False Positive Rate of Protein Target Discovery Using A

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 N1- normalized 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 N1- normalized 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%.

4 Table S-1. Peptide and protein hits identified in the three control experiments. Peptide Sequence Protein Control 1A vs. 1B ELMQQIENFEK YGL253W/P04807 IVDMSTSK1,2 YJR047C/P19211 EYLDKM(ox)GFK YOR074C/P06785 MTPSGHNWVSGQGAGPR YLR249W/P16521 MVLIGPPGAGK YDR226W/P07170

Control 2A vs. 2B GPYDNFMQK2 YMR085W/Q6B308 TVTELVM(ox)NAFAK YOL008W/Q08058

Control 3A vs. 3B VIEQPITSETAMK YOL127W/P04456

1Only assayed in one control experiment. 2 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).

5 Table S-2. Summary of N1 normalization values and standard deviations for non- methionine containing peptides in the control and manassantin A binding experiments performed in this work.

Experiment 113 114 115 116 117 118 119 121 Control 1A 1.29 1.36 1.00 1.34 0.71 0.52 0.83 0.52 (0.52) (0.54) (0.42) (0.50) (0.40) (0.37) (0.47) (0.41) Control 1B 1.21 1.11 0.93 1.07 0.69 0.67 0.95 1.10 (0.56) (0.53) (0.44) (0.47) (0.43) (0.38) (0.42) (0.52) Control 2A 1.47 1.37 1.02 1.07 0.68 0.60 1.08 0.65 (0.49) (0.46) (0.39) (0.36) (0.30) (0.31) (0.44) (0.37) Control 2B 1.33 1.08 0.92 1.14 0.69 0.76 1.02 1.05 (0.52) (0.43) (0.33) (0.38) (0.31) (0.35) (0.36) (0.43) Control 3A 1.47 1.33 1.00 1.09 0.67 0.57 1.08 0.64 (0.46) (0.43) (0.34) (0.37) (0.29) (0.30) (0.40) (0.39) Control 3B 1.36 1.08 0.91 1.16 0.70 0.69 1.04 1.00 (0.48) (0.35) (0.30) (0.34) (0.28) (0.30) (0.32) (0.38) ManA (-) 1 0.43 1.20 0.94 1.14 0.94 1.04 1.13 1.17 (0.22) (0.17) (0.14) (0.13) (0.15) (0.13) (0.20) (0.17) ManA (+) 1 0.78 1.04 1.03 1.02 0.98 1.06 0.98 1.10 (0.24) (0.14) (0.13) (0.12) (0.13) (0.15) (0.18) (0.15) ManA (-) 2 0.41 1.22 0.92 1.23 0.87 1.11 1.04 1.20 (0.18) (0.16) (0.14) (0.14) (0.14) (0.13) (0.15) (0.16) ManA (+) 2 0.57 1.04 1.10 1.07 1.02 1.15 0.99 1.06 (0.19) (0.14) (0.13) (0.12) (0.14) (0.15) (0.15) (0.14)

6 Table S-3. Peptide and protein hits identified using the minus ligand data sets generated in the manassantin A binding experiment.

Peptide Sequence Protein GVLM(ox)YGPPGTGK1 YDL126C/P25694 SM(ox)VEEAEASGR1 YGR155W/P32582 ETM(ox)YSVVQK1 YDL185W/P17255 YIAAPSGSVM(ox)DK1 YMR120C/P38009 ELYGNIVMSGGTTMFPGIAER1 YFL039C/P60010 SAIGEGMTR YBR127C/P16140 NAGMYGER1 YLR027C/P23542 ILMVGLDGAGK 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 Protein Replicate 1 APSLFGGM(ox)GQTGPK1 YBR106W/P38264 GYIPLQAPVMM(ox)NK1 YDR023W/P07284 YDSASDNVYM(ox)NAEQEEK1 YHR179W/Q03558 IYEVEGM(ox)R2 YLR044C/P06169 LSFQDLAFAIMR2,3 YCR053W/P16120 SDVM(ox)SVDIDKK2 YMR116C/P38011 SAIGEGMTR2,3 YBR127C/P16140

Replicate 2 NVEVVALNDPFISNDYSAYMFK1 YJR009C/P00358 SKLGANAILGVSM(ox)AAAR1 YHR174W/P00925 EQAIIDMAK1 YDR368W/Q12458 VKADRDESSPYAAM(ox)LAAQDVAAK1,3 YCR031C/P06367 GLPGTHDMK2,3 YGR205W/P42938 AQNPMR2,3 YGR085C/Q3E757

1Only assayed in one replicate. 2Hit 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).