Supporting Information for “A High-throughput Targeted Proteomic Approach for Comprehensive

Profiling of Methylglyoxal-induced Perturbations of Human Kinome”

Weili Miao, Yongsheng Xiao, Lei Guo, Xiaogang Jiang, Ming Huang and Yinsheng Wang*

Correspondence author: Yinsheng Wang. Telephone: (951)827-2700; E-mail:

[email protected]

(Anal. Chem., 2016)

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Materials and Methods

The desthiobiotinylated nucleotide affinity probes were prepared according to previously published procedures.1,2

Labeling of ATP-binding proteins, tryptic digestion, and affinity purification of desthiobiotin-C3-labeled peptides

Approximately 2×107 cells were harvested, washed with cold PBS for three times, and lysed in a 1 mL lysis buffer, which contained 0.7% CHAPS, 50 mM HEPES (pH 7.4), 0.5 mM EDTA, 100 mM NaCl, and 10 µL (1:100) protease inhibitor cocktail on ice for 30 min. The cell lysates were centrifuged at 16,000g at 4°C for 30 min and the resulting supernatants were collected. Endogenous nucleotides in the resulting protein extract were removed by gel filtration using a NAP-25 column (Amersham Biosciences). Cell lysates were subsequently eluted into a buffer containing 50 mM HEPES (pH 7.4), 75 mM NaCl, and 5% glycerol. The amounts of proteins in the lysates were quantified using Quick Start Bradford Protein Assay (Bio-Rad). Prior to the

labeling reaction, MgCl2, MnCl2, and CaCl2 were added to the concentrated cell lysate until their final concentrations reached 50, 5 and 5 mM, respectively.

Approximately 1.0 mg cell lysate in a 1.0-mL solution was treated with the light desthiobiotin-C3-ATP affinity probe (Figure 1a) at a final concentration of 100 µM. Labeling reactions were conducted at room temperature with gentle shaking for 2.5 h. After the reaction, the remaining probes in the cell lysates were removed by buffer exchange with 50 mM

NH4HCO3 (pH 8.5) using Amicon Ultra-4 filters (10,000 NMWL, Millipore). After addition of 8 M urea for protein denaturation, dithiothreitol and iodoacetamide for cysteine reduction and alkylation, the labeled proteins were digested with modified sequencing-grade trypsin (Roche

Applied Science) at an enzyme/substrate ratio of 1:100 in 50 mM NH4HCO3 (pH 8.5) at 37°C overnight. The peptide mixture was subsequently dried in a Speed-vac and redissolved in 1.0 mL PBS buffer (100 mM potassium phosphate and 0.15 M NaCl, pH 7.5), to which solution was subsequently added 300 µL avidin-agarose resin (Sigma-Aldrich). The mixture was then incubated at room temperature for 1 h with gentle shaking. The agarose resin was washed

sequentially with 3 mL PBS buffer and 3 mL H2O to remove unbound peptides, and the

desthiobiotin-conjugated peptides were subsequently eluted with 1% TFA in CH3CN/H2O (7:3, v/v) at 75°C. The resulting enriched peptide samples were desalted by employing OMIX C18

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pipet tips (Agilent Technologies) and analyzed on an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) for discovery-mode analysis.

LC-MS/MS analysis on an LTQ-Orbitrap Velos mass spectrometer

Probe pull-down samples were analyzed by on-line 2D-LC-MS/MS and synthetic peptides were analyzed by 1D-LC-MS/MS on an LTQ-Orbitrap Velos mass spectrometer equipped with a nanoelectrospray ionization source. The fully automated 8-cycle on-line two-dimensional LC- MS/MS was set up as described3 with minor modifications. Briefly, samples were automatically loaded from a 48-well microplate autosampler using an EASY-nLC II system (Thermo Fisher Scientific) at 3 µL/min onto a biphasic precolumn (150 µm i.d.) comprised of a 3.5-cm column packed with 5 µm C18 120 Å reversed-phase material (ReproSil-Pur 120 C18-AQ, Dr. Maisch) for 1D-LC-MS/MS, and packed for another 3.5-cm with Luna 5 µm SCX 100 Å strong cation- exchange resin (Phenomenex, Torrance, CA) for on-line 2D-LC-MS/MS. The biphasic trapping column was connected to a 20-cm fused-silica analytical column (PicoTip Emitter, New Objective, 75 µm i.d.) with 3 µm C18 beads (ReproSil-Pur 120 C18-AQ, Dr. Maisch). For on- line 2D separation, ammonium acetate at concentrations of 0, 5, 10, 20, 50, 100, 200, 500 and 1000 mM were then sequentially injected using a 48-well autosampler from the sample vial to elute bound peptides from the precolumn to the analytical column with reversed-phase separation. The peptides were then separated with a 180-min linear gradient of 2-35% acetonitrile in 0.1% formic acid and at a flow rate of 250 nL/min. The LTQ-Orbitrap Velos was operated in a data-dependent scan mode. Full-scan mass spectra were acquired using the Orbitrap mass analyzer with a resolution of 60,000 with lock mass option enabled for the ion of m/z 445.120025.4 Up to 20 most abundant ions found in MS with charge state of 2 and above were sequentially isolated and sequenced in the linear ion-trap with a normalized collision energy of 35, an activation Q value of 0.25, and an activation time of 10 ms.

Construction of MRM spectral library for the kinase peptides

Skyline (version 3.5)5 was used to generate the spectral libraries for kinome peptides. All raw files obtained from discovery-mode proteomic analysis on the LTQ-Orbitrap Velos were searched by using MaxQuant (Version 1.3.0.5)6 against the human IPI protein database version 3.68 (87,062 entries). Initial precursor mass tolerance of 10 ppm and fragment mass deviation of 0.8 Th were set as the search criteria. The maximum number of miss-cleavages for trypsin was

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set at two per peptide. Cysteine carbamidomethylation was considered as a fixed modification, whereas methionine oxidation and light desthiobiotinylation of lysine (+281.17394 Da, respectively) were included as variable modifications. The resulting MS/MS files were then imported into Skyline and filtered with a threshold score of 0.95. An interactive Skyline spectral library file containing tandem mass spectra of all desthiobiotin-C3-labeled kinase peptides was then generated.

Retention time (RT) extraction and iRT calculation for kinase peptides

To calculate the iRT score7 for each kinase peptide in the MRM kinome library, 10 peptides derived from the tryptic digestion mixture of bovine serum albumin (BSA) were selected to constitute the reference peptides for a new iRT scale. iRT values of these 10 standard peptides from BSA were calculated using their empirically measured RT from shotgun LC-MS/MS analysis by setting iRT scores of peptides AEFVEVTK and DAFLGSFLYEYSR as 0 and 100, respectively. The BSA peptide mixture was then added to desthiobiotin-C3-labeled kinase peptide mixture from the lysates of GM04429, GM00637, HEK293T, GM15876A, DU-145, HCT116, MCF7, U2OS and CEM cells and measured by 1D-LC-MS/MS on the LTQ Orbitrap Velos with a 130-min linear gradient. RTs were extracted for all BSA standard peptides as well as kinase peptides using the Skyline MS1 filtering workflow.8 The transformed iRT values for all the newly identified kinase peptides were calculated based on linear regression of iRT and experimentally measured RT of peptides with previously determined iRT score. With the accumulation of iRT scores for targeted kinase peptides, it is not necessary to include the BSA standard mixture for RT extraction and iRT calculation. Any common kinase peptides that yield a regression with a R2 value of 0.99 or higher were used as standard peptides to calculate the new iRT score for newly identified kinase peptides by Skyline.

LC-MRM Analysis

All LC-MRM experiments were carried out on a TSQ Vantage triple-quadrupole mass spectrometer equipped with a nanoelectrospray ionization source coupled to an EASY-nLC II system (Thermo Scientific). Samples were automatically loaded onto a 4-cm trapping column (150 µm i.d.) packed with 5 µm 120 Å reversed phase C18 material (ReproSil-Pur 120 C18-AQ, Dr. Maisch) at 3 µL/min. The trapping column was coupled to a 20-cm fused silica analytical column (PicoTip Emitter, New Objective, 75 µm i.d.) with 3 µm C18 beads (ReproSil-Pur 120

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C18-AQ, Dr. Maisch). The peptides were then separated with a 130-min linear gradient of 2- 35% acetonitrile in 0.1% formic acid and at a flow rate of 230 nL/min. The spray voltage was 1.9 kV. Q1 and Q3 resolutions were 0.7 Da and the cycle time was 5 s.

To enable automated, multiplexed MRM analyses, we generated an interactive Skyline spectral library file containing tandem mass spectra of all unique desthiobiotin-C3 conjugated kinase peptides from 15 cell line samples and synthetic peptides, along with their iRT values,7 which were acquired from discovery-mode analysis on an LTQ Orbitrap Velos mass spectrometer, using Skyline (version 3.5).5 Collision energies were calculated using a linear equation that was specific to the TSQ Vantage instrument according to the Skyline default setting. Tryptic peptide mixture of BSA was analyzed in unscheduled MRM-mode prior to the analysis of the enriched desthiobiotin-C3 conjugated peptides. The linear predictor of empirical RT from iRT for targeted kinase peptides was then determined by the linear regression of RTs of BSA standard peptides obtained for the current chromatography setup. This linear predictor was rechecked between every five MRM sample analyses by injecting another BSA standard mixture. Three or four pairs of transitions were monitored for each light/heavy desthiobiotin-C3- labeled peptide in quantification experiments. These targeted transitions were monitored in five separate injections for each sample in scheduled MRM mode with a retention time window of 8 min.

All raw files were processed using Skyline (version 3.5) for the generation of extracted-ion chromatograms and peak integration. The targeted peptides were first manually checked to ensure the overlaid chromatographic profiles of multiple fragment ions derived from light and heavy forms of the same peptide. The data were then processed to ensure that the distribution of the relative intensities of multiple transitions associated with the same precursor ion correlates with the theoretical distribution from kinome MS/MS spectral library entry. The sum of peak area from all transitions of light- or heavy-labeled peptides was used for quantification.

Western blot

HEK293T cells were cultured in 75 cm2 cell culture flasks until 40-50% confluency and treated with methylglyoxal. Cells were lysed following the aforementioned procedures. The concentrations of the resulting protein lysates were determined by Bradford Assay (Bio-Rad). The whole cell lysate for each sample (10 μg) was denatured by boiling in Laemmli loading

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buffer and then resolved by using SDS-PAGE. Subsequently, the proteins were transferred onto the nitrocellulose membrane at 4°C overnight. The resulting membrane was blocked with PBS- T (PBS with 0.1% Tween 20) containing 5% milk (Bio-Rad) at 4°C for 6 h. Next, the membrane was incubated with primary antibody at 4°C overnight and then secondary antibody at room temperature for 1 h. After thorough washing with PBS-T buffer, the HRP signals were detected with Pierce ECL Western Blotting Substrate (Thermo).

Real-time PCR

HEK293T cells were seeded in 12-well plates at 50% confluence level. Total RNA was extracted from cells using TRI Reagent (Sigma). Approximately 1 μg RNA was reverse transcribed by employing M-MLV reverse transcriptase (Promega) and an oligo(dT)16 primer. After incubating at 42°C for 60 min, the reverse transcriptase was deactivated by heating at 85°C for 5 min. Quantitative real-time PCR was performed using iQ SYBR Green Supermix kit (Bio- Rad) on a Bio-Rad iCycler system (Bio-Rad), and the running conditions were at 95°C for 3 min and 45 cycles at 95°C for 15 sec, 55°C for 30 sec, and 72°C for 45 sec. The comparative cycle threshold (Ct) method (ΔΔCt) was used for the relative quantification of gene expression by qRT-PCR. Primers are listed in Table S1. The mRNA level of each gene was normalized to that of the internal control (GAPDH).

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Table S1. Sequences for qRT-PCR primers.

Gene Name Forward Primer Reverse Primer EGFR 5’-AAAGTTTGCCAAGGCACGAGT-3’ 5’- AGGACATAACCAGCCACCTCCT-3’ IGF2R 5’-CCATGCAGAACCAGAGCAGAAT-3’ 5’- TCCATTTTTATCCACTGCACACACT-3’ FGFR1 5’-AAGTCGGACGCAACAGAGAAAGAC-3’ 5’- CTTGGAGGCATACTCCACGA-3’ GAPDH 5’- GTGGAGTCCACTGGCGTCTTC-3’ 5’-CTGATGATCTTGAGGCTGTTGTCA-3’

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Table S2. List of kinases included in kinome library.

Kinase Gene IPI Number UniprotKB Kinase Group Description Name dual-specificity tyrosine-(Y)- IPI00000352 Q9Y463 DYRK1B Protein kinase, CMGC phosphorylation regulated kinase 1B IPI00000685 P50613 CDK7 Protein kinase, CMGC cyclin-dependent kinase 7 neurotrophic tyrosine kinase, IPI00000824 P41212 NTRK3 Protein kinase, TK receptor, type 3 megakaryocyte-associated tyrosine IPI00000868 P42679 MATK Protein kinase, TK kinase IPI00000878 P42680 TEC Protein kinase, TK tec protein tyrosine kinase IPI00000879 P42681 TXK Protein kinase, TK TXK tyrosine kinase IPI00000885 P42685 FRK Protein kinase, TK fyn-related kinase mitogen-activated protein kinase IPI00000977 Q16584 MAP3K11 Protein kinase, STE kinase kinase 11 discoidin domain receptor tyrosine IPI00001477 Q08345 DDR1 Protein kinase, TK kinase 1 p21 protein (Cdc42/Rac)-activated IPI00001814 Q9P286 PAK7 Protein kinase, STE kinase 7 serine/threonine kinase 24 (STE20 IPI00002212 Q9Y6E0 STK24 Protein kinase, STE homolog, yeast) IPI00002232 Q7L7X3 TAOK1 Protein kinase, STE TAO kinase 1 3-phosphoinositide dependent protein IPI00002538 O15530 PDPK1 Protein kinase, AGC kinase-1 phosphoinositide-3-kinase, class 2, IPI00002580 O00443 PIK3C2A Lipid kinase alpha polypeptide phosphatidylinositol 4-kinase, IPI00002591 Q9UBF8 PI4KB Lipid kinase catalytic, beta IPI00002773 P52333 JAK3 Protein kinase, TK Janus kinase 3 IPI00002803 Q16512 PKN1 Protein kinase, AGC protein kinase N1 IPI00002804 Q16513 PKN2 Protein kinase, AGC protein kinase N2 IPI00002857 Q16539 MAPK14 Protein kinase, CMGC mitogen-activated protein kinase 14 IPI00003145 P45983 MAPK8 Protein kinase, CMGC mitogen-activated protein kinase 8 phosphoribosyl pyrophosphate IPI00003168 O60256 PRPSAP2 Other kinase synthetase-associated protein 2 neurotrophic tyrosine kinase, IPI00003366 Q16620 NTRK2 Protein kinase, TK receptor, type 2 IPI00003431 Q16659 MAPK6 Protein kinase, CMGC mitogen-activated protein kinase 6 IPI00003479 P28482 MAPK1 Protein kinase, CMGC mitogen-activated protein kinase 1 mitogen-activated protein kinase IPI00003814 P52564 MAP2K6 Protein kinase, STE kinase 6 tight junction protein 2 (zona IPI00003843 Q9UDY2 TJP2 Other kinase occludens 2) serine threonine kinase 39 IPI00004363 Q9UEW8 STK39 Protein kinase, STE (STE20/SPS1 homolog, yeast) discoidin domain receptor tyrosine IPI00004409 Q16832 DDR2 Protein kinase, TK kinase 2 IPI00004420 O15016 TRIM66 Protein kinase, Atypical tripartite motif-containing 66 IPI00004497 P11274 BCR Protein kinase, Atypical breakpoint cluster region IPI00004560 O15075 DCLK1 Protein kinase, CMGC doublecortin-like kinase 1 IPI00004566 Q08881 ITK Protein kinase, TK IL2-inducible T-cell kinase

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Kinase Gene IPI Number UniprotKB Kinase Group Description Name v-crk sarcoma virus CT10 oncogene IPI00004839 P46109 CRKL Protein kinase, CMGC homolog (avian)-like IPI00005014 P11309 PIM1 Protein kinase, CAMK pim-1 oncogene conserved helix-loop-helix IPI00005104 O15111 CHUK Protein kinase, Other ubiquitous kinase IPI00005142 P11362 FGFR1 Protein kinase, TK fibroblast growth factor receptor 1 IPI00005184 O15164 TRIM24 Protein kinase, Atypical tripartite motif-containing 24 protein kinase, AMP-activated, IPI00005367 Q9UGJ0 PRKAG2 Protein kinase, AGC gamma 2 non-catalytic subunit IPI00005487 Q9H477 RBKS Other kinase ribokinase IPI00005722 P36888 FLT3 Protein kinase, TK fms-related tyrosine kinase 3 IPI00005732 P36896 ACVR1B Protein kinase, TKL activin A receptor, type IB transforming growth factor, beta IPI00005733 P36897 TGFBR1 Protein kinase receptor 1 mitogen-activated protein kinase- IPI00005777 Q16644 MAPKAPK3 Protein kinase, CAMK activated protein kinase 3 LATS, large tumor suppressor, IPI00005858 O95835 LATS1 Protein kinase, AGC homolog 1 (Drosophila) IPI00006283 Q9UL54 TAOK2 Protein kinase, STE TAO kinase 2 TNNI3 interacting kinase; fucose-1- IPI00006465 Q59H18 TNNI3K Protein kinase, TKL phosphate guanylyltransferase maternal embryonic leucine zipper IPI00006471 Q14680 MELK Protein kinase, CAMK kinase ribosomal protein S6 kinase, 90kDa, IPI00007123 Q9UK32 RPS6KA6 Protein kinase, AGC polypeptide 6 IPI00007128 P05129 PRKCG Protein kinase, AGC protein kinase C, gamma interleukin-1 receptor-associated IPI00007641 Q9NWZ3 IRAK4 Protein kinase, TKL kinase 4 IPI00007811 P11802 CDK4 Protein kinase, CMGC cyclin-dependent kinase 4 IPI00008290 P54756 EPHA5 Protein kinase, TK EPH receptor A5 IPI00008315 P54762 EPHB1 Protein kinase, TK EPH receptor B1 IPI00008318 P54764 EPHA4 Protein kinase, TK EPH receptor A4 IPI00008883 Q9H093 NUAK2 Protein kinase, CAMK NUAK family, SNF1-like kinase, 2 IPI00009334 Q9BZL6 PRKD2 Protein kinase, AGC protein kinase D2 inositol 1,3,4,5,6-pentakisphosphate IPI00009371 Q9H8X2 IPPK Other kinase 2-kinase IPI00009464 Q01780 EXOSC10 Protein kinase, AGC exosome component 10 phosphatidylinositol-5-phosphate 4- IPI00009688 P48426 PIP4K2A Lipid kinase kinase, type II, alpha IPI00009790 Q01813 PFKP Other kinase phosphofructokinase, platelet serum/glucocorticoid regulated IPI00009887 Q9HBY8 SGK2 Protein kinase, AGC kinase 2 IPI00010067 Q9UPE1 SRPK3 Protein kinase, CMGC SFRS protein kinase 3 IPI00010080 O95747 OXSR1 Protein kinase, STE oxidative-stress responsive 1 IPI00010156 P31152 MAPK4 Protein kinase, CMGC mitogen-activated protein kinase 4 receptor tyrosine kinase-like orphan IPI00010405 Q01973 ROR1 Protein kinase, TK receptor 1 IPI00010466 P05771 PRKCB Protein kinase, AGC protein kinase C, beta IPI00010680 P21802 FGFR2 Protein kinase, TK fibroblast growth factor receptor 2

