Cumulative Examinations

Department of Chemistry Cumulative Examinations April 20, 2019 You may choose to answer any exam from any area covered in the examination booklet. Each exam may contain multiple parts. You may answer more than one exam but each exam is scored separately and is treated as an individual examination result. Thus, answering parts of two exams with а score of 50% wou/d not yield а 100% grade for this cumulative exam. lnstead you would receive 50% оп each examination attempted. This booklet contains five examinations. 1) 2) 3) 4) 5) Analytical Cumulative Examination, Biochemistry Cumulative Examination, lnorganic Cumulative Examination, Organic Cumulative Examination, Physical Cumulative Examination, Pages 1-7 Page 8 Pages 9-10 Page 11 Page 12 Оп your examination booklet: 1) 2) 3) 4) Print your student 1D number. Print the Exam Booklet number. Print the question number you are answering. Print the Exam Date. Do not write vour пате anywhere оп the examination booklet. Each exam will Ье scored anonymously. lf you attempt more than one exam, уои must use а separate examination booklet for each examination. When you complete the examination, return the examination and your answer booklet to the proctor. Exam results will Ье posted оп bulletin board #2В оп the north side of the hall near BRWN 2124. lmatinib-lnduced Changes in Protein Expression and ATP-Binding Affinities of Кinases in Chronic My elocy tic Leukemia Cells О Sttpporting Info1·mation AВSTRACT: Кinases are cгucial components in numerous cell signaling patl1ways. Abeпant expression and activation of proteiп kiпases are known to TKL . Ье accompanied Ьу many types of cancer, and more than 30 small-molecпle kinase Е inblЬitors have been approved Ьу tЬе Food апd Drug Admiпistration (FDA) for cancer cl1emotЬerapy. Biological and cliпical applications of small-molecule kinase inblЬitors reqпire compreЬensive cЬaracterizations about Ьоw tl1ese inhiЬitors modulate tl1e protein expгession and activities of kinases at tЬе entire proteome scale. Iп this stпdy, we developed а parallel-reaction monitoring (PRМ)-based targeted proteomic metЬod to monitor tЬе alterations in proteiп expressioп of kinases in К-562 cЬronic myelocytic leпkemia (СМL) cells elicited Ьу treatmeпt witЬ imatinib, ап АВL kinase inblЬitor approved Ъу tЬе FDA for СМL treatmeпt. • PRM Ubrary • MRM Ubrary Ву employiпg isotope-coded АТР affinity probes togetЬer witЬ liquid • PRM & MRM Ubrary cЬromatograpЬy-mпltiple-гeaction monitoring (LC-MRМ) analysis, ,ve also examined tЬе modпlatioп of tЬе ATP-Ьinding affinities of kinases induced Ьу imatinib tгeatment. Tl1e results revealed profound incгeases in protein expression levels of а large nпmber of kinases in К -562 cells upon treatment witЬ imatinib, wrucl1 is accompanied Ьу substantial decreases in ATP-Ьinding capacities of many kinases. Apart fгom АВL kinases, we identified а пumber of otЬer kinases ,vЬose ATP-Ьinding affinities are markedly diminished проn imatinib treatment, inclпding CHKl, а �Ьeckpoint kiпase iпvolved in DNA damage response signaling. TogetЬer, our targeted qпaпtitative proteomic metЬods enaЬled, for tЬе first time, dual assessmeпts of small-molecule kinase inruЬitor-induced cЬanges in proteiп expression and ATP-Ьinding affinities of kiпases in live cells. . Т.