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Figure 1. Combining TMT labeling with TiO2-based phosphopeptide enrichment

workflow.

(A) Schematic representation of the workflow. TMT-labeled phosphopeptides from both

methods were mixed in equal amounts and analyzed by LC/MS/MS. (B) Distribution of reporter

ion intensities of identified phosphopeptides. TMT quantitation was performed only when the

signals were detected in all 6 TMT channels. The box itself spans the interquartile range. The

whiskers represent 5% and 95% quantiles. The thick horizontal line in each box indicates the

median. (C) Relationship between reporter ion intensity ratios and the number of

phosphorylations or acidic residues (Glu, Asp) on each phosphopeptide. The monophosphorylated peptides were harder to capture in the TMT-TiO2 workflow. The box itself

spans the interquartile range. The whiskers represent 5% and 95% quantiles. The thick

horizontal line in each box indicates the median. (D) Frequency distribution of relative standard

deviation (RSD) of identified phosphopeptide reporter ion intensities.

Figure 2. pH optimization of StageTip-based solid-phase TMT reaction.

(A) Peak area ratio of a synthetic phosphopeptide, YLpSFTPPEK. The peptide was

categorized into three classes according to the number of TMT-tags. Not labeled: The peptide

with no TMT-tag. Partially labeled: The peptide with a TMT-tag, but either the lysine side chain

or peptide N-terminus is not labeled. Fully labeled: The peptide completely labeled with TMT.

(B) Extracted ion chromatogram of synthetic phosphopeptide in each category.

Figure 3. Sorbent optimization for StageTip-based solid-phase TMT labeling.

(A) Screening of reversed-phase particles for solid-phase labeling. Each sorbent was

evaluated in duplicate experiments. The sum of the phosphopeptide peak area is shown. Not

labeled: The peptide with no TMT-tag. Partially labeled: The peptide with a TMT-tag but either

the lysine side chain or peptide N-terminus is not labeled. Fully labeled: The peptide

completely labeled with TMT. (B) GRAVY index distribution of identified phosphopeptides

labeled with or without TMT from 5 different sorbents. (C) Phosphopeptide ID numbers from

solid-phase labeling using different sorbents. Error bars show the standard deviation (n = 3).

Figure 4. Ion-pairing reagent optimization for StageTip-based solid-phase TMT labeling.

(A) Comparison of distributions of reporter ion intensity ratios between solution-phase and

solid-phase labeling method with each ion-pairing reagent (n = 5, average). The box itself

spans the interquartile range. The whiskers represent 5% and 95% quantiles. The thick

horizontal line in each box indicates the median. (B-D) Relationship between reporter ion

intensity ratio of phosphopeptides and their elution ACN composition in gradient analysis. (B)

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Comparison between solid-phase TMT labeling with TFA and solution-phase labeling. (C)

Comparison between solid-phase TMT labeling with HFBA and solution-phase labeling. (D)

Comparison between solid-phase TMT labeling with HFBA and solid-phase TMT labeling with

TFA. The box itself spans the interquartile range. The whiskers represent 5% and 95%

quantiles. The thick horizontal line in each box indicates the median.

Figure 5. Differential analysis of selumetinib-treated HeLa phosphoproteomes.

(A) Schematic representation of the workflow. Control and selumetinib-treated HeLa cells were

stimulated with EGF (15 min) and collected. After protein digestion, phosphopeptide

enrichment and solid-phase TMT labeling, samples were analyzed in triplicate LC/MS/MS runs.

(B) Phosphoproteome differential analysis. The plot shows the intensity ratio and the p-value

of the identified phosphopeptides (n = 7524). 305 and 165 phosphopeptides showed

significant down- and up-regulation, respectively (highlighted in blue/red: S0 = 0.05 and FDR

= 0.01, n = 5). (C) The result of Kinase-Set Enrichment Analysis (KSEA). All identified

phosphopeptides (n=7524) were utilized for the analysis. The z-scores of kinases with four

and more substrates are shown. A negative score corresponds to a decrease of the kinase

activity. The kinases with |z-score| > 1 are colored blue/red.