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Kinase Gene IPI Number UniprotKB Kinase Group Description Name lymphocyte antigen 6 complex, locus IPI00010865 P67870 CSNK2B Protein kinase, CK1 G5B; casein kinase 2, beta polypeptide IPI00011488 Q13043 STK4 Protein kinase, STE serine/threonine kinase 4 IPI00011689 O00418 EEF2K Protein kinase, Atypical eukaryotic elongation factor-2 kinase serine/threonine kinase 25 (STE20 IPI00012093 O00506 STK25 Protein kinase, STE homolog, yeast) non-metastatic cells 3, protein IPI00012315 Q13232 NME3 Other kinase expressed in IPI00012465 Q9BXA7 TSSK1B Protein kinase, CAMK testis-specific serine kinase 1B IPI00012485 Q9BXA6 TSSK6 Protein kinase, CAMK testis-specific serine kinase 6 IPI00012497 P25098 ADRBK1 Protein kinase, AGC adrenergic, beta, receptor kinase 1 protein phosphatase 4 (formerly X), IPI00012833 P60510 PPP4C Protein kinase catalytic subunit v-akt murine thymoma viral IPI00012866 P31749 AKT1 Protein kinase, AGC oncogene homolog 1 v-akt murine thymoma viral IPI00012870 P31751 AKT2 Protein kinase, AGC oncogene homolog 2 IPI00012885 Q05397 PTK2 Protein kinase, TK PTK2 protein tyrosine kinase 2 phosphorylase kinase, gamma 2 IPI00012891 P15735 PHKG2 Protein kinase, CAMK (testis) 2,4-dienoyl CoA reductase 2, IPI00012972 Q9NUI1 DECR2 Other kinase peroxisomal IPI00013212 P41240 CSK Protein kinase, TK c-src tyrosine kinase IPI00013219 Q13418 ILK Protein kinase, TKL integrin-linked kinase similar to hCG1820375; PRP4 pre- IPI00013721 Q13523 PRPF4B Other kinase mRNA processing factor 4 homolog B (yeast) IPI00013735 Q9NYY8 FASTKD2 Protein kinase, Other FAST kinase domains 2 receptor (TNFRSF)-interacting IPI00013773 Q13546 RIPK1 Protein kinase, TKL serine-threonine kinase 1 deoxythymidylate kinase (thymidylate kinase); similar to IPI00013862 P23919 DTYMK Other kinase Deoxythymidylate kinase (thymidylate kinase) v-yes-1 Yamaguchi sarcoma viral IPI00013981 P07947 YES1 Protein kinase, TK oncogene homolog 1 IPI00013983 P07949 RET Protein kinase, TK ret proto-oncogene p21 protein (Cdc42/Rac)-activated IPI00014068 O96013 PAK4 Protein kinase, STE kinase 4 protein kinase CHK2-like; CHK2 IPI00014072 O96017 CHEK2 Protein kinase, CAMK checkpoint homolog (S. pombe); similar to hCG1983233 dual-specificity tyrosine-(Y)- IPI00014344 Q13627 DYRK1A Protein kinase, CMGC phosphorylation regulated kinase 1A IPI00014414 P25440 BRD2 Protein kinase, Atypical bromodomain containing 2 IPI00014629 Q504Y2 PKDCC Protein kinase, CAMK protein kinase-like protein SgK493 IPI00014873 Q15131 CDK10 Protein kinase, CMGC cyclin-dependent kinase 10 IPI00014878 Q15139 PRKD1 Protein kinase, AGC protein kinase D1 IPI00015538 O94806 PRKD3 Protein kinase, AGC protein kinase D3

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Kinase Gene IPI Number UniprotKB Kinase Group Description Name ribonuclease L (2',5'- IPI00015864 Q05823 RNASEL Protein kinase, Other oligoisoadenylate synthetase- dependent) IPI00015892 Q8IZL9 CCRK Protein kinase, CMGC cell cycle related kinase IPI00015927 Q13882 PTK6 Protein kinase, TK PTK6 protein tyrosine kinase 6 endoplasmic reticulum to nucleus IPI00015974 O75460 ERN1 Protein kinase, Other signaling 1 threonine synthase-like 1 (S. IPI00016287 Q8IYQ7 THNSL1 Other kinase cerevisiae) v-erb-a erythroblastic leukemia viral IPI00016371 Q15303 ERBB4 Protein kinase, TK oncogene homolog 4 (avian) casein kinase 2, alpha 1 polypeptide IPI00016613 P68400 CSNK2A1 Protein kinase, CK1 pseudogene; casein kinase 2, alpha 1 polypeptide IPI00016639 P41743 PRKCI Protein kinase, AGC protein kinase C, iota IPI00016645 Q15375 EPHA7 Protein kinase, TK EPH receptor A7 LATS, large tumor suppressor, IPI00016690 Q9NRM7 LATS2 Protein kinase, AGC homolog 2 (Drosophila) Gardner-Rasheed feline sarcoma IPI00016871 P09769 FGR Protein kinase, TK viral (v-fgr) oncogene homolog calcium/calmodulin-dependent IPI00017083 Q8N5S9 CAMKK1 Protein kinase, CAMK protein kinase kinase 1, alpha ribosomal protein S6 kinase, 90kDa, IPI00017305 Q15418 RPS6KA1 Protein kinase, AGC polypeptide 1 IPI00017330 P43250 GRK6 Protein kinase, AGC G protein-coupled receptor kinase 6 IPI00017567 P17813 ENG Other kinase endoglin mitogen-activated protein kinase IPI00017801 Q99759 MAP3K3 Protein kinase, STE kinase kinase 3 hypothetical LOC100271831; IPI00018195 P27361 MAPK3 Protein kinase, CMGC mitogen-activated protein kinase 3 IPI00018246 P19367 HK1 Other kinase hexokinase 1 epidermal growth factor receptor IPI00018274 P00533 EGFR Protein kinase, TK (erythroblastic leukemia viral (v-erb- b) oncogene homolog, avian) polymerase (RNA) II (DNA directed) IPI00018288 P19387 POLR2C Protein kinase polypeptide C, 33kDa v-mos Moloney murine sarcoma viral IPI00018290 P00540 MOS Protein kinase, Other oncogene homolog fms-related tyrosine kinase 1 (vascular endothelial growth IPI00018335 P17948 FLT1 Protein kinase, TK factor/vascular permeability factor receptor) IPI00018342 P00568 AK1 Other kinase adenylate kinase 1 IPI00018597 P43405 SYK Protein kinase, TK spleen tyrosine kinase IPI00019353 Q53H12 AGK Lipid kinase acylglycerol kinase IPI00019383 P51570 GALK1 Other kinase galactokinase 1 IPI00019450 P10155 TROVE2 Other kinase TROVE domain family, member 2 eukaryotic translation initiation IPI00019463 P19525 EIF2AK2 Protein kinase, Other factor 2-alpha kinase 2 IPI00019640 Q99986 VRK1 Protein kinase, CK1 vaccinia related kinase 1 IPI00019926 P35626 ADRBK2 Protein kinase, AGC adrenergic, beta, receptor kinase 2

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Kinase Gene IPI Number UniprotKB Kinase Group Description Name N-acetylneuraminate pyruvate lyase IPI00020124 Q9BTU6 PI4K2A Lipid kinase 2 (putative) mitogen-activated protein kinase IPI00020258 Q92918 MAP4K1 Protein kinase, STE kinase kinase kinase 1 MAP/microtubule affinity-regulating IPI00020326 Q96L34 MARK4 Protein kinase, CAMK kinase 4 IPI00020454 P27707 DCK Other kinase deoxycytidine kinase v-raf murine sarcoma 3611 viral IPI00020578 P10398 ARAF Protein kinase, TKL oncogene homolog casein kinase 2, alpha prime IPI00020602 P19784 CSNK2A2 Protein kinase, CK1 polypeptide ribosomal protein S6 kinase, 90kDa, IPI00020898 P51812 RPS6KA3 Protein kinase, AGC polypeptide 3 IPI00020899 P51813 BMX Protein kinase, TK BMX non-receptor tyrosine kinase IPI00020904 P51817 PRKX Protein kinase, AGC protein kinase, X-linked IPI00021156 O60229 KALRN Protein kinase, CAMK kalirin, RhoGEF kinase Cdc2-related kinase, arginine/serine- IPI00021175 Q9NYV4 CRKRS Protein kinase, CMGC rich IPI00021248 P53350 PLK1 Protein kinase, Other polo-like kinase 1 (Drosophila) IPI00021250 P53355 DAPK1 Protein kinase, CAMK death associated protein kinase 1 IPI00021267 P29317 EPHA2 Protein kinase, TK EPH receptor A2 IPI00021274 P29322 EPHA8 Protein kinase, TK EPH receptor A8 IPI00021275 P29323 EPHB2 Protein kinase, TK EPH receptor B2 NIMA (never in mitosis gene a)- IPI00021331 P51955 NEK2 Protein kinase, Other related kinase 2 NIMA (never in mitosis gene a)- IPI00021333 P51957 NEK4 Protein kinase, Other related kinase 4 v-raf-1 murine leukemia viral IPI00021786 P04049 RAF1 Protein kinase, TKL oncogene homolog 1 phosphatidylinositol-4-phosphate 5- IPI00022149 Q99755 PIP5K1A Lipid kinase kinase, type I, alpha similar to Mast/stem cell growth factor receptor precursor (SCFR) (Proto-oncogene tyrosine-protein IPI00022296 P10721 KIT Protein kinase, TK kinase Kit) (c-kit) (CD117 antigen); v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog IPI00022353 P29597 TYK2 Protein kinase, TK tyrosine kinase 2 citron (rho-interacting, IPI00022465 O14578 CIT Protein kinase, STE serine/threonine kinase 21) dual-specificity tyrosine-(Y)- IPI00022521 Q92630 DYRK2 Protein kinase, CMGC phosphorylation regulated kinase 2 ribosomal protein S6 kinase, 90kDa, IPI00022536 O75676 RPS6KA4 Protein kinase, AGC polypeptide 4 cyclin-dependent kinase-like 2 IPI00022541 Q92772 CDKL2 Protein kinase, CMGC (CDC2-related kinase) similar to Rho-associated, coiled-coil containing protein kinase 1; Rho- IPI00022542 Q13464 ROCK1 Protein kinase, AGC associated, coiled-coil containing protein kinase 1 IPI00022633 Q13470 TNK1 Protein kinase, TK tyrosine kinase, non-receptor, 1

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Kinase Gene IPI Number UniprotKB Kinase Group Description Name IPI00022827 Q9H2G2 SLK Protein kinase, STE STE20-like kinase (yeast) IPI00022872 P53671 LIMK2 Protein kinase, TKL LIM domain kinase 2 IPI00022977 P12277 CKB Other kinase creatine kinase, brain glucosamine (UDP-N-acetyl)-2- IPI00023162 Q9Y223 GNE Other kinase epimerase/N-acetylmannosamine kinase IPI00023283 P07108 TTN Protein kinase, CAMK titin IPI00023503 Q00526 CDK3 Protein kinase, CMGC cyclin-dependent kinase 3 cyclin-dependent kinase-like 1 IPI00023527 Q00532 CDKL1 Protein kinase, CMGC (CDC2-related kinase) IPI00023529 Q00534 CDK6 Protein kinase, CMGC cyclin-dependent kinase 6 IPI00023530 Q00535 CDK5 Protein kinase, CMGC cyclin-dependent kinase 5 mitogen-activated protein kinase IPI00023636 O14733 MAP2K7 Protein kinase, STE kinase 7 CHK1 checkpoint homolog (S. IPI00023664 O14757 CHEK1 Protein kinase, CAMK pombe) IPI00024403 O75131 CPNE3 Protein kinase, Other copine III IPI00024539 Q02156 PRKCE Protein kinase, AGC protein kinase C, epsilon p21 protein (Cdc42/Rac)-activated IPI00024584 Q9NQU5 PAK6 Protein kinase, STE kinase 6 mitogen-activated protein kinase IPI00024674 P45985 MAP2K4 Protein kinase, STE kinase 4 natriuretic peptide receptor IPI00024685 P20594 NPR2 Protein kinase, RGC B/guanylate cyclase B (atrionatriuretic peptide receptor B) collagen, type IV, alpha 3 IPI00024701 Q9Y5P4 COL4A3BP Protein kinase, Atypical (Goodpasture antigen) binding protein inhibitor of kappa light polypeptide IPI00024709 O14920 IKBKB Protein kinase gene enhancer in B-cells, kinase beta IPI00025489 P20794 MAK Protein kinase, CMGC male germ cell-associated kinase IPI00025679 P57059 SIK1 Protein kinase, CAMK salt-inducible kinase 1 IPI00025698 P53671 LIMK2 Protein kinase, TKL LIM domain kinase 2 IPI00025803 P06213 INSR Protein kinase, TK insulin receptor IPI00025830 P30291 WEE1 Protein kinase, Other WEE1 homolog (S. pombe) mitogen-activated protein kinase- IPI00026054 P49137 MAPKAPK2 Protein kinase, CAMK activated protein kinase 2 cell division cycle 2, G1 to S and G2 IPI00026689 P06493 CDC2 Protein kinase, CMGC to M IPI00027174 P22607 FGFR3 Protein kinase, TK fibroblast growth factor receptor 3 natriuretic peptide receptor IPI00027200 P16066 NPR1 Protein kinase, RGC A/guanylate cyclase A (atrionatriuretic peptide receptor A) IPI00027212 P14616 INSRR Protein kinase, TK insulin receptor-related receptor IPI00027232 P08069 IGF1R Protein kinase, TK insulin-like growth factor 1 receptor IPI00027251 Q15208 STK38 Protein kinase, STE serine/threonine kinase 38 IPI00027487 P06732 CKM Other kinase creatine kinase, muscle platelet-derived growth factor IPI00027721 P16234 PDGFRA Protein kinase, TK receptor, alpha polypeptide

S13

Kinase Gene IPI Number UniprotKB Kinase Group Description Name polymerase (RNA) II (DNA directed) IPI00027808 P30876 POLR2B Protein kinase polypeptide B, 140kDa IPI00028061 P49759 CLK1 Protein kinase, CMGC CDC-like kinase 1 IPI00028071 P49760 CLK2 Protein kinase, CMGC CDC-like kinase 2 calcium/calmodulin-dependent IPI00028296 Q14012 CAMK1 Protein kinase, CAMK protein kinase I IPI00028344 Q8IWQ3 BRSK2 Protein kinase, CAMK BR serine/threonine kinase 2 IPI00028566 O60285 NUAK1 Protein kinase, CAMK NUAK family, SNF1-like kinase, 1 IPI00028570 P49841 GSK3B Protein kinase, CMGC glycogen synthase kinase 3 beta microtubule associated IPI00028932 O60307 MAST3 Protein kinase, AGC serine/threonine kinase 3 phosphatidylinositol-4-phosphate 5- IPI00029009 O60331 PIP5K1C Lipid kinase kinase, type I, gamma inhibitor of kappa light polypeptide IPI00029045 Q14164 IKBKE Protein kinase gene enhancer in B-cells, kinase epsilon Bruton agammaglobulinemia IPI00029132 Q06187 BTK Protein kinase, TK tyrosine kinase cell division cycle 2-like 5 IPI00029162 Q14004 CDC2L5 Protein kinase, CMGC (cholinesterase-related cell division controller) IPI00029196 Q04759 PRKCQ Protein kinase, AGC protein kinase C, theta IPI00029219 Q04771 ACVR1 Protein kinase, TKL activin A receptor, type I IPI00029263 P16591 FER Protein kinase, TK fer (fps/fes related) tyrosine kinase met proto-oncogene (hepatocyte IPI00029273 P08581 MET Protein kinase, TK growth factor receptor) sterile alpha motif and leucine zipper IPI00029643 Q9NYL2 ZAK Protein kinase, TKL containing kinase AZK IPI00029702 Q14289 PTK2B Protein kinase, TK PTK2B protein tyrosine kinase 2 beta excision repair cross-complementing IPI00029728 P18074 ERCC2 Protein kinase, CMGC rodent repair deficiency, complementation group 2 IPI00029769 P08631 HCK Protein kinase, TK hemopoietic cell kinase macrophage stimulating 1 receptor IPI00030273 Q04912 MST1R Protein kinase, TK (c-met-related tyrosine kinase) general transcription factor IIH, IPI00030380 P32780 GTF2H1 Protein kinase polypeptide 1, 62kDa IPI00030389 Q12979 ABR Protein kinase, Atypical active BCR-related gene IPI00030887 Q06418 TYRO3 Protein kinase, TK TYRO3 protein tyrosine kinase IPI00031016 O60674 JAK2 Protein kinase, TK Janus kinase 2 phosphoinositide-3-kinase, catalytic, IPI00031386 P42336 PIK3CA Lipid kinase alpha polypeptide mechanistic target of rapamycin IPI00031410 P42345 MTOR Protein kinase, Atypical (serine/threonine kinase) polymerase (RNA) II (DNA directed) IPI00031627 P24928 POLR2A Protein kinase polypeptide A, 220kDa IPI00031681 P24941 CDK2 Protein kinase, CMGC cyclin-dependent kinase 2 v-akt murine thymoma viral IPI00031747 Q9Y243 AKT3 Protein kinase, AGC oncogene homolog 3 (protein kinase B, gamma) IPI00032263 Q9Y2K2 SIK3 Protein kinase, CAMK serine/threonine-protein kinase QSK