Т iпases play пucial roles iп cell sigпaliпg, 1 and small­ .l'-.molecule kinase inruЬitors Ьаvе Ьееп widely employed as valuaЬle tools for delineatiпg kinase-mediated cell signaling patЬways for decades.2 Iп addition, aberrant activation of kiпase signaling is frequeпtly accompanied Ьу and sometimes leads to tЬе development of mапу types of саnсег.3'4 As а result, kinase inblЬitors Ьаvе receпtly become а ve1y important class of dшgs in anticancer tЬerapy, ,vЬere more tЬan 30 small­ molecнle kinase inruЬitors Ьаvе been appl'Oved Ьу tЬе Food and Drug Administratioп (FDA) for treatiпg different types of maligпaпcies.2 Appropriate use of small-molecпle kiпase inruЬitors in cell sigпaliпg researcЬ and in сапсеr cЬemotЬerapy гequires tЬе knowledge about wЬetЬer otl1er kiпases are also taгgeted Ьу tЬese inblЬitors. Iп trus 1·espect, most kinase iпruЬitors are designed to disrupt, directly 01· indirectly, tЬе ATP-Ьiпding capaЬilities of tЬе taгget kinases, wblcl1 possess ЬigЫу conseгved ATP-Ьiпding domains.2' 5 Hence, despite the substantial efforts iп optimizing the stшctшes of kinase inruЬitors for selective Ьinding to,vard tЬе iпtended kinase, tЬе inblЬitors may also Ьind to tЬе ATP-Ьinding pocket апd suppгess tЬе ATP-Ьiпdiпg capacities of otЬer kiпases, wblcЬ may lead to off-target effects.2'5 In addition, cancer cells may respond to inruЬitor treatmeпt Ъу repl'Ogramming tЬeir kinome tЬrougЬ alteгing tЬе protein expressioп levels of kinases.6 ТЬе knowledge about tЬе proteome-wide alterations in pl'Otein expression and ATP-Ьindiпg affinities of kinases elicited Ьу small-molecпle iпruЬitors is importaпt for uпder­ standiпg more completely tЬе mecЬanisms underlying tЬerapeutic efficacy, гesistance, and side effects associated ,vitЬ tЬе kinase inruЬitor. SпсЬ knowledge is also crucial for tЬе ассшаtе iпterpretation of data wЬen tЬese inruЬitors are used in cell signaling геsеаrсЬ. FшtЬermore, on tЬе grouпds that tЬе drug safety and pЬa1шacological properties of tЬese FDA­ approved kiпase iпЬiЬitors are well-documented,7 revealing previously uшecognized kinase targets for tЬese inblЬitors may facilitate novel applications of tЬese inruЬitors in treating otЬer Ьшnаn diseases. ТЬе Ьшnаn kinome is encoded with 518 genes, 8 and many kinases are expressed at very low levels. Thus, tЬе iпvestigatioпs about tЬе alterations in pl'Otein expression and tЬе ATP-Ьinding capaЬilities of kiпases iп cells uроп iпruЬitor treatment entail rugЬ-tЬrougЬput and rugbly seпsitive aпalytical metЬods. In tЬе present study, we address tЬе aforemeпtioned analytical cЬallenge Ьу developing а parallel-reaction monitor- 2 iпg (РRМ) metlюd (Figure S1),9-11 iп сопjппсtiоп with staЬ\e isotope labeliпg Ьу amiпo acids iп cell шlture (SILAC),12 for the proteome-wide iпterrogatioп of tl1e proteiп expressioп levels of kiпases ппdеr the same treatmeпt coпditioпs. We also employed the previoпsly reported isotope-coded АТР affiпity probes (Figure la), together witl1 the mпltiple-reactioп а OestNoblorin HN J /jH АТР llrker О Х Х О О О он он он �х х х 11 о 11 11 ч N :: JC N /; , но Х = Н, Ught; X=D, Heavy ь N iLf}!y 0-�-О-�-О-�-о LJ� / We first establis\1ed а РRМ kiпome library based 011 data collected from shotguп pюteomic aпalysis. То tbls епd, we first collected LC-MS/MS data from