unfolding/folding transition with a pre- and post-transition baseline or no
transition).
Prior to visual inspection, the distributions of the normalized iTRAQ®
reporter ion intensities obtained at the highest and lowest denaturant concentrations in
each ligand binding experiment were used to determine the normalized reporter ion
intensity value that best separated the pre- and post-transition baselines in the SPROX
data. This normalized reporter ion intensity was 1.0 in all the experiments. The transition
midpoint was assigned to be the chemical denaturant concentration at which the
normalized iTRAQ® intensity values transitioned from the pre- to the post-transition
baseline. If there was a normalized iTRAQ® reporter ion intensity value of 1.0 ± 0.1 at
the transition, then the denaturant concentration corresponding to that iTRAQ ® reporter
ion was taken as the midpoint. Otherwise the denaturant concentrations corresponding
to the iTRAQ® reporter ions flanking the transition were averaged and this average
value was assigned as the transition midpoint. In our visual inspection, peptides with
SPROX data in which more than one data point was inconsistent with the structure of a
SPROX data set (e.g., in the case of a non-oxidized methionine-containing peptide a
data point that was < 1.0 or >1.0 in the pre- or post-transition baselines, respectively)
3
were classified as uninterpretable and not included in the analysis. In cases where only
a single normalized reporter ion intensity was inconsistent with the expected pre- and
post-transition baseline values, the outlying value was removed from the data set and
the remaining seven values were used in the visual inspection to assign the transition
midpoint.
Peptides with shifted transition midpoints in the manassantin A binding
experiment were initially identified by examining the differences between the normalized
iTRAQ® reporter ion intensities observed for a given methionine-containing peptide in
the samples being compared (e.g., the two (-) ligand samples, or the (-) and (+) ligand
samples in the manassantin A binding experiment). Normalized iTRAQ® reporter ion
differences greater than 0.2 or less than -0.2 were deemed significant based on the
distribution of all the reporter ion differences, which revealed that 69 – 76% of the
differences from all the reporter ions from all the peptides were within 0.2 of the average
difference of approximately 0.
The chemical denaturation data sets of the peptides identified in the difference
analysis used in the manassantin A binding experiments were then visually inspected,
as described above, to assign a transition midpoint and identify those that had transition
midpoints shifts of >0.5 M.
Using the difference analysis prior to visual inspection
reduced the number of chemical denaturation data sets that had to be visually inspected
by about 50%.
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Table S-1. Peptide and protein hits identified in the three control experiments.
Peptide Sequence
Control 1A vs. 1B
ELMQQIENFEK
IVDMSTSK1,2
EYLDKM(ox)GFK
MTPSGHNWVSGQGAGPR
MVLIGPPGAGK
Protein
YGL253W/P04807
YJR047C/P19211
YOR074C/P06785
YLR249W/P16521
YDR226W/P07170
Control 2A vs. 2B
GPYDNFMQK2
TVTELVM(ox)NAFAK
YMR085W/Q6B308
YOL008W/Q08058
Control 3A vs. 3B
VIEQPITSETAMK
YOL127W/P04456
1Only
assayed in one control experiment.
Hit peptide with at least one product ion mass spectrum from low purity ions (i.e.,
<50% pure, which included the bottom 19% of the data from all the peptides).
2
5
Table S-2. Summary of N1 normalization values and standard deviations for nonmethionine containing peptides in the control and manassantin A binding experiments
performed in this work.