S14

Kinase Gene IPI Number UniprotKB Kinase Group Description Name TAF9 RNA polymerase II, TATA IPI00032879 Q16594 TAF9 Other kinase box binding protein (TBP)-associated factor, 32kDa IPI00033606 Q9NSY1 BMP2K Protein kinase, Other BMP2 inducible kinase IPI00044584 Q96PN8 TSSK3 Protein kinase, CAMK testis-specific serine kinase 3 NIMA (never in mitosis gene a)- IPI00044749 Q96PY6 NEK1 Protein kinase, Other related kinase 1 IPI00045068 Q96QS6 PSKH2 Protein kinase, CAMK protein serine kinase H2 interleukin-1 receptor-associated IPI00060149 P51617 IRAK1 Protein kinase, TKL kinase 1 protein kinase, AMP-activated, alpha IPI00061282 Q13131 PRKAA1 Protein kinase, AGC 1 catalytic subunit IPI00062264 Q96KG9 SCYL1 Protein kinase SCY1-like 1 (S. cerevisiae) branched chain ketoacid IPI00062344 O14874 BCKDK Protein kinase, Atypical dehydrogenase kinase IPI00064935 Q96L96 ALPK3 Protein kinase, Atypical alpha-kinase 3 IPI00065671 Q9BZX2 UCK2 Other kinase uridine-cytidine kinase 2 bromodomain adjacent to zinc finger IPI00069817 Q9UIG0 BAZ1B Protein kinase, Atypical domain, 1B YSK4 Sps1/Ste20-related kinase IPI00074966 Q56UN5 MAP3K19 Protein kinase, STE homolog (S. cerevisiae) IPI00099756 Q9UQB9 AURKC Protein kinase, Other aurora kinase C IPI00100329 Q13572 ITPK1 Lipid kinase inositol 1,3,4-triphosphate 5/6 kinase hexokinase 2 pseudogene; IPI00102864 P52789 HK2 Other kinase hexokinase 2 budding uninhibited by IPI00141933 O60566 BUB1B Protein kinase, Other benzimidazoles 1 homolog beta (yeast) IPI00142487 Q5TCX8 MLK4 Protein kinase, TKL mixed lineage kinase 4 IPI00145805 Q9UKE5 TNIK Protein kinase, STE TRAF2 and NCK interacting kinase IPI00149048 Q13164 MAPK7 Protein kinase, CMGC mitogen-activated protein kinase 7 mitogen-activated protein kinase IPI00149094 Q12851 MAP4K2 Protein kinase, STE kinase kinase kinase 2 WNK lysine deficient protein kinase IPI00151141 Q96J92 WNK4 Protein kinase, Other 4 transient receptor potential cation IPI00151366 Q9BX84 TRPM6 Protein kinase, Atypical channel, subfamily M, member 6 phosphatidylinositol-5-phosphate 4- IPI00152303 Q8TBX8 PIP4K2C Lipid kinase kinase, type II, gamma NIMA (never in mitosis gene a)- IPI00152658 Q8TDX7 NEK7 Protein kinase, Other related kinase 7 mitogen-activated protein kinase IPI00158248 Q13163 MAP2K5 Protein kinase, STE kinase 5 mitogen-activated protein kinase- IPI00160672 Q8IW41 MAPKAPK5 Protein kinase, CAMK activated protein kinase 5 IPI00161013 Q8N2I9 STK40 Protein kinase, CAMK serine/threonine kinase 40 U2AF homology motif (UHM) IPI00163376 Q8TAS1 UHMK1 Protein kinase, Other kinase 1 eukaryotic translation initiation IPI00163851 Q9P2K8 EIF2AK4 Protein kinase, Other factor 2 alpha kinase 4 IPI00165249 O94921 CDK14 Protein kinase, CMGC PFTAIRE protein kinase 1

S15

Kinase Gene IPI Number UniprotKB Kinase Group Description Name IPI00165955 Q8TD08 MAPK15 Protein kinase, CMGC mitogen-activated protein kinase 15 IPI00166098 Q8N165 PDIK1L Protein kinase, Other PDLIM1 interacting kinase 1 like FYN oncogene related to SRC, FGR, IPI00166845 P06241 FYN Protein kinase, TK YES IPI00167089 Q07912 TNK2 Protein kinase, TK tyrosine kinase, non-receptor, 2 IPI00167096 Q8N752 CSNK1A1L Protein kinase, CK1 casein kinase 1, alpha 1-like serine/threonine kinase 3 (STE20 IPI00168325 Q13188 STK3 Protein kinase, STE homolog, yeast) IPI00168813 Q13308 PTK7 Protein kinase, TK PTK7 protein tyrosine kinase 7 IPI00168907 Q8NFU5 IPMK Other kinase inositol polyphosphate multikinase IPI00169383 P00558 PGK1 Other kinase phosphoglycerate kinase 1 calcium/calmodulin-dependent IPI00170508 Q8IU85 CAMK1D Protein kinase, CAMK protein kinase ID calcium/calmodulin-dependent IPI00170509 Q96RR4 CAMKK2 Protein kinase, CAMK protein kinase kinase 2, beta IPI00171336 Q9BRS2 RIOK1 Protein kinase, Atypical RIO kinase 1 (yeast) IPI00171791 Q8N4C6 NIN Protein kinase ninein (GSK3B interacting protein) calcium/calmodulin-dependent IPI00172450 Q13555 CAMK2G Protein kinase, CAMK protein kinase II gamma IPI00172460 P54819 AK2 Other kinase adenylate kinase 2 calcium/calmodulin-dependent IPI00172636 Q13557 CAMK2D Protein kinase, CAMK protein kinase II delta IPI00175649 Q5S007 LRRK2 Protein kinase, TKL leucine-rich repeat kinase 2 chaperone, ABC1 activity of bc1 IPI00176469 Q8NI60 ADCK3 Protein kinase, Atypical complex homolog (S. pombe) IPI00176642 Q96GD4 AURKB Protein kinase, Other aurora kinase B IPI00178315 Q96D53 ADCK4 Protein kinase, Atypical aarF domain containing kinase 4 IPI00179193 Q8WXR4 MYO3B Protein kinase, STE myosin IIIB IPI00180143 Q9HAZ1 CLK4 Protein kinase, CMGC CDC-like kinase 4 IPI00180781 Q8NB16 MLKL Protein kinase, TKL mixed lineage kinase domain-like IPI00181294 Q9Y6M4 CSNK1G3 Protein kinase, CK1 casein kinase 1, gamma 3 serine/threonine protein kinase IPI00182383 Q9P289 STK26 Protein kinase, TK MST4 MAP/microtubule affinity-regulating IPI00183118 P27448 MARK3 Protein kinase, CAMK kinase 3 SMG1 homolog, IPI00183368 Q96Q15 SMG1 Protein kinase, Atypical phosphatidylinositol 3-kinase-related kinase (C. elegans) IPI00183400 P48729 CSNK1A1 Protein kinase, CK1 casein kinase 1, alpha 1 twinfilin, actin-binding protein, IPI00183508 Q12792 TWF1 Protein kinase homolog 1 (Drosophila) IPI00184572 P24723 PRKCH Protein kinase, AGC protein kinase C, eta IPI00185036 Q8NEV4 MYO3A Protein kinase, STE myosin IIIA MAP/microtubule affinity-regulating IPI00185037 Q9P0L2 MARK1 Protein kinase, CAMK kinase 1 mitogen-activated protein kinase IPI00186536 Q9Y6R4 MAP3K4 Protein kinase, STE kinase kinase 4 IPI00186826 P54760 EPHB4 Protein kinase, TK EPH receptor B4 membrane protein, palmitoylated 1, IPI00215610 Q9UQ80 PA2G4 Other kinase 55kDa

S16

Kinase Gene IPI Number UniprotKB Kinase Group Description Name calcium/calmodulin-dependent IPI00215715 Q9UQM7 CAMK2A Protein kinase, CAMK protein kinase II alpha IPI00215901 P54819 AK2 Other kinase adenylate kinase 2 homeodomain interacting protein IPI00215949 Q9H2X6 HIPK2 Protein kinase, CMGC kinase 2; similar to homeodomain interacting protein kinase 2 IPI00215958 Q09013 DMPK Protein kinase, AGC dystrophia myotonica-protein kinase ribosomal protein S6 kinase, 70kDa, IPI00216132 P23443 RPS6KB1 Protein kinase, AGC polypeptide 1 phosphatidylinositol-5-phosphate 4- IPI00216470 P78356 PIP4K2B Lipid kinase kinase, type II, beta P00519, c-abl oncogene 1, receptor tyrosine IPI00216969 ABL1,ABL2 Protein kinase, TK P42684 kinase mitogen-activated protein kinase IPI00217024 Q8IVH8 MAP4K3 Protein kinase, STE kinase kinase kinase 3 ribosomal protein S6 kinase, 70kDa, IPI00217069 Q9UBS0 RPS6KB2 Protein kinase, AGC polypeptide 2 IPI00217158 Q8IVW4 CDKL3 Protein kinase, CMGC cyclin-dependent kinase-like 3 IPI00217437 P05787 KRT8 Protein kinase tau tubulin kinase 2 IPI00217690 Q8IY84 NIM1K Protein kinase, CAMK serine/threonine-protein kinase NIM1 IPI00217776 Q9UPZ9 ICK Protein kinase, CMGC intestinal cell (MAK-like) kinase protein kinase, cAMP-dependent, IPI00217960 P17612 PRKACA Protein kinase, AGC catalytic, alpha mitogen-activated protein kinase IPI00218566 O43318 MAP3K7 Protein kinase, STE kinase kinase 7 mitogen-activated protein kinase IPI00218857 P46734 MAP2K3 Protein kinase, STE kinase 3 FYN oncogene related to SRC, FGR, IPI00219012 P06241 FYN Protein kinase, TK YES IPI00219072 Q15831 STK11 Protein kinase, STE serine/threonine kinase 11 hormonally up-regulated Neu- IPI00219553 P57058 HUNK Protein kinase, CAMK associated kinase IPI00219568 P07205 PGK2 Other kinase phosphoglycerate kinase 2 IPI00219585 P08237 PFKM Other kinase phosphofructokinase, muscle protein kinase, cAMP-dependent, IPI00219592 P22612 PRKACG Protein kinase, AGC catalytic, gamma IPI00219601 Q13164 MAPK7 Protein kinase, CMGC mitogen-activated protein kinase 7 mitogen-activated protein kinase IPI00219604 Q02750 MAP2K1 Protein kinase, STE kinase 1 phosphoribosyl pyrophosphate IPI00219616 P60891 PRPS1 Other kinase synthetase 1; phosphoribosyl pyrophosphate synthetase 1-like 1 phosphoribosyl pyrophosphate IPI00219617 P11908 PRPS2 Other kinase synthetase 2 IPI00219823 Q9HCP0 CSNK1G1 Protein kinase, CK1 casein kinase 1, gamma 1 cytidine monophosphate (UMP- IPI00219953 P30085 CMPK1 Other kinase CMP) kinase 1, cytosolic IPI00220253 P22607 FGFR3 Protein kinase, TK fibroblast growth factor receptor 3 phosphorylase kinase, gamma 1 IPI00220396 Q16816 PHKG1 Protein kinase, CAMK (muscle) IPI00221127 Q9H1R3 MYLK2 Protein kinase, CAMK myosin light chain kinase 2

S17

Kinase Gene IPI Number UniprotKB Kinase Group Description Name IPI00221255 Q15746 MYLK Protein kinase, CAMK myosin light chain kinase IPI00234368 P55263 ADK Other kinase adenosine kinase serine/threonine kinase 36, fused IPI00235407 Q9NRP7 STK36 Protein kinase, Other homolog (Drosophila) IPI00237011 Q9Y2H1 STK38L Protein kinase, AGC serine/threonine kinase 38 like NIMA (never in mitosis gene a)- IPI00243995 Q6P3R8 NEK5 Protein kinase, Other related kinase 5 obscurin, cytoskeletal calmodulin IPI00288940 Q5VST9 OBSCN Protein kinase, CAMK and titin-interacting RhoGEF c-ros oncogene 1 , receptor tyrosine IPI00288965 P08922 ROS1 Protein kinase, TK kinase muscle, skeletal, receptor tyrosine IPI00289243 O15146 MUSK Protein kinase, TK kinase IPI00289329 P54753 EPHB3 Protein kinase, TK EPH receptor B3 IPI00289357 O75385 ULK1 Protein kinase, Other unc-51-like kinase 1 (C. elegans) IPI00289715 Q96PF2 TSSK2 Protein kinase, CAMK testis-specific serine kinase 2 p21 protein (Cdc42/Rac)-activated IPI00289746 Q13153 PAK1 Protein kinase, STE kinase 1 IPI00289819 P11717 IGF2R Protein kinase, TK insulin-like growth factor 2 receptor similar to Serine-protein kinase ATM (Ataxia telangiectasia mutated) (A-T, IPI00289986 Q13315 ATM Protein kinase, Atypical mutated); ataxia telangiectasia mutated transient receptor potential cation IPI00290032 Q96QT4 TRPM7 Protein kinase, Atypical channel, subfamily M, member 7 MAP/microtubule affinity-regulating IPI00290158 Q7KZI7 MARK2 Protein kinase, CAMK kinase 2 IPI00290305 Q9Y3C4 TPRKB Protein kinase, Other TP53 regulating kinase IPI00290308 Q96RU8 TRIB1 Protein kinase, CAMK tribbles homolog 1 (Drosophila) IPI00290439 Q96SB4 SRPK1 Protein kinase, CMGC SFRS protein kinase 1 IPI00290684 Q96T60 PNKP Other kinase polynucleotide kinase 3'-phosphatase phosphatidylinositol 4-kinase type 2 IPI00291068 Q8TCG2 PI4K2B Lipid kinase beta polymerase (RNA) II (DNA directed) IPI00291093 P19388 POLR2E Protein kinase polypeptide E, 25kDa ribosomal protein S6 kinase, 52kDa, IPI00291351 Q96S38 RPS6KC1 Protein kinase, AGC polypeptide 1 IPI00291457 Q8TDR2 STK35 Protein kinase, Other serine/threonine kinase 35 IPI00292228 P49840 GSK3A Protein kinase, CMGC glycogen synthase kinase 3 alpha IPI00293271 P37023 ACVRL1 Protein kinase, TKL activin A receptor type II-like 1 IPI00293303 Q99538 LGMN Protein kinase legumain IPI00293565 P35916 FLT4 Protein kinase, TK fms-related tyrosine kinase 4 IPI00293613 Q9UHD2 TBK1 Protein kinase, Other TANK-binding kinase 1 inhibitor of kappa light polypeptide IPI00293735 O95163 IKBKAP Protein kinase gene enhancer in B-cells, kinase complex-associated protein golgi autoantigen, golgin subfamily IPI00294065 Q8TBA6 GOLGA5 Protein kinase a, 5 IPI00294250 P21709 EPHA1 Protein kinase, TK EPH receptor A1 IPI00294344 P07332 FES Protein kinase, TK feline sarcoma oncogene

S18

Kinase Gene IPI Number UniprotKB Kinase Group Description Name menage a trois homolog 1, cyclin H IPI00294701 P51948 MNAT1 Protein kinase, CMGC assembly factor (Xenopus laevis) mitogen-activated protein kinase IPI00294842 Q9Y4K4 MAP4K5 Protein kinase, STE kinase kinase kinase 5 non-metastatic cells 7, protein IPI00294997 Q9Y5B8 NME7 Other kinase expressed in (nucleoside-diphosphate kinase) similar to protein kinase, DNA- activated, catalytic polypeptide; IPI00296337 P78527 PRKDC Protein kinase, AGC protein kinase, DNA-activated, catalytic polypeptide IPI00296526 Q9UJ70 NAGK Other kinase N-acetylglucosamine kinase thioredoxin domain containing 3 IPI00296545 Q8N427 NME8 Other kinase (spermatozoa) IPI00296992 P30530 AXL Protein kinase, TK AXL receptor tyrosine kinase serum/glucocorticoid regulated IPI00297590 O00141 SGK1 Protein kinase, AGC kinase 1 IPI00297767 Q8NCQ2 CSNK1G2 Protein kinase, CK1 casein kinase 1, gamma 2 IPI00298105 P29320 EPHA3 Protein kinase, TK EPH receptor A3 IPI00298199 O14730 RIOK3 Protein kinase, Atypical RIO kinase 3 (yeast) similar to Serine-protein kinase ATM (Ataxia telangiectasia mutated) (A-T, IPI00298306 Q13315 ATM Protein kinase, Atypical mutated); ataxia telangiectasia mutated phosphoinositide-3-kinase, catalytic, IPI00298410 O00329 PIK3CD Lipid kinase delta polypeptide v-yes-1 Yamaguchi sarcoma viral IPI00298625 P07948 LYN Protein kinase, TK related oncogene homolog IPI00298896 P49761 CLK3 Protein kinase, CMGC CDC-like kinase 3 aurora kinase A; aurora kinase A IPI00298940 O14965 AURKA Protein kinase, Other pseudogene 1 IPI00298949 O14976 GAK Protein kinase, Other cyclin G associated kinase IPI00299214 P04183 TK1 Protein kinase, TK thymidine kinase 1, soluble IPI00299755 Q8NEB9 PIK3C3 Lpid kinase phosphoinositide-3-kinase, class 3 ribosomal protein S6 kinase, 90kDa, IPI00300321 Q15349 RPS6KA2 Protein kinase, AGC polypeptide 2; hypothetical LOC100127984 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, IPI00300384 P04626 ERBB2 Protein kinase, TK neuro/glioblastoma derived oncogene homolog (avian) IPI00300700 Q7RTN6 STRADA Protein kinase, STE STE20-related kinase adaptor alpha carbamoyl-phosphate synthetase 2, IPI00301263 P27708 CAD Protein kinase, TK aspartate transcarbamylase, and dihydroorotase NIMA (never in mitosis gene a)- IPI00301609 Q8TD19 NEK9 Protein kinase, Other related kinase 9 IPI00301923 P50750 CDK9 Protein kinase, CMGC cyclin-dependent kinase 9 homeodomain interacting protein IPI00302130 Q8NE63 HIPK4 Protein kinase, CMGC kinase 4

S19

Kinase Gene IPI Number UniprotKB Kinase Group Description Name homeodomain interacting protein IPI00302302 Q86Z02 HIPK1 Protein kinase, CMGC kinase 1 IPI00302351 Q9BYT3 STK33 Protein kinase, CAMK serine/threonine kinase 33 IPI00302552 Q8NER5 ACVR1C Protein kinase, TKL activin A receptor, type IC IPI00302787 Q9NYY3 PLK2 Protein kinase, Other polo-like kinase 2 (Drosophila) eukaryotic translation initiation IPI00303053 Q9NZJ5 EIF2AK3 Protein kinase, Other factor 2-alpha kinase 3 v-raf murine sarcoma viral oncogene IPI00303797 P15056 BRAF Protein kinase, TKL homolog B1 phosphatidylinositol-4-phosphate 5- IPI00304025 O14986 PIP5K1B Lipid kinase kinase, type I, beta IPI00304186 Q9H4B4 PLK3 Protein kinase, Other polo-like kinase 3 (Drosophila) IPI00304578 P22455 FGFR4 Protein kinase, TK fibroblast growth factor receptor 4 IPI00304742 O94804 STK10 Protein kinase, STE serine/threonine kinase 10 receptor tyrosine kinase-like orphan IPI00304999 Q01974 ROR2 Protein kinase, TK receptor 2 IPI00306406 Q9BVS4 RIOK2 Protein kinase, Atypical RIO kinase 2 (yeast) IPI00306522 Q38SD2 LRRK1 Protein kinase, TKL leucine-rich repeat kinase 1 IPI00306708 Q96KB5 PBK Protein kinase, Other PDZ binding kinase Rho-associated, coiled-coil IPI00307155 O75116 ROCK2 Protein kinase, AGC containing protein kinase 2 protein kinase, AMP-activated, alpha IPI00307755 P54646 PRKAA Protein kinase, AGC 2 catalytic subunit eukaryotic translation initiation IPI00328149 Q9BQI3 EIF2AK1 Protein kinase, Other factor 2-alpha kinase 1 NIMA (never in mitosis gene a)- IPI00328397 Q86SG6 NEK8 Protein kinase, Other related kinase 8 IPI00328835 Q86UX6 STK32C Protein kinase, AGC serine/threonine kinase 32C v-src sarcoma (Schmidt-Ruppin A-2) IPI00328867 P12931 SRC Protein kinase, TK viral oncogene homolog (avian) IPI00329236 Q05655 PRKCD Protein kinase, AGC protein kinase C, delta IPI00329275 Q86Y07 VRK2 Protein kinase, CK2 vaccinia related kinase 2 CDC42 binding protein kinase alpha IPI00329326 Q5VT25 CDC42BPA Protein kinase, CMGC (DMPK-like) transforming growth factor beta IPI00329625 Q969Z0 TBRG4 Protein kinase regulator 4 sterile alpha motif and leucine zipper IPI00329638 Q9NYL2 ZAK Protein kinase, TKL containing kinase AZK zeta-chain (TCR) associated protein IPI00329789 P43403 ZAP70 Protein kinase, TK kinase 70kDa IPI00332371 P17858 PFKL Other kinase phosphofructokinase, liver IPI00332692 Q9P1W9 PIM2 Protein kinase, CAMK pim-2 oncogene IPI00333420 P78362 SRPK2 Protein kinase, CMGC SFRS protein kinase 2 IPI00333559 Q9UKI8-2 TLK1 Protein kinase, Other tousled-like kinase 1 ribosomal protein S6 kinase, 90kDa, IPI00335101 O75582 RPS6KA5 Protein kinase, AGC polypeptide 5 serum/glucocorticoid regulated IPI00335929 Q96BR1 SGK3 Protein kinase, AGC kinase family, member 3 IPI00337659 Q86UE8 TLK2 Protein kinase, Other tousled-like kinase 2