shotguп proteomic aпalysis of ilie tryptic digestion mixtures of the ATP-Ьiпdiпg proteiпs eпriched from 8 humaп cell liпes a11d the tryptic digestioп mixtures of the whole cell lysates of 3 hпmап cell liпes. А Maxquaпt search of these data led to the identificatioп of 11 879 ршtеiп groups.10•1 1 We sпbsequeпtly i11cшporated the ta11dem mass spectra апd reteпtioп time iпformatio11 for all uпmodified kiпase peptides iпto Skyliпe21 for coпstructi11g the ki11ome РRМ library. Because some kiпases share ЫgЫу similar sequeпces, ,ve maппally checked all kiпase peptides апd iпcorporated опlу those peptides iliat сап Ье u11iquely assigпed to specific kiпases iпto the library, with а maximum of four ппique peptides being iпcluded for а11у given ki11ase. Iп doing so, the сuп·епt РRМ kiпome libra1-y co11tained 1050 uпique peptides represe11ti11g 478 11011redu11daпt huma11 kiпases, wblch eпcompassed 395 proteiп kiпases, 21 lipid kiпases, апd 62 otheг kiпases (ТаЬ\е S1). The 395 protei11 kiпases iпclude members fi:om all 7 groпps of the humaп ki11ase family (see kiпome deпdrogram iп Figure lb, ТаЬ\е S1). Thпs, our РRМ kiпome library coпtaiпs appшximately 80% of the humaп kiпome tl1at inclпdes а total of 518 ргоtеiп ki11ases.22 То acbleve blgh-throughpпt detectioп of kiпase peptides, ,ve adopted scheduled РRМ aпalysis, where the mass spectrometer was progгammed to acquire the MS/MS fог the p1·ecursor ioпs of а limited пumber of peptides in each predefiпed 8 nuп retention time wiпdow. Tl1e1·efore, accurate pгedictioп of retention time (RT) for each ki11ase peptide was required for our PRМ-based kiпome assay. То this епd, we calculated the пormalized RT (iRT) value for each peptide оп our taгget list follo,viпg а previously ·puЬ\ished ·method with the use of а tryptic digestioп mixture of boviп'e serum albun1i11 (BSA) as the staпdard.23 The liпear RT · vs iRT relatioпsblp was redefi11ed betweeп every eight LC-PRМ ruпs Ьу reiпjectiпg the tryptic digestioп mixture of BSA. Siпce the iRT valпe гepresents an intrinsic attribute (i.e., l1ydroph0Ыcity) of а peptide, we also employed it as а пiterioп to validate t\1e data obtaiпed from the РRМ assay, where а marked deviatio11 of the observed RT fi:om the predicted RT is coпsidered а false­ positive detectioп. Ош РRМ- апd MRМ-based ki11ome spectral libгaries commoпly сопtаiп 350 пoпreduпdaпt kiпases (Figure lc,d). А comЬiпatioп of the РRМ апd МRМ data, therefore, permits iпdepeпdeпt assessme11ts about how the expressio11 levels a11d ATP-Ьi11di11g capacities of ki11ases are modпlated Ьу imatiпib. The PRМ-based targeted proteomic method led to the qua11tification of 295 uпique kiпases iп К-562 cells ,vith or ,vithout imatinib treatmeпt, inclпding ~250 proteiп kiпases, wblcl1 cover approximately 50% of the humaп kinome (Figпre 2а and ТаЬ\е S2). Simila1·ly, the MRМ-based targeted proteomic method resulted iп the quantificatio11 of the ATP­ Ьindi.пg affiпities of 332 unique kinases (Figure 2Ь and ТаЫе S2). All transitioпs from РRМ (4-6 transitions) and МRМ (3 transitioпs) used for quantificatioп of each kinase peptide displayed the same rete11tio11 time with the dot product (dotp) value being >0.7 (Figure S2).24 Furtl1ermore, over 80% of the kinases quantified ,vith the РRМ approach апd all the kinases quantified with the МRМ method appeared in both forward and reverse labeling experiments (Figure S3a). The ratios of t\1e qпantified peptides obtained from forward апd reverse experiments exhiblted а li11ear fit (Figure S3b), wblch confirms 1 /,/1 N он 1---: ТК ,� � :. ' '· -�-; CrAGC��� ...