Experiment
Control 1A
Control 1B
Control 2A
Control 2B
Control 3A
Control 3B
ManA (-) 1
ManA (+) 1
ManA (-) 2
ManA (+) 2
113
1.29
(0.52)
1.21
(0.56)
1.47
(0.49)
1.33
(0.52)
1.47
(0.46)
1.36
(0.48)
0.43
(0.22)
0.78
(0.24)
0.41
(0.18)
0.57
(0.19)
114
1.36
(0.54)
1.11
(0.53)
1.37
(0.46)
1.08
(0.43)
1.33
(0.43)
1.08
(0.35)
1.20
(0.17)
1.04
(0.14)
1.22
(0.16)
1.04
(0.14)
115
1.00
(0.42)
0.93
(0.44)
1.02
(0.39)
0.92
(0.33)
1.00
(0.34)
0.91
(0.30)
0.94
(0.14)
1.03
(0.13)
0.92
(0.14)
1.10
(0.13)
116
1.34
(0.50)
1.07
(0.47)
1.07
(0.36)
1.14
(0.38)
1.09
(0.37)
1.16
(0.34)
1.14
(0.13)
1.02
(0.12)
1.23
(0.14)
1.07
(0.12)
117
0.71
(0.40)
0.69
(0.43)
0.68
(0.30)
0.69
(0.31)
0.67
(0.29)
0.70
(0.28)
0.94
(0.15)
0.98
(0.13)
0.87
(0.14)
1.02
(0.14)
118
0.52
(0.37)
0.67
(0.38)
0.60
(0.31)
0.76
(0.35)
0.57
(0.30)
0.69
(0.30)
1.04
(0.13)
1.06
(0.15)
1.11
(0.13)
1.15
(0.15)
119
0.83
(0.47)
0.95
(0.42)
1.08
(0.44)
1.02
(0.36)
1.08
(0.40)
1.04
(0.32)
1.13
(0.20)
0.98
(0.18)
1.04
(0.15)
0.99
(0.15)
121
0.52
(0.41)
1.10
(0.52)
0.65
(0.37)
1.05
(0.43)
0.64
(0.39)
1.00
(0.38)
1.17
(0.17)
1.10
(0.15)
1.20
(0.16)
1.06
(0.14)
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Table S-3. Peptide and protein hits identified using the minus ligand data sets
generated in the manassantin A binding experiment.
Peptide Sequence
GVLM(ox)YGPPGTGK1
SM(ox)VEEAEASGR1
ETM(ox)YSVVQK1
YIAAPSGSVM(ox)DK1
ELYGNIVMSGGTTMFPGIAER1
SAIGEGMTR
NAGMYGER1
ILMVGLDGAGK
Protein
YDL126C/P25694
YGR155W/P32582
YDL185W/P17255
YMR120C/P38009
YFL039C/P60010
YBR127C/P16140
YLR027C/P23542
YDL137W/P19146
1Hit
peptide with at least one product ion mass spectrum from low purity ions (i.e., <70%
pure, which included the bottom 22% of the data from all the peptides).
7
Table S-4. Peptide and protein hits identified in the two technical replicates of the
manassantin A binding experiment.
Peptide Sequence
Replicate 1
APSLFGGM(ox)GQTGPK1
GYIPLQAPVMM(ox)NK1
YDSASDNVYM(ox)NAEQEEK1
IYEVEGM(ox)R2
LSFQDLAFAIMR2,3
SDVM(ox)SVDIDKK2
SAIGEGMTR2,3
Protein
YBR106W/P38264
YDR023W/P07284
YHR179W/Q03558
YLR044C/P06169
YCR053W/P16120
YMR116C/P38011
YBR127C/P16140
Replicate 2
NVEVVALNDPFISNDYSAYMFK1
SKLGANAILGVSM(ox)AAAR1
EQAIIDMAK1
VKADRDESSPYAAM(ox)LAAQDVAAK1,3
GLPGTHDMK2,3
AQNPMR2,3
YJR009C/P00358
YHR174W/P00925
YDR368W/Q12458
YCR031C/P06367
YGR205W/P42938
YGR085C/Q3E757
1Only
assayed in one replicate.
was eliminated in the technical replicate.
3Hit peptide with at least one product ion mass spectrum from low purity ions (i.e., <70%
pure, which included the bottom 22% of the data from all the peptides).
2Hit
8