S20

Kinase Gene IPI Number UniprotKB Kinase Group Description Name protein kinase, cAMP-dependent, IPI00376119 P22694 PRKACB Protein kinase, AGC catalytic, beta IPI00376955 Q00537 CDK17 Protein kinase, CMGC PCTAIRE protein kinase 2 protein kinase, membrane associated IPI00377017 Q99640 PMYT1 Protein kinase, Other tyrosine/threonine 1 receptor (TNFRSF)-interacting IPI00383377 Q9Y572 RIPK3 Protein kinase, TKL serine-threonine kinase 3 bromodomain adjacent to zinc finger IPI00383565 Q9NRL2 BAZ1A Protein kinase, Atypical domain, 1A IPI00384276 Q86V86 PIM3 Protein kinase, CAMK pim-3 oncogene IPI00385449 P17252 PRKCA Protein kinase, AGC protein kinase C, alpha IPI00385887 P04183 TK Protein kinase, TK tyrosine kinase lymphocyte-specific protein tyrosine IPI00394952 P06239 LCK Protein kinase, TK kinase protein kinase, cAMP-dependent, IPI00395654 P22694 PRKACB Protein kinase, AGC catalytic, beta MAP kinase interacting IPI00396114 Q9HBH9 MKNK2 Protein kinase, CAMK serine/threonine kinase 2 phosphoinositide kinase, FYVE IPI00396145 Q9Y2I7 PIKFYVE Lipid kinase finger containing IPI00396218 Q6P3W7 SCYL2 Protein kinase SCY1-like 2 (S. cerevisiae) microtubule associated IPI00396230 Q96GX5 MASTL Protein kinase, AGC serine/threonine kinase-like dual-specificity tyrosine-(Y)- IPI00396286 Q9NR20 DYRK4 Protein kinase, CMGC phosphorylation regulated kinase 4 protein kinase, cAMP-dependent, IPI00396630 P17612 PRKACA Protein kinase, AGC catalytic, alpha dual serine/threonine and tyrosine IPI00396963 Q6XUX3 DSTYK Protein kinase, Other protein kinase SH3-binding domain kinase family, IPI00396997 P0C263 SBK2 Protein kinase, Other member 2 IPI00397836 Q8TF76 GSG2 Protein kinase, Other germ cell associated 2 (haspin) IPI00398648 Q9UQ07 MOK Protein kinase, CMGC renal tumor antigen PAS domain containing IPI00398824 Q96RG2 PASK Protein kinase, CAMK serine/threonine kinase IPI00399180 Q52WX2 SBK1 Protein kinase, Other SH3-binding domain kinase 1 protein kinase, AMP-activated, alpha IPI00410287 Q13131 PRKAA1 Protein kinase, AGC 1 catalytic subunit IPI00410344 O00444 PLK4 Protein kinase, Other polo-like kinase 4 (Drosophila) cytidine monophosphate (UMP- IPI00410404 Q5EBM0 CMPK2 Other kinase CMP) kinase 2, mitochondrial IPI00411818 Q6PHR2 ULK3 Protein kinase, Other unc-51-like kinase 3 (C. elegans) serine/threonine kinase 3 (STE20 IPI00411984 Q13188 STK3 Protein kinase, STE homolog, yeast) IPI00412099 Q86TW2 ADCK1 Protein kinase, Atypical aarF domain containing kinase 1 ataxia telangiectasia and Rad3 related; similar to ataxia IPI00412298 Q13535 ATR Protein kinase, Atypical telangiectasia and Rad3 related protein IPI00412344 Q96SB4 SRPK1 Protein kinase, CMGC SFRS protein kinase 1

S21

Kinase Gene IPI Number UniprotKB Kinase Group Description Name mitogen-activated protein kinase IPI00412433 Q99683 MAP3K5 Protein kinase, STE kinase kinase 5 mitogen-activated protein kinase IPI00412740 Q6ZN16 MAP3K15 Protein kinase, STE kinase kinase 15 pregnancy up-regulated non- IPI00413306 Q6P2M8 PNCK Protein kinase, Other ubiquitously expressed CaM kinase IPI00413778 P62942 FKBP1A Protein kinase FK506 binding protein 1A, 12kDa IPI00413780 Q6P5Z2 PKN3 Protein kinase, AGC protein kinase N3 IPI00414058 P34947 GRK5 Protein kinase, AGC G protein-coupled receptor kinase 5 IPI00414440 Q6SA08 TSSK4 Protein kinase, CAMK testis-specific serine kinase 4 IPI00414612 Q2TB90 HKDC1 Other kinase hexokinase domain containing 1 IPI00414973 Q7L8L6 FASTKD5 Protein kinase, Other FAST kinase domains 5 mitogen-activated protein kinase IPI00418221 O95382 MAP3K6 Protein kinase, STE kinase kinase 6 IPI00418366 Q3MIX3 ADCK5 Protein kinase, Atypical aarF domain containing kinase 5 IPI00419204 Q9BXU1 STK31 Protein kinase, Other serine/threonine kinase 31 p21 protein (Cdc42/Rac)-activated IPI00419979 Q13177 PAK2 Protein kinase, STE kinase 2 calcium/calmodulin-dependent IPI00430411 Q16566 CAMK4 Protein kinase, CAMK protein kinase IV IPI00438229 Q13263 TRIM28 Protein kinase, Atypical tripartite motif-containing 28 IPI00440727 O60885 BRD4 Protein kinase, Atypical bromodomain containing 4 triple functional domain (PTPRF IPI00442035 O75962 TRIO Protein kinase, CAMK interacting) IPI00442129 Q00536 CDK16 Protein kinase, CMGC PCTAIRE protein kinase 1 cell division cycle 7 homolog (S. IPI00448121 O00311 CDC7 Protein kinase, CMGC cerevisiae) IPI00452669 Q92551 IP6K1 Other kinase inositol hexakisphosphate kinase 1 CDC42 binding protein kinase IPI00454910 Q6DT37 CDC42BPG Protein kinase, CMGC gamma (DMPK-like) IPI00465256 Q9UIJ7 AK3 Other kinase adenylate kinase 3 IPI00465291 Q9H0K1 SIK2 Protein kinase, CAMK salt-inducible kinase 2 dual-specificity tyrosine-(Y)- IPI00465459 O43781 DYRK3 Protein kinase, CMGC phosphorylation regulated kinase 3 family with sequence similarity 20, IPI00470607 Q8IXL6 FAM20C Protein kinase member C IPI00470811 Q9NRH2 SNRK Protein kinase, CAMK SNF related kinase protein kinase, AMP-activated, IPI00473047 P54619 PRKAG1 Protein kinase, AGC gamma 1 non-catalytic subunit CDC42 binding protein kinase beta IPI00477763 Q9Y5S2 CDC42BPB Protein kinase, CMGC (DMPK-like) IPI00479399 Q8IYT8 ULK2 Protein kinase, Other unc-51-like kinase 2 (C. elegans) IPI00479760 Q2M2I8 AAK1 Protein kinase, Other AP2 associated kinase 1 PX domain containing IPI00479912 Q7Z7A4 PXK Protein kinase, Other serine/threonine kinase mitogen-activated protein kinase IPI00513803 Q9Y2U5 MAP3K2 Protein kinase, STE kinase kinase 2 PAN3 poly(A) specific ribonuclease IPI00513919 Q58A45 PAN3 Protein kinase subunit homolog (S. cerevisiae)

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Kinase Gene IPI Number UniprotKB Kinase Group Description Name calcium/calmodulin-dependent serine IPI00514301 O14936 CASK Protein kinase, CAMK protein kinase (MAGUK family) IPI00604756 Q9UHY1 NRBP1 Protein kinase, Other nuclear receptor binding protein 1 triple functional domain (PTPRF IPI00657953 O75962 TRIO Protein kinase, CAMK interacting) IPI00658151 Q15772 SPEG Protein kinase, CAMK SPEG complex locus IPI00737545 Q9H792 PEAK1 Protein kinase, AGC NKF3 kinase family member IPI00742193 Q6ZMQ8 AATK Protein kinase, TK apoptosis-associated tyrosine kinase IPI00743623 Q9Y6K8 AK5 Other kinase adenylate kinase 5 IPI00746301 O76039 CDKL5 Protein kinase, CMGC cyclin-dependent kinase-like 5 budding uninhibited by IPI00783305 O43683 BUB1 Protein kinase, Other benzimidazoles 1 homolog (yeast) IPI00783950 Q8WZ42 TTN Protein kinase, CAMK titin IPI00784013 P23458 JAK1 Protein kinase, TK Janus kinase 1 microtubule associated IPI00788168 O15021 MAST4 Protein kinase, AGC serine/threonine kinase family member 4 IPI00852660 Q9C098 DCLK3 Protein kinase, CMGC doublecortin-like kinase 3 mitogen-activated protein kinase IPI00855985 Q13233 MAP3K1 Protein kinase, STE kinase kinase 1 IPI00871409 Q7Z2Y5 NRK Protein kinase, STE Nik related kinase integrin, alpha E (antigen CD103, IPI00871807 P38570 ITGAE Protein kinase human mucosal lymphocyte antigen 1; alpha polypeptide) cell division cycle 2-like 6 (CDK8- IPI00909706 P49336 CDK8 Protein kinase, CMGC like) ribosomal protein S6 kinase, 70kDa, IPI00941637 Q8IU60 DCP2 Other kinase polypeptide 1

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Table S3. iRT comparison between desthiobiotin-labeled and desthiobiotin-C3-labeled peptides.

Peptide sequence iRT of desthiobiotin-labeled iRT of desthiobiotin-C3- peptides labeled peptides AVIMGAPGSGK#GTVSSR 30.27 25.66 EAFSLFDK#DGDGTITTK 79.45 75.09 GGPNIITLADIVK#DPVSR 110.31 106.18 GTFGK#VILVK 63.41 59.80 GTFGK#VILVR 66.77 63.14 NIIHGSDSVK#SAEK 14.27 7.92 TFIAIK#PDGVQR 57.95 52.31 VMLGETNPADSK#PGTIR 39.61 37.72 DIK#GANILR 50.62 46.37 AVDDGVNTFK#VLTR 70.70 65.79

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Table S4. List of kinases quantified in 5 replicates and their ratios in MG-treated over untreated HEK293T cells. Samples F/R1/R2 were forward and reverse replicate samples prepared in parallel. Sample FE/RE were forward and reverse replicate samples prepared in parallel on a different day. The probe-conjugated lysines were highlighted in blue.

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.GIIPETATLFKSALMPAQLFF Phosphatidylinositol 3-kinase PIK3C3 0.290 0.244 0.373 0.303 0.065 K.T [602, 623] (missed 1) (rank 1) catalytic subunit type 3 R.VLGVIDKVLLVMDTR.T Ribosomal protein S6 kinase RPS6KC1 0.560 0.281 0.450 0.430 0.141 [341, 355] (missed 1) (rank 1) delta-1 R.DLKLDNVMLDSEGHIK.I PRKCA Protein kinase C alpha type 0.810 0.340 0.326 0.293 0.442 0.246 [462, 477] (missed 1) (rank 1) R.ESSYACYYDEKR.S [213, LGMN Legumain 0.880 0.355 0.203 0.479 0.355 224] (missed 1) (rank 1) R.DLKPENILLDSVGHVVLTDF Serine/threonine-protein SGK3 0.540 0.485 0.483 0.503 0.032 GLCK.E [285, 308] (rank 1) kinase Sgk3 R.AAKPVTVAVKMLK.D [498, Fibroblast growth factor FGFR3 0.660 0.510 0.395 0.522 0.133 510] (missed 1) receptor 3 K.TDTGKESCDVVLLDENLLK. 1-phosphatidylinositol 3- PIP5K3 0.670 0.541 0.385 0.532 0.143 M [1962, 1980] (missed 1) (rank 2) phosphate 5-kinase K.DIISELLTSDDMKNAYK.L FAST kinase domain- FASTKD2 0.720 0.463 0.313 0.704 0.550 0.197 [522, 538] (missed 1) (rank 1) containing protein 2 K.EITEGKTVQIPVYDFVSHSR. UCK2 Uridine-cytidine kinase 2 0.590 0.760 0.617 0.280 0.562 0.202 K [99, 118] (missed 1) (rank 2) K.LFGHSGSGKTTLVESLK.C Death-associated protein DAPK1 0.770 0.450 0.508 0.426 0.662 0.563 0.148 [691, 707] (missed 1) kinase 1 K.IGDFGLAKAVPEGHEYYR.V Non-receptor tyrosine-protein TYK2 0.540 0.524 0.667 0.577 0.078 [1038, 1055] (missed 1) (rank 1) kinase TYK2 K.TFIGKIPIMLR.S [146, 156] DNA-directed RNA POLR2B 0.690 0.565 0.498 0.621 0.593 0.082 (missed 1) (rank 1) polymerase II subunit RPB2 R.LFPNADSKALDLLDK.M Mitogen-activated protein MAPK1 0.530 0.400 0.794 0.654 0.594 0.168 [277, 291] (missed 1) (rank 2) kinase 1 K.ITDFGLAKLLGAEEK.E [852, Epidermal growth factor EGFR 0.600 0.625 0.581 0.581 0.597 0.021 866] (missed 1) (rank 2) receptor K.LNVTDSVINKVTEK.S [124, Serine/threonine-protein RIOK1 0.570 0.741 0.481 0.597 0.132 137] (missed 1) (rank 2) kinase RIO1

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Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.GELVAVKAAR.Q [137, 146] Mitogen-activated protein MAP3K11 0.510 0.610 0.680 0.600 0.086 (missed 1) (rank 2) kinase kinase kinase 11 R.AGFTAAYSEK.G [1535, 1544] Insulin-Like Growth Factor 2 IGF2R 0.680 0.790 0.526 0.529 0.481 0.601 0.130 (rank 2) Receptor K.GSFGKVMLADR.K [347, 357] PRKCA Protein kinase C alpha type 0.610 0.410 0.680 0.719 0.613 0.607 0.119 (missed 1) (rank 2) R.DLKLDNLLLDTEGYVK.I Serine/threonine-protein PKN1 0.940 0.538 0.465 0.500 0.611 0.222 [739, 754] (missed 1) (rank 2) kinase N1 R.DIKPENLLLGFR.G [165, 176] AURKC Aurora kinase C 0.580 0.599 0.662 0.614 0.043 K.TLALIKPDAISKAGEIIEIINK. Nucleoside diphosphate kinase NME7 0.580 0.730 0.565 0.625 0.091 A [93, 114] (missed 1) (rank 1) 7 R.DLKPQNILLSYANR.R [130, Serine/threonine-protein ULK2 0.650 0.650 0.585 0.628 0.038 143] (rank 1) kinase ULK2 R.DVAIKVIDK.M [600, 608] Serine/threonine-protein PRKD3 0.547 0.600 0.664 0.694 0.676 0.636 0.061 (missed 1) (rank 2) kinase D3 R.FGSDLQKIYEANAK.R [133, Serine/threonine-protein VRK1 0.610 0.772 0.531 0.638 0.123 146] (missed 1) (rank 2) kinase VRK1 Eukaryotic translation R.DLKPSNIFFTMDDVVK.V EIF2AK3 initiation factor 2-alpha kinase 0.570 0.719 0.658 0.617 0.641 0.063 [936, 951] (rank 1) 3 R.DLKPENILLDDYGHIR.I [310, GRK5 GPCR kinase 5 0.690 0.680 0.617 0.578 0.641 0.053 325] (rank 1) R.ELGEGAFGKVFLAECYNLC BDNF/NT-3 growth factors NTRK2 0.540 0.671 0.725 0.645 0.095 PEQDK.I [542, 565] (missed 1) receptor R.NLIPMDPNGLSDPYVKLK.L PRKCG Protein kinase C gamma type 0.850 0.606 0.578 0.562 0.649 0.135 [181, 198] (missed 1) (rank 1) K.KGSEQESVK.E [8, 16] (missed cAMP-dependent protein PRKACA 0.740 0.710 0.565 0.599 0.633 0.649 0.074 1) (rank 1) kinase catalytic subunit alpha R.KIVSLLAASEAEVEQLLSER. LGMN Legumain 0.530 0.694 0.735 0.653 0.109 A [350, 369] (missed 1) (rank 2) K.AKQEADEEK.H [578, 586] EPHA4 Ephrin type-A receptor 4 0.694 0.744 0.711 0.466 0.654 0.127 (missed 1) (rank 1) 5'-AMP-activated protein R.SLDVVGKIK.R [53, 61] PRKAA2 kinase catalytic subunit alpha- 0.620 0.680 0.662 0.654 0.031 (missed 1) (rank 1) 2