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Supporting Information

Nano-scale

solid-phase

phosphoproteomics

isobaric

labeling

for

multiplexed

quantitative

Kosuke Ogata1, Chia-Feng Tsai1, Yasushi Ishihama1,2,*

1) Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical

Sciences, Kyoto University, Kyoto 606–8501, Japan

2) Laboratory of Clinical and Analytical Chemistry, National Institute of Biomedical

Innovation, Health and Nutrition, Ibaraki, Osaka, 567-0085, Japan.

*Corresponding author:

Tel: +81-75-753-4555, Fax: +81-75-753-4601, E-mail: yishiham@pharm.kyoto-u.ac.jp

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Table of content

Figure S1. Recovery of TMT-labeled phosphopeptides in TiO2-based phosphopeptide

enrichment.

Figure S2. Photograph of a StageTip used for solid-phase TMT labeling.

Figure S3. Applicable range of peptide amounts in the optimized solid-phase TMT labeling

method.

Figure S4. Labeling efficiency of solid-phase TMTpro labeling.

Figure S5. STRING network analysis of proteins having decreased phosphopeptides upon

selumetinib treatment.

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Figure S1. Recovery of TMT-labeled phosphopeptides in TiO2-based phosphopeptide

enrichment.

Analysis of the flow-through fraction from TiO2-based phosphopeptide enrichment. Tandem

phosphopeptide enrichment experiments were performed to analyze phosphopeptides in the

flow-through fraction. The peak areas of identified phosphopeptides were compared between

eluted and flow-through fractions.

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Figure S2. Photograph of a StageTip used for solid-phase TMT labeling.

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Figure S3. Applicable range of peptide amounts in the optimized solid-phase TMT labeling

method. Phosphopeptides were enriched by TiO2 chromatography from HeLa digests, and

were divided into the amounts indicated in (A). After labeling with TMT10plex reagents by the

optimized solid-phase TMT labeling method. The indicated amounts of peptides were mixed

and analyzed by LC/MS/MS. An Orbitrap Exploris 480 mass spectrometer equipped with

FAIMS pro and an UltiMate 3000 RSLCnano pump (Thermo Fisher Scientific) was employed

for the analysis. (B) The bar chart shows the number of quantifiable phosphopeptides.

Phosphopeptides from 1 μg with peaks detected in all ten reporter ion channels were

considered as quantifiable. Phosphopeptides from 5 μg, 10 μg, and 50 μg with eight reporter

ions (127C, 128N, 128C, 129N, 129C, 130N, 130C, and 131), six reporter ions (128C, 129N,

129C, 130N, 130C, and 131), and four reporter ions (129C, 130N,130C, and 131), respectively

were considered quantifiable. (C) The box plot shows the upper quartile, median, and lower

quartile for the TMT reporter ion intensity ratios to channel 131. Outliers were identified using

box-plot statistics (threshold: 1.5 x the interquartile range (IQR)). Dashed lines represent

expected ratios. The phosphopeptides quantifiable from 1 μg were plotted.

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Figure S4. Labeling efficiency of solid-phase TMTpro labeling.

The bar graph showed the peak area ratio of TMTpro-labeled phosphopeptides from HeLa

cell digests using the optimized solid-phase TMT labeling protocol. The samples were

prepared in triplicate. The peptide was categorized into three classes according to the number

of TMT-tags. Not labeled: The peptide with no TMT-tag. Partially labeled: The peptide with a

TMT-tag but either the lysine side chain or peptide N-terminus is not labeled. Fully labeled:

The peptide completely labeled with TMT. Over 98% of the phosphopeptide peak area was

due to fully labeled peptides.

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Figure S5. STRING network analysis of proteins having decreased phosphopeptides

upon selumetinib treatment.

STRING network (v11.0) of high-confidence interactions (minimum confidence score of 0.700)

among proteins with decreased phosphosites upon selumetinib treatment. Colors on nodes

indicate the clusters based on STRING MCL clustering (inflation parameter: 1.6).

Disconnected nodes are not shown.

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