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Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation Microtubule-associated K.LYAVKVVK.K [57, 64] MASTL serine/threonine-protein 0.910 0.613 0.368 0.735 0.657 0.228 (missed 1) (rank 1) kinase like R.KVNAEGSVDSVFSQVCTHL DALK.- [171, 193] (missed 1) AK1 Adenylate kinase isoenzyme 1 0.940 0.515 0.578 0.602 0.659 0.191 (rank 2) K.FADIVSILDKLIR.A [863, 875] EPHA2 Ephrin type-A receptor 2 0.700 0.478 0.613 0.855 0.662 0.158 (missed 1) (rank 3) R.LQAKDEQGPIR.C [434, 444] NTKL N-terminal kinase-like protein 0.730 0.526 0.781 0.621 0.665 0.114 (missed 1) (rank 2) K.KIIQQVLQGLDYLHTK.C SRPK1 SRSF protein kinase 1 0.665 0.677 0.672 0.646 0.665 0.013 [190, 205] (missed 1) (rank 2) R.EIKILQLLK.H [65, 73] (missed CDK9 Cyclin-dependent kinase 9 0.590 0.710 0.641 0.719 0.665 0.061 1) (rank 3) R.DLKPENIVLQDVGGK.I [143, Inhibitor of nuclear factor CHUK 0.800 0.617 0.556 0.694 0.667 0.105 157] (rank 1) kappa-B kinase subunit alpha R.DLKPENLLLDEK.N [140, 151] Serine/threonine-protein BRSK2 0.680 0.641 0.633 0.714 0.667 0.038 (rank 1) kinase BRSK2 K.IFAMKVLK.K [118, 125] Ribosomal protein S6 kinase STK14A 0.920 0.526 0.559 0.668 0.219 (missed 1) (rank 1) beta-1 R.VTKVAVK.M [507, 513] Fibroblast growth factor FGFR1 0.630 0.710 0.641 0.633 0.741 0.671 0.051 (missed 1) receptor 1 R.DIKAANVLLSEHGEVK.L Serine/threonine-protein STK24 0.650 0.690 0.671 0.676 0.672 0.016 [155, 170] (missed 1) (rank 1) kinase 24 K.TLSIFMEYMPGGSIKDQLK.A Mitogen-activated protein MAP3K2 0.820 0.585 0.500 0.787 0.673 0.155 [431, 449] (missed 1) (rank 2) kinase kinase kinase 2 R.FVHSENQHLVSPEALDFLDK Casein kinase 2 isoform alpha LLR.Y [283, 305] (missed 1) (rank CSNK2A1 0.800 0.680 0.559 0.680 0.121 1 2) K.SLLQEAQNNLKIFK.N [198, Inositol-pentakisphosphate 2- IPPK 0.690 0.630 0.719 0.680 0.046 211] (missed 1) (rank 1) kinase K.IEKEAEILSVLSHR.N [49, 62] Mitogen-activated protein ZAK 0.700 0.694 0.645 0.680 0.030 (missed 1) (rank 2) kinase kinase kinase MLT R.EHPFLVKGGEDLR.Q [3746, DNA-dependent protein PRKDC 0.770 0.667 0.592 0.699 0.682 0.074 3758] (missed 1) (rank 3) kinase catalytic subunit R.DLKSNNILLLQPIESDDMEH Mitogen-activated protein MAP3K11 0.550 0.810 0.769 0.633 0.649 0.682 0.106 K.T [240, 260] (missed 1) (rank 1) kinase kinase kinase 11

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Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.KGGSWIQEINVAEK.N [4022, DNA-dependent protein PRKDC 0.890 0.565 0.585 0.690 0.682 0.149 4035] (missed 1) (rank 2) kinase catalytic subunit R.DLKPANIFISTEDLVLK.I Mitogen-activated protein MAPK4 0.920 0.662 0.518 0.641 0.685 0.169 [148, 164] (rank 1) kinase 4 R.DLKPENIMLNHQGHVK.L Ribosomal protein S6 kinase STK14A 0.680 0.769 0.613 0.688 0.078 [217, 232] (rank 2) beta-1 Receptor-interacting K.DLKPENILVDNDFHIK.I [137, RIPK1 serine/threonine-protein 0.673 0.798 0.600 0.690 0.100 152] (rank 1) kinase 1 K.ALYATKTLR.K [728, 736] Serine/threonine-protein LATS1 0.770 0.840 0.581 0.613 0.662 0.693 0.109 (missed 1) (rank 1) kinase LATS1 K.TPQGLSNLAKTIGQVLAR.M EKC/KEOPS complex subunit PRPK 0.630 0.830 0.658 0.685 0.701 0.089 [134, 151] (missed 1) (rank 2) TPRKB R.DLKPSNLLVNENCELK.I [42, Mitogen-activated protein MAPK7 0.900 0.794 0.348 0.800 0.711 0.246 57] (rank 1) kinase 7 R.ITSVAMKSGGTQLK.L [257, Extracellular serine/threonine FAM20C 0.730 0.710 0.752 0.758 0.613 0.713 0.059 270] (missed 1) protein kinase FAM20C K.YASLCGKSGR.L [1655, 1664] Serine/threonine-protein MTOR 0.707 0.707 0.728 0.761 0.726 0.026 (missed 1) (rank 1) kinase Mtor K.TGAKLPVK.W [1240, 1247] Hepatocyte growth factor MET 0.880 0.650 0.685 0.621 0.820 0.731 0.113 (missed 1) (rank 1) receptor R.VKAAQAGR.Q [3, 10] (missed Serine/threonine-protein kinase VRK1 0.930 0.830 0.633 0.658 0.610 0.732 0.141 1) (rank 1) VRK1 R.TGEVVAIKK.I [34, 42] Mitogen-activated protein MAPK15 0.429 0.833 0.814 0.875 0.738 0.208 (missed 1) (rank 1) kinase 15 R.GNEFFCEVDEDYIQDKFNLT GLNEQVPHYR.Q [17, 46] CSNK2B Casein kinase 2 isoform beta 0.830 0.806 0.699 0.621 0.739 0.097 (missed 1) (rank 2) [3-methyl-2-oxobutanoate R.QLLDDHKDVVTLLAEGLR.E BCKDK dehydrogenase [lipoamide]] 0.830 0.800 0.588 0.739 0.132 [156, 173] (missed 1) (rank 2) kinase, mitochondrial Eukaryotic translation R.YLNEFEELAILGKGGYGR.V EIF2AK1 initiation factor 2-alpha kinase 0.790 0.840 0.571 0.794 0.749 0.120 [162, 179] (missed 1) (rank 1) 1 K.LVSDEMVVELIEKNLETPLC AK2 Adenylate kinase isoenzyme 2 1.010 0.658 0.709 0.645 0.756 0.172 K.N [72, 92] (missed 1) (rank 2)

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Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.YAVTDDYQLSKQVLGLGVN MAP kinase-activated protein MAPKAPK3 0.860 0.700 0.885 0.667 0.671 0.757 0.107 GK.V [36, 56] (missed 1) (rank 2) kinase 3 R.STEIGKLLSSYLQK.K [45, 58] TYMK Thymidylate kinase 1.000 0.658 0.781 0.595 0.759 0.179 (missed 1) (rank 3) Phosphatidylinositol 5- K.FKEYCPQVFR.N [99, 108] PIP4K2C phosphate 4-kinase type-2 0.930 1.050 0.671 0.552 0.595 0.760 0.219 (missed 1) (rank 1) gamma K.KIQGLELQQIK.T [255, 265] PX domain-containing protein PXK 0.721 0.803 0.763 0.762 0.041 (missed 1) kinase-like protein R.MLDVLEYIHENEYVHGDIK Serine/threonine-protein AANLLLGYK.N [149, 176] VRK2 0.800 0.763 0.725 0.763 0.038 kinase VRK2 (missed 1) (rank 1) K.ELLVDCYKPTEAFISGLLDK. LIMK2 LIM domain kinase 2 0.813 0.794 0.829 0.702 0.678 0.763 0.068 I [653, 672] (rank 2) K.DIKEIAK.K [239, 245] (missed ADK Adenosine kinase 0.567 0.693 0.920 0.949 0.698 0.765 0.164 1) (rank 2) R.NISHLDLKPQNILLSSLEKPH Serine/threonine-protein ULK3 0.780 0.850 0.730 0.699 0.775 0.767 0.057 LK.L [131, 153] (rank 2) kinase ULK3 R.DLKSSNILLLEK.I [262, 273] Mitogen-activated protein MLK4 0.980 0.685 0.658 0.774 0.179 (missed 1) (rank 1) kinase kinase kinase MLK4 K.DLFGKSDPYLEFHK.Q [153, CPNE3 Copine-3 0.773 0.747 0.806 0.682 0.867 0.775 0.069 166] (missed 1) (rank 2) R.DLKSNNIFLHEDLTVK.I [575, Serine/threonine-protein BRAF 0.920 0.910 0.685 0.610 0.752 0.775 0.137 590] (missed 1) (rank 1) kinase B-raf 5'-AMP-activated protein R.SLDVVGKIR.R [55, 63] PRKAA1 kinase catalytic subunit alpha- 0.760 0.970 0.685 0.565 0.909 0.778 0.165 (missed 1) (rank 2) 1 R.DIKGANIFLTSSGLIK.L Mitogen-activated protein MAP3K4 0.580 0.910 0.763 0.813 0.826 0.779 0.123 [1462, 1477] (missed 1) (rank 1) kinase kinase kinase 4 Calcium/calmodulin- R.FTDDYQLFEELGKGAFSVVR CAMK2G dependent protein kinase type 0.740 0.901 0.694 0.806 0.785 0.090 .R [9, 28] (missed 1) (rank 2) 2G R.AACLLDGVPVALKK.V [50, Serine/threonine-protein NEK7 0.667 0.987 0.794 0.698 0.786 0.144 63] (missed 1) (rank 1) kinase Nek7 PAB-dependent poly(A)- R.IQKSSNFGYITSCYK.A [347, PAN3 specific ribonuclease subunit 0.800 0.643 0.920 0.787 0.139 361] (missed 1) (rank 2) PA

S29

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.KNPQAVLDVLEFYNSK.K Serine/threonine-protein PAK1 0.638 1.008 0.730 0.792 0.193 [118, 133] (missed 1) (rank 1) kinase PAK1 R.DIKPDNILIDLDGHIK.L [790, Serine/threonine-protein LATS2 0.769 0.874 0.734 0.792 0.073 805] (rank 1) kinase LATS2 Testis-specific K.KKASDDYLNK.F [58, 67] TSSK4 serine/threonine-protein 0.710 0.640 0.952 0.833 0.826 0.792 0.121 (missed 2) (rank 1) kinase 4 Leucine-rich repeat R.KILLSK.G [433, 438] (missed LRRK2 serine/threonine-protein 0.771 0.824 0.829 0.794 0.776 0.799 0.027 1) (rank 1) kinase 2 K.YSVKVLPWLSPEVLQQNLQ STE20-related kinase adapter GYDAK.S [235, 258] (missed 1) LYK5 0.910 0.820 0.685 0.787 0.801 0.093 protein alpha (rank 1) R.DTVTIHSVGGTITILPTKTKP Serine/threonine-protein K.K [1486, 1507] (missed 1) (rank SMG1 0.860 0.649 0.893 0.801 0.132 kinase SMG1 1) R.NKTLTEDEIATILQSTLK.G Serine/threonine-protein STK4 0.980 0.730 0.781 0.826 0.690 0.801 0.112 [117, 134] (missed 1) (rank 2) kinase 4 R.DIKPENLLLGSAGELK.I [255, AURKA Aurora kinase A 0.870 0.850 0.800 0.741 0.769 0.806 0.054 270] (rank 1) R.DLKPANLFINTEDLVLK.I Mitogen-activated protein MAPK6 0.880 0.775 0.769 0.808 0.062 [151, 167] (rank 1) kinase 6 R.TKNTLQSR.I [551, 558] GOGA5 Golgin subfamily A member 5 0.634 0.924 0.863 0.890 0.755 0.813 0.118 (missed 1) (rank 1) R.DLKVENILLHDR.G [175, 186] AP2-associated protein kinase AAK1 0.750 0.870 0.735 0.901 0.814 0.084 (missed 1) (rank 1) 1 R.VLQEGKAR.G [521, 528] Cation-independent mannose- IGF2R 0.767 0.711 0.867 0.915 0.815 0.093 (missed 1) (rank 1) 6-phosphate receptor R.SAGKWPLK.W [496, 503] Tyrosine-protein kinase ZAP- ZAP70 0.760 0.769 0.917 0.816 0.088 (missed 1) (rank 1) 70 R.DLKPENVLLSSQEEDCLIK.I Serine/threonine-protein CHEK2 0.698 0.961 0.792 0.817 0.133 [346, 364] (rank 1) kinase Chk2 K.LTHTDLKPENILFVK.S [281, Dual specificity protein kinase CLK4 0.850 0.740 0.893 0.794 0.819 0.067 295] (rank 1) CLK4 K.ELGSGQFGVVQLGKWK.G Cytoplasmic tyrosine-protein BMX 0.770 0.840 0.952 0.714 0.819 0.103 [421, 436] (missed 1) kinase BMX

S30

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.FDPSTKIYEISNR.W [710, EXOSC10 Exosome component 10 0.815 0.946 0.699 0.774 0.867 0.820 0.093 722] (missed 1) (rank 3) R.DLKLDNVLLDSEGHIK.L PRKCI Protein kinase C iota type 0.890 0.813 0.826 0.758 0.822 0.054 [377, 392] (missed 1) (rank 1) R.VPCAYDKTALALEVGDIVK. CRKL Crk-like protein 0.830 0.920 0.826 0.758 0.781 0.823 0.062 V [246, 264] (missed 1) (rank 1) R.YLNEIKPPIIHYDLKPGNILL Serine/threonine-protein VDGTACGEIK.I [594, 624] (rank TLK1 0.780 0.820 0.847 0.847 0.826 0.824 0.028 kinase tousled-like 1 1) R.DLKTQNILLDK.H [127, 137] Serine/threonine-protein NEK8 0.800 0.929 0.919 0.651 0.825 0.130 (missed 1) (rank 1) kinase Nek8 K.ELFDELVKK.F [46, 54] ADK Adenosine kinase 0.794 0.834 0.871 0.810 0.827 0.033 (missed 1) (rank 1) R.LPVIDPESGNTLYILTHKR.I 5'-AMP-activated protein PRKAG1 0.815 0.874 0.808 0.818 0.829 0.030 [161, 179] (missed 1) (rank 1) kinase subunit gamma-1 K.ISIEGNIAAGKSTFVNILK.Q DCK Deoxycytidine kinase 0.960 0.833 0.699 0.831 0.130 [23, 41] (missed 1) (rank 1) K.EVPVAIKTLK.A [639, 648] EPHA2 Ephrin type-A receptor 2 0.717 0.994 0.783 0.831 0.145 (missed 1) (rank 2) Testis-specific K.GTVAIKVVDR.R [35, 44] TSSK6 serine/threonine-protein 0.890 0.850 0.813 0.781 0.826 0.832 0.041 (missed 1) kinase 6 R.DLKPSNLLINTTCDLK.I [165, Mitogen-activated protein MAPK3 0.810 0.710 0.926 0.885 0.833 0.095 180] (rank 1) kinase 3 R.DIKGDNVLINTYSGVLK.I Mitogen-activated protein MAP3K5 0.830 0.840 0.826 0.840 0.834 0.007 [802, 818] (missed 1) (rank 1) kinase kinase kinase 5 R.VKELLVDCYKPTEAFISGLL DK.I [651, 672] (missed 1) (rank LIMK2 LIM domain kinase 2 0.828 0.856 0.769 0.900 0.838 0.055 1) Microtubule-associated R.DLKPDNLLITSLGHIK.L [489, MAST3 serine/threonine-protein 0.940 0.800 0.719 0.901 0.840 0.100 504] (rank 1) kinase 3 R.DIKPDNFLMGTGR.H [135, Casein kinase I isoform alpha- CSNK1A1L 0.665 0.901 0.786 0.914 0.937 0.841 0.114 147] like R.VPHTQAVVLNSKDK.A [380, Phosphatidylinositol 4-kinase PI4KB 0.890 0.760 0.962 0.752 0.841 0.102 393] (missed 1) (rank 2) beta

S31

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.SYPYLFKGLEDLHLDER.I Serine/threonine-protein SMG1 0.777 0.900 0.850 0.850 0.844 0.051 [1518, 1534] (missed 1) (rank 2) kinase SMG1 R.NCTIVSPDAGGAKR.V [163, Ribose-phosphate PRPS1 0.890 0.820 0.826 0.845 0.039 176] (missed 1) (rank 1) pyrophosphokinase 1 K.IPVAIKELR.E [739, 747] Epidermal growth factor EGFR 0.743 0.827 0.977 0.735 0.957 0.848 0.115 (missed 1) (rank 1) receptor K.KQILR.T [527, 531] (missed 1) JAK1 Tyrosine-protein kinase JAK1 0.240 1.010 0.952 0.971 1.075 0.850 0.344 (rank 1) R.DIKPQNLLVDPDTAVLK.L Glycogen synthase kinase-3 GSK3A 0.762 0.918 0.870 0.850 0.080 [243, 259] (rank 1) alpha R.KDGLVSLLTTSEGADEPQR. Cyclin-G-associated-like GALK1 0.849 0.741 0.967 0.955 0.753 0.853 0.107 L [98, 116] (missed 1) (rank 1) kinase 1 Phosphatidylinositol 5- K.ELPTLKDMDFLNK.N [235, PIP4K2C phosphate 4-kinase type-2 0.890 0.920 0.877 0.813 0.769 0.854 0.061 247] (missed 1) (rank 2) gamma R.DIKPDNILLDR.C [194, 204] DMPK Myotonin-protein kinase 0.806 0.881 0.885 0.846 0.854 0.037 (rank 1) R.DLKPENIVLQQGEQR.L [144, Inhibitor of nuclear factor IKBKB 0.940 0.833 0.800 0.858 0.073 158] (rank 1) kappa-B kinase subunit beta R.DVKAGNILLSEPGLVK.L Serine/threonine-protein TAOK2 1.030 0.970 0.741 0.725 0.826 0.858 0.137 [150, 165] (missed 1) (rank 1) kinase TAOK2 Calcium/calmodulin- R.DIKPSNLLVGEDGHIK.I [311, CAMKK2 dependent protein kinase 0.924 0.900 0.929 0.688 0.860 0.115 326] (rank 1) kinase 2 R.VGMIPVPYVEKLVR.S [169, CRKL Crk-like protein 0.994 0.667 0.767 0.897 0.982 0.861 0.142 182] (missed 1) (rank 2) R.DLKPQNLLINR.N [125, 135] CDK5 and ABL1 enzyme CABLES1 0.970 0.840 0.806 0.833 0.863 0.073 (rank 1) substrate 1 Phosphatidylinositol 5- K.DLPTFKDNDFLNEGQK.L PIP4K2B phosphate 4-kinase type-2 0.890 0.850 0.893 0.855 0.826 0.863 0.028 [233, 248] (missed 1) (rank 1) beta R.DLKPQNLLINEK.G [312, 323] CDK17 Cyclin-dependent kinase 17 0.784 0.921 0.885 0.863 0.071 (rank 1) R.DIKPQNLLLDPDTAVLK.L Glycogen synthase kinase-3 GSK3B 0.781 0.960 0.849 0.864 0.090 [180, 196] (rank 1) beta K.ETGQIVAIKQVPVESDLQEII Serine/threonine-protein STK4 0.824 0.884 0.902 0.845 0.864 0.036 K.E [50, 71] (missed 1) (rank 1) kinase 4

S32

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.LGDYGLAKK.L [191, 199] Serine/threonine-protein NEK9 0.950 0.940 0.826 0.787 0.833 0.867 0.073 (missed 1) (rank 1) kinase Nek9 K.EKELTELR.N [38, 45] (missed Serine/threonine-protein CARK 0.871 0.859 0.909 0.842 0.870 0.029 1) (rank 1) kinase TNNI3K R.LSKGDGLEFK.R [442, 451] Serine/threonine-protein CHEK1 1.100 0.910 0.800 0.769 0.781 0.872 0.139 (missed 1) (rank 1) kinase Chk1 R.LATLPTKEQK.T [338, 347] Phosphatidylinositol 4-kinase PI4KB 0.890 0.870 0.893 0.877 0.840 0.874 0.021 (missed 1) (rank 1) beta R.DLKPANLLISASGQLK.I [126, CDK20 Cyclin-dependent kinase 20 0.900 0.890 0.840 0.943 0.800 0.875 0.056 141] (rank 1) R.DLKPENILLDR.D [171, 181] cAMP-dependent protein PRKX 0.920 0.862 0.855 0.870 0.877 0.030 (rank 1) kinase catalytic subunit PRKX K.IGEGSFGKAILVK.S [9, 21] Serine/threonine-protein NEK1 0.900 0.833 0.916 0.811 0.923 0.877 0.051 (missed 1) (rank 2) kinase Nek1 R.GKLQLVVLR.D [374, 382] TJP2 Tight junction protein ZO-2 1.210 0.820 0.719 0.758 0.877 0.877 0.196 (missed 1) (rank 1) R.IKNCLNPQFSK.T [50, 60] CPNE3 Copine-3 0.880 0.920 0.840 0.917 0.826 0.877 0.043 (missed 1) (rank 1) R.GLEAGEESTPVALKTVNELA Insulin receptor-related INSRR 0.890 0.850 0.962 0.893 0.794 0.878 0.062 SPR.E [999, 1021] (missed 1) protein R.AESIHTGLEVAIKMIDK.K Serine/threonine-protein PLK4 0.920 0.850 0.870 0.880 0.036 [28, 44] (missed 1) kinase PLK4 R.DLKSNNIFLHEGLTVK.I [431, Serine/threonine-protein ARAF 1.030 1.020 0.625 0.709 1.031 0.883 0.199 446] (missed 1) (rank 1) kinase A-Raf R.DLKPENILLDEIGHIK.L [197, Ribosomal protein S6 kinase RSK4 0.909 0.841 0.936 0.846 0.883 0.047 212] (rank 1) alpha-6 Eukaryotic translation K.IGDFGLVTSLKNDGK.R [429, EIF2AK2 initiation factor 2-alpha kinase 0.862 0.973 0.815 0.883 0.081 443] (missed 1) (rank 2) 2 TFIIH basal transcription -.MATSSEEVLLIVKK.V [0, 13] BTF2 factor complex helicase XPD 0.768 0.884 0.837 0.778 1.152 0.884 0.157 (missed 1) (rank 1) subunit K.LVALKVIR.L [159, 166] CDK14 Cyclin-dependent kinase 14 0.825 0.941 0.899 0.888 0.059 (missed 1) (rank 2) R.FLSGLELVKQGAEAR.V [31, EKC/KEOPS complex subunit PRPK 0.913 0.855 0.914 0.873 0.889 0.029 45] (missed 1) (rank 1) TPRKB

S33

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.VAVKNIK.C [258, 264] Megakaryocyte-associated MATK 0.847 0.820 1.097 0.724 0.961 0.890 0.143 (missed 1) tyrosine-protein kinase Calcium/calmodulin- K.IPTGQEYAAKIINTK.K [33, CAMK2D dependent protein kinase type 0.813 0.895 0.886 0.876 0.979 0.890 0.059 47] (missed 1) (rank 1) 2D TFIIH basal transcription R.TVPEIEKVIEELR.K [75, 87] ERCC2 factor complex helicase XPD 0.900 0.909 0.862 0.890 0.025 (missed 1) (rank 1) subunit R.IEIAQKHPDIYAVPIK.T TJP2 Tight junction protein ZO-2 0.806 0.894 0.967 0.775 1.026 0.894 0.105 [1107, 1122] (missed 1) (rank 2) R.FLEDDTSDPTYTSALGGKIPI EPHB2 Ephrin type-B receptor 2 0.827 0.893 0.926 0.926 0.898 0.894 0.041 R.W [769, 790] (missed 1) (rank 1) Phosphorylase b kinase R.ATGHEFAVKIMEVTAER.L PHKG2 gamma catalytic chain, 0.900 0.953 0.987 0.932 0.725 0.899 0.103 [44, 60] (missed 1) (rank 1) liver/testis isoform K.SAIEKLTHDYINLFHFPPLIK. Mitogen-activated protein ZAK 0.830 1.000 0.909 0.758 1.000 0.899 0.106 D [401, 421] (missed 1) (rank 1) kinase kinase kinase MLT Dual specificity mitogen- R.DVKPSNMLVNTR.G [282, MAP2K5 activated protein kinase kinase 0.980 1.150 0.826 0.704 0.840 0.900 0.170 293] (rank 1) 5 K.CLVIKYAK.D [49, 56] (missed TK1 Thymidine kinase, cytosolic 0.862 0.952 0.863 0.873 0.954 0.901 0.048 1) (rank 1) K.TIELSDDDFLGECECTLGQIV SSKK.L [85, 109] (missed 1) (rank CPNE3 Copine-3 0.920 0.820 0.980 0.917 0.870 0.901 0.060 3) K.GNLVAVKR.V [560, 567] Atrial natriuretic peptide NPR1 0.879 0.860 0.924 0.851 0.996 0.902 0.060 (missed 1) (rank 1) receptor 1 Phosphatidylinositol 4- K.DLVTEDGADPNPYVKTYLL phosphate 3-kinase C2 PDNHK.T [1582, 1605] (missed 1) PIK3C2A 0.870 0.930 0.971 0.893 0.847 0.902 0.049 domain-containing subunit (rank 1) alpha Phosphatidylinositol 4,5- R.IMSSAKR.P [770, 776] (missed PIK3CA bisphosphate 3-kinase 1.030 1.020 0.893 0.787 0.781 0.902 0.121 1) (rank 1) catalytic subunit alpha isoform R.DIKPENILVDR.T [220, 230] CIT Citron Rho-interacting kinase 1.100 1.170 0.694 0.714 0.833 0.902 0.220 (rank 2)

S34

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.DMKAANVLITR.D [148, 158] CDK9 Cyclin-dependent kinase 9 0.842 1.019 0.850 0.904 0.100 (missed 1) (rank 1) Calcium/calmodulin- R.DIKPSNLLLGDDGHVK.I CAMKK1 dependent protein kinase 0.912 0.900 0.914 0.899 0.906 0.008 [274, 289] (rank 1) kinase 1 R.DLKPENLLVQR.N [476, 486] Serine/threonine-protein DCLK3 1.010 0.950 0.855 0.840 0.877 0.906 0.072 (rank 1) kinase DCLK3 R.DLKPSNLLLNTTCDLK.I Mitogen-activated protein MAPK1 0.920 0.888 0.919 0.906 0.908 0.015 [148, 163] (rank 1) kinase 1 K.LKGAVLAAVSSHK.F [303, Peripheral plasma membrane CASK 0.908 0.841 0.927 0.825 1.042 0.908 0.086 315] (missed 1) (rank 2) protein CASK R.DIKSQNIFLTK.D [127, 137] Serine/threonine-protein NEK1 0.855 0.983 0.880 0.854 0.983 0.911 0.067 (missed 1) (rank 1) kinase Nek1 K.INEVQTDVGVDTKHQTLQG VAFPISR.E [192, 217] (missed 1) TWF1 Twinfilin-1 0.890 0.920 1.020 0.893 0.833 0.911 0.069 (rank 2) PAB-dependent poly(A)- R.VMDPTKILITGK.T [493, 504] PAN3 specific ribonuclease subunit 0.840 0.800 1.111 0.935 0.877 0.913 0.122 (missed 1) (rank 1) PA Serine/threonine-protein K.ESEVKALCAK.A [21, 30] PP4C phosphatase 4 catalytic 1.058 0.803 0.878 0.913 0.131 (missed 1) (rank 1) subunit Phosphatidylinositol 5- K.ELPTLKDNDFINEGQK.I PIP4K2A phosphate 4-kinase type-2 0.930 0.880 0.971 0.935 0.855 0.914 0.046 [228, 243] (missed 1) (rank 1) alpha R.DLKPENVVFFEK.Q [138, 149] Serine/threonine-protein SNRK 1.050 1.520 0.775 0.746 0.488 0.916 0.392 (rank 1) kinase SNRK R.DVAVAIKTLK.V [658, 667] EPHA7 Ephrin type-A receptor 7 0.890 0.920 0.952 0.901 0.917 0.916 0.024 (missed 1) R.DIKGDNVLINTFSGLLK.I Mitogen-activated protein MAP3K6 0.931 0.846 1.000 0.839 0.970 0.917 0.073 [770, 786] (missed 1) (rank 1) kinase kinase kinase 6 R.DLKLENIMVK.S [237, 246] Serine/threonine-protein STK33 0.897 0.889 0.940 0.920 0.940 0.917 0.024 (missed 1) (rank 1) kinase 33 Dual specificity mitogen- R.DIKPSNILLDR.S [239, 249] MAP2K4 activated protein kinase kinase 1.000 0.980 0.847 0.813 0.952 0.919 0.083 (rank 1) 4

S35

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.DIKPGNLLLTTGGTLK.I [175, Serine/threonine-protein STK11 1.110 0.990 0.833 0.862 0.806 0.920 0.127 190] (rank 1) kinase STK11 R.DLKAGNILFTLDGDIK.L STE20-like serine/threonine- SLK 0.960 0.970 0.885 0.820 0.971 0.921 0.067 [154, 169] (missed 1) (rank 1) protein kinase R.FCTGLTQIETLFKSK.D [252, CKB Creatine kinase B-type 0.938 0.854 1.011 0.941 0.865 0.922 0.064 266] (missed 1) (rank 2) R.DLKPSNILYVDESGNPECLR. MAPKAPK1 Ribosomal protein S6 kinase 0.890 0.950 0.952 0.917 0.901 0.922 0.028 I [534, 553] (rank 1) A alpha-1 R.VTMNEFEYLKLLGK.G [144, RAC-alpha serine/threonine- AKT1 0.925 0.840 0.880 0.809 1.161 0.923 0.140 157] (missed 1) (rank 1) protein kinase 1 R.DLKPNNLLLDENGVLK.L CDK7 Cyclin-dependent kinase 7 0.886 0.950 0.986 0.870 0.923 0.055 [136, 151] (rank 1) Eukaryotic translation R.DLKPVNIFLDSDDHVK.I EIF2AK4 initiation factor 2-alpha kinase 0.836 0.986 0.986 0.897 0.915 0.924 0.064 [847, 862] (rank 1) 4 R.VIEDDPEAVYTTTGGKIPVR. EPHA7 Ephrin type-A receptor 7 0.930 0.910 0.893 0.935 0.952 0.924 0.023 W [781, 800] (missed 1) (rank 1) R.DIKAQNIFLSK.N [127, 137] Serine/threonine-protein NEK5 0.966 0.874 0.907 0.967 0.911 0.925 0.040 (missed 1) kinase Nek5 R.AIEKLAVEALSSLDGDLAGR CKB Creatine kinase B-type 0.932 0.874 0.953 0.943 0.923 0.925 0.031 .Y [152, 171] (missed 1) (rank 1) K.IPVAIKVLR.E [747, 755] Receptor tyrosine-protein ERBB2 0.837 0.920 0.724 1.072 1.077 0.926 0.152 (missed 1) (rank 1) kinase erbB-2 Testis-specific R.DLKCENLLLDK.D [135, 145] TSSK2 serine/threonine-protein 0.837 1.075 0.860 0.935 0.927 0.107 (missed 1) kinase 2 K.NKSELLPTVR.L [1323, 1332] CAD CAD protein 0.950 0.920 0.962 0.917 0.885 0.927 0.030 (missed 1) (rank 2) R.DLKTENLLLDGNMDIK.L Serine/threonine-protein SIK1 0.940 0.920 0.870 0.980 0.927 0.046 [148, 163] (missed 1) kinase SIK1 R.ITKNVPK.Q [1234, 1240] ATM Serine-protein kinase ATM 0.938 0.900 0.942 0.922 0.935 0.928 0.017 (missed 1) (rank 1) R.ALLYKR.H [7955, 7960] OBSCN Obscurin 0.941 0.971 0.890 0.872 0.966 0.928 0.045 (missed 1) (rank 1) R.QPVDAVKLGLPDYHK.I [100, Bromodomain-containing BRD2 0.965 0.849 0.942 0.959 0.925 0.928 0.047 114] (missed 1) (rank 1) protein 2

S36

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.MLVAVKALK.D [566, 574] NTRK3 NT-3 growth factor receptor 0.930 0.879 0.898 0.898 1.037 0.929 0.063 (missed 1) R.VLEDDPEATYTTSGGKIPIR. EPHA2 Ephrin type-A receptor 2 0.983 0.819 0.838 0.929 1.077 0.929 0.106 W [762, 781] (missed 1) (rank 1) R.SLETSLVPLSDPKLAVLITNS Cyclin-G-associated-like NVR.H [234, 257] (missed 1) GALK1 0.997 0.858 0.916 0.949 0.930 0.058 kinase 1 (rank 2) R.DIKTLNIFLTK.A [175, 185] Serine/threonine-protein NEK9 0.857 0.988 0.988 0.866 0.955 0.931 0.065 (missed 1) (rank 2) kinase Nek9 Calcium/calmodulin- R.DLKPENLLYATPAPDAPLK.I CAMK4 dependent protein kinase type 0.871 0.958 0.957 0.903 0.980 0.934 0.045 [163, 181] (rank 1) 4 R.DLKPQNILLSNPAGR.R [137, Serine/threonine-protein ULK1 1.090 0.890 0.833 0.935 0.926 0.935 0.095 151] (rank 1) kinase ULK1 K.IDDKAFSLKQR.E [4489, TTN Titin 1.028 0.797 0.909 0.890 1.051 0.935 0.105 4499] (missed 2) (rank 1) R.ELASKQVQFDPDSPETSK.E Mitogen-activated protein MAP3K3 0.943 0.884 0.888 0.918 1.045 0.935 0.066 [386, 403] (missed 1) (rank 1) kinase kinase kinase 3 K.VAVKSLK.Q [313, 319] LCK Tyrosine-protein kinase Lck 0.971 0.853 1.024 0.846 0.983 0.935 0.081 (missed 1) (rank 1) R.DLKLDNLLLDTEGFVK.I Serine/threonine-protein PKN2 0.960 0.930 0.926 0.917 0.943 0.935 0.017 [781, 796] (missed 1) (rank 1) kinase N2 Dual specificity mitogen- R.DVKPSNVLINALGQVK.M MAP2K6 activated protein kinase kinase 0.860 0.970 0.971 0.943 0.936 0.052 [178, 193] (rank 1) 6 K.QLMTLENKLK.A [476, 485] Serine/threonine-protein TAOK1 0.949 0.888 0.941 0.935 0.971 0.937 0.030 (missed 1) (rank 2) kinase TAOK1 R.DLKPQNLLISDTGELK.L CDK14 Cyclin-dependent kinase 14 0.950 0.877 0.909 1.020 0.939 0.062 [255, 270] (rank 1) R.DLKPSNLAVNEDCELK.I Mitogen-activated protein MAPK14 0.992 0.883 0.916 0.968 0.940 0.049 [149, 164] (rank 1) kinase 14 R.DLKPQNLLINTEGAIK.L CDK2 Cyclin-dependent kinase 2 0.985 0.862 1.000 0.915 0.942 0.941 0.056 [126, 141] (rank 2) K.NLVPMDPNGLSDPYVKLK.L PRKCB Protein kinase C beta type 0.960 0.940 0.962 0.926 0.917 0.941 0.020 [181, 198] (missed 1) (rank 1)

S37

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation Eukaryotic translation R.DLKPSNIFLVDTK.Q [413, EIF2AK2 initiation factor 2-alpha kinase 0.921 0.951 1.028 0.866 0.941 0.068 425] (rank 1) 2 R.DLKPQNLLIDDK.G [133, 144] CCNB1 G2/mitotic-specific cyclin-B1 0.954 0.895 0.970 0.918 0.970 0.942 0.034 (rank 1) K.YPCVLDLKMGTR.Q [218, Inositol hexakisphosphate IP6K1 0.930 0.940 0.952 0.971 0.917 0.942 0.021 229] (missed 1) (rank 2) kinase 1 R.GLQVVAVK.K [787, 794] ITGAE Integrin alpha-E 0.790 0.830 0.917 0.885 1.316 0.948 0.212 R.DTLLALAHLHSQGLVHLDV Membrane-associated KPANIFLGPR.G [215, 243] (rank PKMYT1 tyrosine- and threonine- 0.992 0.950 0.945 0.992 0.863 0.948 0.052 1) specific cdc2-inhibitory kinase K.TSVPVLDAELFIPPKINR.R IKBKAP Elongator complex protein 1 1.250 0.910 0.775 0.862 0.949 0.208 [1304, 1321] (missed 1) (rank 2) R.NKYYGR.G [276, 281] (missed Inositol hexakisphosphate IP6K1 0.940 0.960 0.962 0.935 0.952 0.950 0.012 1) (rank 1) kinase 1 R.KVEAPFIPKFK.G [301, 311] cAMP-dependent protein PRKACA 1.100 0.745 1.153 0.934 0.822 0.951 0.175 (missed 2) (rank 1) kinase catalytic subunit alpha K.KSLAYVDNILK.K [76, 86] Serine/threonine-protein PKN2 0.930 0.940 0.962 0.971 0.952 0.951 0.016 (missed 1) (rank 2) kinase N2 K.VAVKTLKK.G [1976, 1983] Proto-oncogene tyrosine- ROS1 0.980 0.930 0.962 0.971 0.917 0.952 0.027 (missed 2) protein kinase ROS R.DLKPENILVTSGGTVK.L CCND1 G2/mitotic-specific cyclin-D1 0.996 0.854 0.815 0.959 1.136 0.952 0.126 [139, 154] (rank 1) Phosphatidylinositol 4,5- K.VNWLAHNVSKDNRQ.- PIK3CD bisphosphate 3-kinase 0.980 0.950 0.962 0.971 0.901 0.953 0.031 [1030, 1043] (missed 2) catalytic subunit delta isoform R.LIKDDEYNPCQGSKFPIK.W FGR Tyrosine-protein kinase Fgr 0.919 0.954 0.886 0.951 1.056 0.953 0.064 [405, 422] (missed 2) (rank 1) R.IHISQEDNVANKQTLASYR.S ATP-dependent chromatin ACF1 1.028 0.850 1.013 1.007 0.875 0.954 0.085 [282, 300] (missed 1) assembly fa… R.DLKTQNVFLTR.T [130, 140] Serine/threonine-protein NEK4 0.861 1.019 0.986 0.895 1.011 0.955 0.072 (missed 1) (rank 1) kinase Nek4 R.DTQTDEIVALKK.V [57, 68] CDK10 Cyclin-dependent kinase 10 0.963 0.922 0.974 0.979 0.937 0.955 0.025 (missed 1) Ovarian-specific R.DLKAGNVLMTLEGDIR.L STK10/LOK serine/threonine-protein 0.966 0.909 0.926 0.893 1.081 0.955 0.075 [156, 171] (missed 1) (rank 1) kinase Lok

S38

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.KVQIFDLMDAK.A [63, 73] Serine/threonine-protein NEK7 0.988 0.838 0.759 0.954 1.237 0.955 0.182 (missed 1) (rank 2) kinase Nek7 R.LVAIKSIR.K [120, 127] NUAK family SNF1-like NUAK2 1.041 0.844 0.917 0.974 1.001 0.955 0.077 (missed 1) (rank 1) kinase 2 R.IYQGSSGSYFVKDPQGR.I Phosphatidylinositol 4-kinase PI4K2A 1.160 0.901 0.806 0.956 0.183 [129, 145] (missed 1) (rank 2) type 2-alpha R.SVKASHILISVDGK.V [194, STE20-related kinase adapter LYK5 1.050 1.120 0.787 0.862 0.962 0.956 0.135 207] (missed 1) (rank 2) protein alpha R.GMVAILIAFMKQR.R [18, 30] Serine/threonine-protein SGK1 0.980 0.990 0.909 0.971 0.935 0.957 0.034 (missed 1) (rank 1) kinase Sgk1 K.LNLLTEYIEGGTLKDFLR.S LIMK2 LIM domain kinase 2 0.829 1.020 0.970 0.786 1.181 0.957 0.158 [400, 417] (missed 1) (rank 1) R.IAKVLGTEELYGYLK.K [245, Casein kinase 2 isoform alpha CSNK2A2 1.180 1.000 0.935 0.840 0.840 0.959 0.141 259] (missed 1) (rank 4) 2 ATP-dependent 6- K.LLAHQKPPK.E [386, 394] PFKL phosphofructokinase, liver 1.280 1.080 0.847 0.769 0.820 0.959 0.215 (rank 2) type K.VLGTGAYGKVFLVR.K [37, Ribosomal protein S6 kinase MSK2 1.030 0.920 0.862 0.971 1.020 0.961 0.070 50] (missed 1) (rank 1) alpha-4 R.DLKPPNLLLVAGGTVLK.I Mitogen-activated protein MAP3K7 0.970 0.950 0.971 0.962 0.952 0.961 0.010 [155, 171] (rank 1) kinase kinase kinase 7 Microtubule-associated R.GAFGKVYLGQK.G [43, 53] MASTL serine/threonine-protein 0.920 0.930 0.962 0.980 1.020 0.962 0.040 (missed 1) (rank 2) kinase like R.KEVAALR.E [610, 616] Serine/threonine-protein DMPK2 1.086 0.814 1.028 0.982 0.902 0.963 0.107 (missed 1) (rank 1) kinase MRCK gamma K.SPASAKR.A [188, 194] Phosphoribosyl pyrophosphate PRPSAP2 0.912 0.937 0.891 1.115 0.964 0.102 (missed 1) (rank 1) synthase-associated protein 2 R.DVKPDNILLDER.G [215, 226] Serine/threonine-protein YANK3 1.001 0.863 0.856 0.865 1.233 0.964 0.162 (rank 2) kinase 32C K.ALVACKIYR.S [651, 659] CHAK1 CHAK1 0.898 1.020 0.811 1.126 0.964 0.138 (missed 1) K.VAIKTLKPGTMMPEAFLQE AQIMK.K [301, 324] (missed 1) YES1 Tyrosine-protein kinase Yes 1.020 1.330 0.592 1.190 0.690 0.964 0.317 (rank 1) R.WCGEDVAVKIFSSR.D [213, Receptor protein ALK7 0.970 0.980 0.943 0.965 0.019 226] (missed 1) serine/threonine kinase 7

S39

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.AGKSTQSR.K [16, 23] (missed TYMK Thymidylate kinase 0.980 0.960 0.971 0.952 0.962 0.965 0.011 1) (rank 1) R.IGKGSFGEVYK.G [25, 35] Serine/threonine-protein YSK1 0.777 1.128 1.073 0.929 0.920 0.965 0.139 (missed 1) (rank 1) kinase 25 K.DKVGILQHPDGTVLK.Q [60, Inositol polyphosphate IPMK 0.970 1.070 0.862 1.053 0.877 0.966 0.096 74] (missed 1) (rank 1) multikinase R.NQAIEELEKSIAVAEAACPGI Serine/threonine-protein TDK.M [312, 335] (missed 1) MASK 0.890 0.920 1.099 0.971 0.952 0.966 0.080 kinase 26 (rank 1) R.MFDGKFVVATFYPLSAYK.R Serine/threonine-protein UHMK1 0.920 0.940 0.971 1.042 0.962 0.967 0.046 [392, 409] (missed 1) kinase Kist R.DVYEEDSYVKR.S [897, 907] Proto-oncogene tyrosine- RET 1.033 0.800 1.059 0.702 1.255 0.970 0.220 (missed 1) protein kinase receptor Ret R.KAILSPK.E [995, 1001] CHAK2 CHAK2 0.944 0.971 1.090 0.928 0.917 0.970 0.070 (missed 1) K.VAIKILDK.T [99, 106] (missed Serine/threonine-protein NIM1K 0.781 1.135 0.881 0.994 1.062 0.970 0.142 1) kinase NIM1K K.EINFEKMPIQEK.E [33, 44] Serine/threonine-protein NEK5 1.061 0.861 0.971 1.040 0.920 0.971 0.083 (missed 1) (rank 1) kinase Nek5 R.ISMADVKFSFQCPGR.M [334, ILK Integrin-linked protein kinase 1.100 1.120 0.885 0.719 1.031 0.971 0.168 348] (missed 1) (rank 1) PAS domain-containing R.DGTTFPLSLKLK.S [304, 315] PASK serine/threonine-protein 0.926 0.990 0.961 1.094 0.885 0.971 0.079 (missed 1) (rank 1) kinase R.FMEELGECAFGKIYK.G [474, Inactive tyrosine-protein kinase ROR1 0.939 0.919 0.954 1.082 0.973 0.074 488] (missed 1) transmembrane receptor ROR1 K.VLGTEELYGYLKK.Y [248, Casein kinase 2 isoform alpha CSNK2A2 1.111 0.812 0.883 1.080 0.982 0.974 0.127 260] (missed 1) (rank 2) 2 R.DLKPQNILVTSSGQIK.L [144, BCL1 Beta-lactamase 0.947 0.964 1.031 0.957 0.975 0.038 159] (rank 1) K.YSLTVAVKTLK.E [263, 273] ABL1 Tyrosine-protein kinase ABL1 0.950 0.970 0.952 0.980 1.020 0.975 0.029 (missed 1) (rank 1) R.DIKPENLLLGLK.G [199, 210] AURKB Aurora kinase B 0.901 0.975 0.942 0.780 1.280 0.976 0.186 (rank 1) K.KYLLSQSSPAPLTAAEEELR. TWF1 Twinfilin-1 0.930 1.030 0.855 1.087 0.980 0.976 0.090 Q [169, 188] (missed 1) (rank 1)

S40

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.IISIFSGTEKGSK.K [52, 64] Serine/threonine-protein PAK2 0.920 0.940 0.962 1.020 1.042 0.977 0.052 (missed 1) (rank 4) kinase PAK2 K.LIDFGLCAKPK.G [147, 157] Maternal embryonic leucine MELK 1.070 1.000 0.885 0.952 0.977 0.078 (rank 2) zipper kinase R.EKFLQEALTMR.Q [465, 475] PTK2 Focal adhesion kinase 1 1.040 1.080 0.893 0.980 0.901 0.979 0.083 (missed 1) (rank 1) R.EYLIEKLLTR.T [345, 354] STE20/SPS1-related proline- SPAK 0.980 0.893 1.064 0.979 0.085 (missed 1) (rank 2) alanine-rich protein kinase Calcium/calmodulin- R.FTDEYQLFEELGKGAFSVVR CAMK2D dependent protein kinase type 0.940 1.000 1.064 0.899 0.993 0.979 0.063 .R [9, 28] (missed 1) (rank 2) 2D R.LGSGKFGQVFR.L [1400, Myosin light chain kinase, MYLK 1.220 0.847 0.870 0.979 0.209 1410] (missed 1) (rank 1) smooth muscle Calcium/calmodulin- R.DLKPENLLLASKLK.G [134, CAMK2A dependent protein kinase type 0.960 0.984 1.017 0.961 0.980 0.027 147] (missed 1) (rank 1) 2A K.MAILQIMKELNK.R [296, Transcription intermediary TRIM28 0.970 1.030 0.962 0.962 0.980 0.981 0.029 307] (missed 1) (rank 4) factor 1-beta Dual specificity mitogen- R.DVKPSNVLINK.E [160, 170] MAP2K3 activated protein kinase kinase 0.973 0.973 0.977 0.916 1.065 0.981 0.053 (rank 1) 3 K.VTMNDFDYLKLLGK.G [146, RAC-alpha serine/threonine- AKT2 1.320 1.140 0.840 0.613 0.990 0.981 0.272 159] (missed 1) (rank 1) protein kinase 2 R.DVKGANLLIDSTGQR.L Mitogen-activated protein MAP3K1 1.117 0.828 1.049 1.016 0.895 0.981 0.117 [1368, 1382] (missed 1) (rank 1) kinase kinase kinase 1 K.GSFGKVMLAR.V [363, 372] PRKCH Protein kinase C eta type 0.980 0.990 0.980 0.990 0.971 0.982 0.008 (missed 1) K.VADKIQLINNMLDK.V [216, PGK1 Phosphoglycerate kinase 1 1.040 0.950 0.893 0.935 1.099 0.983 0.084 229] (missed 1) (rank 6) R.DLKAENLLLDANLNIK.I Serine/threonine-protein SIK3 1.110 1.190 0.813 0.943 0.862 0.984 0.161 [187, 202] (missed 1) (rank 1) kinase SIK3 K.NLALLKAR.S [35, 42] (missed Mitogen-activated protein ERK5 0.920 1.020 0.962 0.980 1.042 0.985 0.048 1) (rank 2) kinase 7 ATP-dependent 6- R.VLPGKYLEEIATQMR.T [481, PFKP phosphofructokinase, platelet 0.960 0.970 0.962 0.980 1.064 0.987 0.044 495] (missed 1) (rank 2) type

S41

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.DIKGANILINDAGEVR.L Mitogen-activated protein kinase kinase MAP4K1 0.944 1.060 0.813 1.134 0.987 0.140 [136, 151] (missed 1) (rank 1) kinase kinase 1 R.DIKPDNLLLDSK.G [211, 222] Serine/threonine-protein STK38 1.050 0.920 1.020 0.980 0.971 0.988 0.050 (rank 1) kinase 38 R.HGALTCLCVAMKR.Q [625, Serine/threonine-protein CDC42BPA 0.980 1.030 1.053 0.893 0.990 0.989 0.061 637] (missed 1) kinase MRCK alpha R.LPVIDPISGNALYILTHKR.I 5'-AMP-activated protein PRKAG2 1.080 0.892 0.971 0.983 1.021 0.989 0.069 [384, 402] (missed 1) (rank 1) kinase subunit gamma-2 R.DLKPHNILISMPNAHGK.I Serine/threonine-protein ERN1 1.154 0.769 0.867 1.300 0.867 0.991 0.225 [687, 703] (rank 1) kinase/endoribonuclease IRE1 R.NTVYAVKR.L [232, 239] Interleukin-1 receptor- IRAK1 1.010 1.070 0.909 0.917 1.053 0.992 0.075 (missed 1) associated kinase 1 K.ASKGKLPIK.W [578, 586] PTK2 Focal adhesion kinase 1 0.950 1.060 0.893 1.087 0.971 0.992 0.080 (missed 2) (rank 2) K.TAQALSSGSGSQETKIPISLV Serine/threonine-protein LR.L [416, 438] (missed 1) (rank OXSR1 1.120 0.970 0.840 0.971 1.064 0.993 0.107 kinase OSR1 1) K.YSKEFFTK.K [585, 592] SRPK1 SRSF protein kinase 1 0.980 1.020 1.010 1.064 0.893 0.993 0.064 (missed 1) (rank 1) K.TSVAVKTCK.E [585, 593] FER/TYK3 Tyrosine-protein kinase Fer 1.050 0.980 0.901 1.075 0.962 0.994 0.070 (missed 1) (rank 2) K.YIKYNSNSGFVR.D [235, 246] Eukaryotic elongation factor 2 EEF2K 0.920 1.050 1.020 0.980 1.000 0.994 0.049 (missed 1) (rank 1) kinase R.KIAEGAQQGDPLSR.Y [264, N-acetyl-D-glucosamine NAGK 0.940 1.060 0.909 1.020 1.042 0.994 0.066 277] (missed 1) (rank 1) kinase R.DLKLENILLDDNCNIK.I [177, NUAK family SNF1-like NUAK1 0.989 0.970 0.825 1.117 1.072 0.994 0.112 192] (missed 1) (rank 1) kinase 1 R.GFKLPDTPQGLLGEAR.M ENG Endoglin 0.880 1.080 0.800 1.053 1.163 0.995 0.150 [288, 303] (missed 1) (rank 1) ATP-dependent 6- K.FTTDDSICVLGISKR.N [710, PFKP phosphofructokinase, platelet 1.040 0.980 1.053 0.943 0.962 0.996 0.048 724] (missed 1) (rank 4) type R.DLKPSNILYVDESGNPESIR.I MAPKAPK1 Ribosomal protein S6 kinase 1.090 1.020 0.943 0.926 1.010 0.998 0.066 [538, 557] (rank 1) B alpha-2 cAMP-dependent protein R.DLKPENLLIDQQGYLQVTDF PRKACG kinase catalytic subunit 0.980 1.020 1.031 0.971 0.990 0.998 0.026 GFAK.R [175, 198] (rank 1) gamma

S42

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.VAIKIISK.R [245, 252] (missed Serine/threonine-protein CHEK2 0.811 1.112 0.977 0.772 1.320 0.998 0.226 1) (rank 3) kinase Chk2 R.GMVAILIAFMKQR.R [18, 30] Serine/threonine-protein SGK2 0.980 1.030 1.042 0.962 0.980 0.999 0.035 (missed 1) (rank 1) kinase Sgk2 R.DIKCSNILLNNR.G [836, 847] CDK13 Cyclin-dependent kinase 13 0.966 1.012 0.976 1.162 0.880 0.999 0.103 (missed 1) (rank 1) R.GDFCIEVGKNLIHGSDSVES Nucleoside diphosphate kinase AR.R [122, 143] (missed 1) (rank NME3 0.938 1.055 0.977 0.929 1.104 1.001 0.076 3 1) R.DIKPDNLLLDAK.G [212, 223] Serine/threonine-protein STK38L 1.020 1.030 0.952 1.020 0.980 1.001 0.033 (rank 1) kinase 38-like K.NSLCFPEDAEISKHAK.N Rho-associated protein kinase ROCK2 0.950 1.040 0.943 1.064 1.010 1.001 0.054 [310, 325] (missed 1) (rank 1) 2 K.EGKIVPVEITISLLK.R [90, CMPK1 UMP-CMP kinase 0.980 1.030 1.075 0.943 0.980 1.002 0.051 104] (missed 1) (rank 1) R.GGKSGAAFYATEDDR.F 1-phosphatidylinositol 3- PIP5K3 1.129 0.848 0.930 1.213 0.890 1.002 0.160 [1858, 1872] (missed 1) (rank 1) phosphate 5-kinase K.DCVGAEVEKACANPAPGSV ILLENLR.F [97, 122] (missed 1) PGK2 Phosphoglycerate kinase 2 0.894 1.094 1.102 1.061 0.860 1.002 0.116 (rank 1) R.HGQKGTCGIQYR.Q [938, DNA-directed RNA POLR2B 0.939 1.069 0.977 1.051 0.983 1.004 0.054 949] (missed 1) (rank 2) polymerase II subunit RPB2 R.GGDVAVKIFSSR.E [227, 238] Receptor protein ALK4 1.104 0.860 1.271 0.837 0.948 1.004 0.182 (missed 1) serine/threonine kinase 4 K.EQKLGLENAEALIR.L [49, 60 kDa SS-A/Ro RO60 1.120 0.700 1.205 0.833 1.163 1.004 0.224 62] (missed 1) (rank 3) ribonucleoprotein R.SIPDGSEKGDFIALDLGGSSF HK1 Hexokinase-1 1.050 0.980 1.031 0.909 1.053 1.005 0.061 R.I [69, 90] (missed 1) (rank 3) R.DLKPENILLDDHGHIR.I [310, GRK6 GPCR kinase 6 0.980 1.040 1.087 1.031 0.893 1.006 0.074 325] (rank 1) R.DLKPENIMLSSQGHIK.L Ribosomal protein S6 kinase STK14B 1.050 0.920 0.847 0.862 1.351 1.006 0.209 [193, 208] (rank 1) beta-2 R.DLKPANILLDEHGHVR.I GRK2 GPCR kinase 2 1.008 0.965 1.046 1.006 0.040 [316, 331] (rank 1) R.DLKPANILLDEHGHAR.I GRK3 GPCR kinase 3 0.996 0.977 1.103 0.795 1.160 1.006 0.140 [316, 331] (rank 1)

S43

Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.DLKLDNVLLDR.D [472, 482] PRKCD Protein kinase C delta type 0.950 1.050 0.926 1.064 1.042 1.006 0.063 (missed 1) (rank 1) R.VAILVDDMADTCGTICHAA Ribose-phosphate DKLLSAGATK.V [214, 242] PRPS2 0.960 1.040 0.971 1.042 1.020 1.007 0.039 pyrophosphokinase 2 (missed 1) (rank 1) R.DIKLENILLDSNGHVVLTDF Ribosomal protein S6 kinase GLSK.E [176, 199] (missed 1) MSK1 0.980 1.030 0.943 1.075 1.010 1.008 0.050 alpha-5 (rank 1) K.TVAVKILK.N [397, 404] SYK Tyrosine-protein kinase SYK 0.740 1.279 0.999 0.962 1.062 1.008 0.194 (missed 1) (rank 1) K.HNLEKVEGER.K [642, 651] Rho-associated protein kinase ROCK1 0.970 0.980 1.075 1.009 0.058 (missed 1) (rank 2) 1 R.DIKPDNFLMGIGR.H [135, CSNK1A1 Casein kinase I isoform alpha 0.980 1.020 0.917 1.087 1.042 1.009 0.064 147] (rank 1) K.SKRDPTEEIEILLR.Y [455, MAPKAPK1 Ribosomal protein S6 kinase 0.980 1.040 0.980 1.042 1.010 1.010 0.030 468] (missed 1) (rank 2) B alpha-2 K.ISDFGLSKALR.A [509, 519] SYK Tyrosine-protein kinase SYK 0.983 1.000 1.279 0.865 0.927 1.011 0.159 (missed 1) (rank 2) K.KNPQAVLDVLKFYDSNTVK. Serine/threonine-protein PAK2 0.980 1.050 0.901 1.087 1.042 1.012 0.073 Q [117, 135] (missed 2) (rank 2) kinase PAK2 Dual specificity mitogen- K.KLEELELDEQQR.K [35, 46] MAP2K1 activated protein kinase kinase 1.120 0.920 1.075 0.971 0.980 1.013 0.082 (missed 1) (rank 2) 1 K.GKGITMLEEIK.V [254, 264] Platelet-derived growth factor PDGFRA 1.013 0.889 1.168 0.994 1.016 0.115 (missed 1) (rank 1) receptor alpha R.LFIQKEEQLQQILK.R [162, CDK-activating kinase MNAT1 1.170 1.070 0.806 0.962 1.087 1.019 0.140 175] (missed 1) (rank 2) assembly factor MAT1 K.LKPANILVMGEGPER.G [27, CDK19 Cyclin-dependent kinase 19 1.055 0.963 0.841 1.057 1.180 1.019 0.126 41] (rank 1) MAPKAPK1 Ribosomal protein S6 kinase R.DLKPSNILYR.D [531, 540] 0.980 1.080 1.075 0.962 1.010 1.021 0.054 C alpha-3 R.ELAVKQVQFDPDSPETSK.E Mitogen-activated protein MAP3K2 0.904 1.130 1.000 0.896 1.177 1.021 0.129 [380, 397] (missed 1) (rank 1) kinase kinase kinase 2 R.IQCAIKSLSR.I [1108, 1117] Macrophage-stimulating RON 1.156 0.868 0.842 1.078 1.164 1.022 0.156 (missed 1) (rank 1) protein receptor K.ESGQVVAIKQVPVESDLQEII Serine/threonine-protein STK3 1.185 0.856 1.066 0.980 1.022 0.139 K.E [47, 68] (missed 1) (rank 1) kinase 3

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Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.KLLASLVK.R [253, 260] Transcription intermediary TRIM28 1.040 1.060 1.020 0.952 1.042 1.023 0.042 (missed 1) (rank 1) factor 1-beta K.FFIGFGGKGANQCVQAAR.L RBKS Ribokinase 1.135 0.914 0.946 0.970 1.159 1.025 0.114 [46, 63] (missed 1) (rank 1) R.DLKPENLLYYSQDEESK.I Calcium/calmodulin-dependent protein CAMK1D 1.037 0.894 1.039 1.135 1.026 0.099 [143, 159] (rank 3) kinase type 1D R.DLKAENLLLDAEANIK.I MAP/microtubule affinity- MARK4 1.000 1.280 0.943 1.010 0.901 1.027 0.148 [210, 225] (missed 1) (rank 1) regulating kinase 4 R.DTVTSELAAVKIVK.L [34, Mitogen-activated protein MAP4K2 1.112 0.956 0.973 1.040 1.060 1.028 0.064 47] (missed 1) (rank 2) kinase kinase kinase kinase 2 R.QEDGGVYSSSGLKQIPIK.W FER/TYK3 Tyrosine-protein kinase Fer 1.100 1.210 0.962 0.870 1.010 1.030 0.131 [707, 724] (missed 1) (rank 1) R.DLKPDNILITER.S [359, 370] Serine/threonine-protein STK35 0.820 0.990 0.990 1.190 1.163 1.031 0.151 (rank 1) kinase 35 R.DIKGANILLTDNGHVK.L Mitogen-activated protein MAP4K3 1.030 0.920 0.952 1.064 1.190 1.031 0.106 [135, 150] (missed 1) (rank 1) kinase kinase kinase kinase 3 K.ATVFLNPAACKGK.A [62, AGK Acylglycerol kinase 1.211 0.814 0.895 0.901 1.339 1.032 0.229 74] (missed 1) (rank 1) K.GTFGKVILVK.E [158, 167] RAC-alpha serine/threonine- AKT1 1.050 1.120 1.124 0.775 1.099 1.034 0.147 (missed 1) (rank 2) protein kinase 1 Mitotic checkpoint K.GPSVPFSIFDEFLLSEKK.N SSK1 serine/threonine-protein 1.070 1.020 1.031 1.075 0.980 1.035 0.039 [522, 539] (missed 1) (rank 1) kinase BUB1 beta Phosphorylase b kinase R.DLKPENILLDDNMQIR.L PHKG2 gamma catalytic chain, 0.888 1.120 1.307 1.109 0.755 1.036 0.216 [152, 167] (rank 2) liver/testis isoform R.DIKSANILLDEAFTAK.I [310, Interleukin-1 receptor- IRAK4 0.960 0.870 1.190 1.087 1.087 1.039 0.125 325] (missed 1) (rank 1) associated kinase 4 K.VAVKTMKPGSMSVEAFLAE HCK Tyrosine-protein kinase HCK 1.315 0.717 0.840 1.287 1.040 0.306 ANVMK.T [265, 288] (missed 1) R.LLDADDAAAVAAKCPR.L TRIB1 Tribbles homolog 1 0.950 0.984 0.732 1.500 1.041 0.326 [33, 48] (missed 1) K.GTYGKVDLVVYK.G [61, 72] Serine/threonine-protein SBK1 1.060 1.120 1.031 0.980 1.020 1.042 0.052 (missed 1) kinase SBK1 R.AEAFGMDPARPDQASTVAV Fibroblast growth factor FGFR4 1.040 1.110 1.031 1.075 0.980 1.047 0.049 KMLK.D [483, 505] (missed 1) receptor 4

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Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.LPGGAAVALKAVDFSGHDL Extracellular tyrosine-protein PKDCC 1.126 0.989 0.983 1.074 1.067 1.048 0.061 GSCVR.E [156, 179] (missed 1) kinase PKDCC K.IPVAIKILNETTGPK.A [745, Receptor tyrosine-protein ERBB4 0.910 1.050 1.190 1.050 0.140 759] (missed 1) kinase erbB-4 R.TVAVKMLK.E [856, 863] Vascular endothelial growth FLT1 1.500 0.870 1.053 0.781 1.051 0.320 (missed 1) factor receptor 1 R.LGGGPDDAKEIMR.H [388, RAC-alpha serine/threonine- AKT3 1.065 1.189 0.914 1.056 0.138 400] (missed 1) (rank 1) protein kinase 3 K.VLGSGAFGKVMNATAYGIS Vascular endothelial growth FLT3 1.176 0.939 1.186 1.099 0.884 1.057 0.138 K.T [614, 633] (missed 1) factor receptor 3 5'-AMP-activated protein K.KICDGIFYTPQYLNPSVISLL AMPK1 kinase catalytic subunit alpha- 1.120 1.170 1.020 0.935 1.061 0.105 K.H [250, 271] (missed 1) (rank 1) 1 R.YVAVKIHQLNK.N [464, 474] Serine/threonine-protein TLK2 1.022 1.115 1.166 0.924 1.083 1.062 0.093 (missed 1) (rank 1) kinase tousled-like 2 R.YDPLQDNTGEVVAVKK.L JAK2 Tyrosine-protein kinase JAK2 1.040 1.075 1.010 1.149 1.069 0.060 [867, 882] (missed 1) (rank 1) K.VTSQVLGLGINGKVLQIFNK. MAP kinase-activated protein MAPKAPK2 0.930 1.420 0.917 0.671 1.408 1.069 0.331 R [64, 83] (missed 1) (rank 1) kinase 2 R.GIFETKFLSQIESDR.L [779, HK1 Hexokinase-1 0.962 1.205 1.091 0.992 1.100 1.070 0.096 793] (missed 1) (rank 2) K.NLYTNEYVAIKLEPIK.S [62, Casein kinase I isoform CSNK1G1 1.360 0.960 1.149 0.781 1.111 1.072 0.217 77] (missed 1) gamma-1 K.FLSAEEYQQKIIPVVVK.M NTKL N-terminal kinase-like protein 1.050 1.020 0.775 1.190 1.370 1.081 0.220 [339, 355] (missed 1) (rank 1) R.DVKAGNILLGEDGSVQIADF Serine/threonine-protein GVSAFLATGGDITR.N [145, OXSR1 1.050 1.090 1.010 1.205 1.064 1.084 0.074 kinase OSR1 178] (missed 1) (rank 2) R.DLKPANVFLDGK.Q [140, Serine/threonine-protein NEK2 1.058 1.050 1.200 1.053 1.101 1.092 0.064 151] (rank 1) kinase Nek2 K.VAVKTLKPGTMSVQAFLEE ANLMK.T [271, 294] (missed 1) LYN Tyrosine-protein kinase Lyn 1.014 1.185 1.104 1.279 0.899 1.096 0.148 (rank 1) Fibroblast growth factor R.ATNFIGVAEK.A [339, 348] FGFR3 1.199 1.045 1.075 1.095 1.104 0.067 receptor 3 R.DIKPENILVSQSGITK.L [124, Cyclin-dependent kinase-like CDKL3 1.033 1.213 1.089 1.042 1.141 1.104 0.075 139] (rank 1) 3

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Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.FLEDDPSDPTYTSSLGGKIPI EPHB3 Ephrin type-B receptor 3 1.040 1.097 0.866 1.412 1.104 0.228 R.W [781, 802] (missed 1) (rank 1) R.GEEVAVKIFSSR.E [225, 236] Receptor protein ALK5 1.096 1.140 0.950 1.140 1.200 1.105 0.094 (missed 1) serine/threonine kinase 5 MAP kinase-activated protein R.DLKPENLLFK.D [147, 156] MAPKAPK5 1.187 1.140 1.174 0.923 1.106 0.124 kinase 5 K.EASSTQDTGKLPVK.W [337, CSK Tyrosine-protein kinase CSK 1.210 1.099 1.010 1.106 0.100 350] (missed 1) (rank 3) R.GLNPKILFSSR.E [728, 738] GAK Cyclin-G-associated kinase 1.155 1.160 1.282 0.832 1.107 0.193 (missed 1) (rank 2) R.VAIKTLKPGTMSPEAFLQEA Proto-oncogene tyrosine- QVMK.K [300, 323] (missed 1) SRC 1.162 1.109 1.121 1.018 1.169 1.116 0.060 protein kinase Src (rank 1) K.TIKVYCQR.M [85, 92] (missed DNA-directed RNA POLR2E 1.273 0.970 0.880 1.245 1.222 1.118 0.180 1) (rank 1) polymerase II subunit RPB5 K.TGDLFAIKVFNNISFLRPVDV Serine/threonine-protein QMR.E [30, 53] (missed 1) (rank TBK1 1.090 1.330 0.935 1.266 0.980 1.120 0.173 kinase TBK1 1) R.FLEENSSDPTYTSSLGGKIPIR EPHB4 Ephrin type-B receptor 4 1.261 0.991 1.388 0.965 1.009 1.123 0.190 .W [763, 784] (missed 1) (rank 1) K.ASKELQSVEQEVR.W [171, TBRG4 Protein TBRG4 1.270 1.510 1.031 0.787 1.150 0.311 183] (missed 1) (rank 3) R.DLKVSNLLMTDK.G [162, CDK10 Cyclin-dependent kinase 10 1.175 1.199 1.143 1.102 1.187 1.161 0.039 173] (missed 1) (rank 1) K.SQVALKIIR.N [180, 188] Dual specificity protein kinase CLK3 1.194 1.090 1.206 1.733 0.763 1.197 0.349 (missed 1) CLK3 R.DTGQIVAIKK.F [25, 34] Cyclin-dependent kinase-like CDKL1 1.450 1.250 1.099 1.053 1.213 0.179 (missed 1) 1 Calcium/calmodulin- R.DLKPENLLYYSLDEDSK.I CAMK1 dependent protein kinase type 1.240 1.180 1.282 1.234 0.051 [140, 156] (rank 2) 1 R.DIKGDNVLVNTYSGVVK.I Mitogen-activated protein MAP3K15 1.380 1.330 1.124 1.163 1.249 0.125 [772, 788] (missed 1) (rank 1) kinase kinase kinase 15 R.DVAVKVIDK.L [575, 583] Serine/threonine-protein PRKD2 1.150 1.200 1.333 1.241 1.395 1.264 0.100 (missed 1) (rank 1) kinase D2 R.DLKLGNLFLNEDLEVK.I Serine/threonine-protein PLK1 1.150 1.600 1.053 1.268 0.292 [175, 190] (missed 1) (rank 1) kinase PLK1

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Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation R.APGLLPYEPFTMVAVKMLK. Muscle, skeletal receptor MUSK 1.090 1.099 1.639 1.276 0.315 E [599, 617] (missed 1) tyrosine-protein kinase R.SDDNEKALK.I [175, 183] AK2 Adenylate kinase isoenzyme 2 0.990 1.587 1.515 1.064 1.289 0.306 (missed 1) (rank 2) R.VIFPTGDSKVVLPCNLLR.M DNA-directed RNA POLR2A 1.395 1.232 1.434 1.171 1.217 1.290 0.117 [967, 984] (missed 1) (rank 2) polymerase II subunit RPB1 R.AAGIGKDFK.E [650, 658] Serine/threonine-protein PRPF4B 1.339 1.328 1.400 1.203 1.271 1.308 0.075 (missed 1) (rank 2) kinase PRP4 homolog R.LLDDFDGTYETQGGKIPIR.W EPHA1 Ephrin type-A receptor 1 1.000 1.688 1.455 1.231 1.231 1.321 0.261 [772, 790] (missed 1) (rank 1) Dual specificity mitogen- R.DVKPSNILLDER.G [242, 253] MAP2K7 activated protein kinase kinase 1.210 1.710 1.064 1.328 0.339 (rank 1) 7 K.DFKENPNLR.D [656, 664] Serine/threonine-protein PRPF4B 1.403 1.336 1.307 1.274 1.406 1.345 0.058 (missed 1) (rank 1) kinase PRP4 homolog Leucine-rich repeat R.QGKSTLLEILQTGR.A [648, LRRK1 serine/threonine-protein 1.180 1.750 1.053 1.429 1.353 0.307 661] (missed 1) kinase 1 R.NVNTGELAAIKVIK.L [34, Mitogen-activated protein MAP4K3 1.260 1.176 1.695 1.377 0.278 47] (missed 1) (rank 2) kinase kinase kinase kinase 3 R.TLYDFPGNDAEDLPFKK.G CRKL Crk-like protein 1.780 1.720 1.075 1.124 1.425 0.377 [129, 145] (missed 1) (rank 3) R.DVKPHCVLLASK.E [140, Peripheral plasma membrane CASK 1.740 1.429 1.282 1.370 1.455 0.199 151] (rank 1) protein CASK K.KNDGQFTVIQLVGMLR.G EPHA5 Ephrin type-A receptor 5 1.410 1.525 1.271 1.649 1.488 1.468 0.140 [767, 782] (missed 1) (rank 1) R.VALKIIK.N [189, 195] (missed Dual specificity protein kinase CLK2 1.677 1.206 1.524 1.469 0.240 1) CLK2 R.DLKLGNFFINEAMELK.V Serine/threonine-protein PLK2 1.552 1.169 1.798 1.383 1.475 0.266 [204, 219] (missed 1) (rank 1) kinase PLK2 R.SSLQPITTLGKSEFGEVFLAK Inactive tyrosine-protein PTK7 1.800 1.980 1.064 1.124 1.492 0.466 .A [663, 683] (missed 1) (rank 1) kinase 7 K.LIKTLFPFEAEAYR.N [297, ATR Serine-protein kinase ATR 1.570 1.695 1.389 1.551 0.154 310] (missed 1) (rank 1) R.DIKCSNILLNNSGQIK.L [858, CDK12 Cyclin-dependent kinase 12 1.770 1.710 1.333 1.408 1.695 1.583 0.198 873] (missed 1) (rank 1)

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Ratio Ratio Ratio Ratio Ratio Average Standard Kinase Peptides Kinase Full Kinase Name F FE R1 R2 RE Ratio Deviation K.MKSMKLEK.T [309, 316] Nucleoside diphosphate kinase NME8 1.420 1.600 1.887 1.493 1.538 1.588 0.180 (missed 2) 8 R.FVALKVVK.S [61, 68] (missed STK23 SRSF protein kinase 3 1.410 1.220 1.408 2.326 1.591 0.498 1) R.GIHSAIDASQTPDVVFASILA AFSKATCK.D [204, 232] (missed AK2 Adenylate kinase isoenzyme 2 1.240 2.083 1.887 1.737 0.441 1) (rank 1) R.KQFEAPTLAEGFSAILEIPFR. Bifunctional polynucleotide PNKP 2.280 2.083 1.282 1.882 0.529 L [483, 503] (missed 1) (rank 1) phosphatase/kinase K.EAVTVAVKMLK.D [509, 519] Fibroblast growth factor FGFR2 1.130 1.667 1.538 3.333 1.917 0.971 (missed 1) receptor 2 K.TGQLAAIKVMDVTGDEEEEI TRAF2 and NCK-interacting KQEINMLK.K [46, 73] (missed 2) TNIK 2.380 2.273 1.333 1.995 0.576 protein kinase (rank 1) R.YQNLKPIGSGAQGIVCAAYD Mitogen-activated protein MAPK8 2.400 1.920 1.754 2.025 0.335 AILER.N [25, 49] (rank 2) kinase 8

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Figure Legends

Figure S1. Experimental strategy for human kinome analysis with the use of ICAP probe and LC-MRM. Shown is the forward labeling experiment, where the light and heavy desthiobiotin- C3-ATP probe were treated with lysate of MG-treated cells and control cells, respectively. Figure S2. (a) Linear fit between iRT values of desthiobiotin- and desthiobiotin-C3-labeled peptides. (b) Venn diagram showing the kinome coverage obtained from DDA and MRM analysis. Figure S3. KEGG pathway analysis of down-regulated kinases and all kinases perturbed upon MG treatment. Figure S4. Representative MRM traces for receptor tyrosine kinases (EGFR, IGF2R, FGFR1 and FGFR3). Owing to chromatographic isotope effect,9 the heavy ICAP reagent-labeled peptides eluted slightly earlier than the corresponding light ICAP reagent-labeled ones. Figure S5. MG induced down-regulation of FGFR activity in HEK293T cells. (a) Western blot result of MG-induced decrease in FGFR activity could be rescued by pre-treatment with NAC, but not AG or MET. (b) Quantification results showing that the MG-induced decrease in FGFR activity could be rescued by pre-treatment with NAC, but not AG or MET (n =3). The p values were calculated by using unpaired, two-tailed t-test, and these values referred to the comparisons with the results obtained from cells treated with MG alone

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References:

(1) Xiao, Y.; Guo, L.; Jiang, X.; Wang, Y. Anal. Chem. 2013, 85, 3198-206. (2) Xiao, Y.; Guo, L.; Wang, Y. Anal. Chem. 2013, 85, 7478-86. (3) Taylor, P.; Nielsen, P. A.; Trelle, M. B.; Hørning, O. B.; Andersen, M. B.; Vorm, O.; Moran, M. F.; Kislinger, T. J. Proteome Res. 2009, 8, 1610-1616. (4) Olsen, J. V.; de Godoy, L. M. F.; Li, G.; Macek, B.; Mortensen, P.; Pesch, R.; Makarov, A.; Lange, O.; Horning, S.; Mann, M. Mol. Cell. Proteomics 2005, 4, 2010-2021. (5) MacLean, B.; Tomazela, D. M.; Shulman, N.; Chambers, M.; Finney, G. L.; Frewen, B.; Kern, R.; Tabb, D. L.; Liebler, D. C.; MacCoss, M. J. Bioinformatics 2010, 26, 966-968. (6) Cox, J.; Mann, M. Nat. Biotechnol. 2008, 26, 1367-1372. (7) Escher, C.; Reiter, L.; MacLean, B.; Ossola, R.; Herzog, F.; Chilton, J.; MacCoss, M. J.; Rinner, O. Proteomics 2012, 12, 1111-1121. (8) Marx, V. Nat. Meth. 2013, 10, 19-22. (9) Szarka, S.; Prokai-Tatrai, K.; Prokai, L. Anal. Chem. 2014, 86, 7033-7040.

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