- t: :·. ТКL ш � MRM Шхаrу PRM l!Ьrary .:�r·:: ,. 8 PRMl!Ьrary 8 MRM liЬfary е Both PRM and,MRM liЬraries MRM liЬrary PRM IJЬrary Figure 1. РRМ- and MRМ-based taгgeted proteomic approaciles for interrogating tile Ьuman kinome. (а) Tile cileшical structures of tile isbtope-coded АТР affinity probes. (Ъ) А kinome dendrogram depicting tl1e kinases tilat are inclнded in РRМ- and MRМ-based kinome spectral iibraries. Tile kinome dendrogram ,vas adapted ,vitil permissioп from Cell Signaling Tecilпology (Ьttp://,V\V\V.cellsignal. com). Green solid circles denote tlюse kinases tilat are present in botil РRМ and МRМ libraries, ,vilereas Ыuе and red solid circles designate tilose kinases tilat are inclнded only in tile РRМ and МRМ libraries, respectively. (с) Venn diagrams displaying tile numbers of kinases (top) апd protein kinases (bottom) included in tile PRМ­ and/or MRМ-based kinome libraries. mo11itoriпg (МRМ)-based targeted proteomic metlюd, 13 -16 for gaпgiпg the alteratioпs iп ATP-Ьiпdiпg affiпities of kiпases in К-562 cells проп а 24 h tгeatmeпt with 1.0 рМ imati11ib. Iп tms veiп, imatiпib, а small-molecпle iпblЬitor for с-АВL kiпase, ,vas approved iп 2001 Ьу the FDA for treatiпg chroпic myelocytic leнkemia ( СМL) iп patieпts carryiпg tl1e BCR-AВL fusion oncoge11e,2 and tms oncogene is also prese11t i11 К -562 cells. 17 It is worth noting that, wblle liqнid chromatography­ mпltiple-reaction monitoring (LC-MRМ) analysis is sпitaЬ\e fш analyzing eпriched samples lilce the above-described affinity-purified destbl0Ьioti11-conjпgated kinase peptides, the same analysis of kinase peptides arisi11g from the t1-yptic digestio11 of whole cell lysate may i11trodпce sнbstantial backgrou11d sigпal.18 РRМ, 011 the other ha11d, allows parallel detectioп of all transitio11s i11 а siпgle aпalysis, wblch е11аЬ\еs mgh-throughput aпalysis, 19 a11d it miпimizes i11terfere11ces owi11g to the пsе of mass a11alyzers ,vith blgh resolutio11 a11d mgh accuracy for mass detectioп.20 Therefore, we employ РRМ for assessiпg kiпase proteiп expressio11 witl1 the use of the tryptic digestioп mixture of whole cell lysate. 3 а (J С!) <( ADRBK1 АКТ2 ССtЮЗ EXOSC10 GRKб LATS1 t.•лsn. РЮ/1 PKt/2 Ркt/3 РRКАА1 РRКАСА PRКACG PRКAG1 PRКAG2 PRKCO PRKCE PRKCI PRКD2 РRКDЗ PRKDC RОСК1 RОСК2 RPS6КA1 RРSбКАЗ RPS6KB1 RPS6KB2 RPS6KC1 STKЗSL ADCK4 АКI дК2 АКЗ РНКА1 РНКА2 РНКВ PHKG2 SIК2 SТК11 sткзз (.) (.? :а: (.) f-��-----, 0.33 ь 3.3 TRIO CStlК1A1 CSIIKID CStlK1G2 CStlK1GЗ CStlК2A1 CStlК2A2 CSNK2B ТТВК2 VRKI VRК2 СОС428Рд СОС42ВРВ CDC428PG СОС7 COKI СОК12 CDK16 СDК2 СDКЗ СОК◄ СDК5 СDК7 СОК9 СLК1 СLКЗ CRКL DCK """ PRPF,Q <( (..) �/JEU< ... Q) :5 о 0.33 З.З о 0.33 � (/) l 00< owo """ '"'" ""' . З.З о.зз ] '"'"' ""-' "" "" "" '"" "" """' EGFR """ С,О :а: (.) ...J :,.:: 1- """' ,rnw Ept\A2 ,rn,., tJ.•PJК11 ,JJJ>э::2 Ш.РJО CS/,l':\,l,I CS';К1All "'" =• CSIIКZAZ ш 1U) '''" С""31..Е51 сос,, (.) (.? :а: (.) о :1..3 RlinatiniЫControl ... Q) .s:: о IJ.>J'�IO IJJJ>U.5 PDGFRA '"'' '"'' С!Ж!7 (;()j(\9 ""' ""'"' сс,о """ "'" "'" с<ю., CIЖU С'-" OJ] 3.3 RlmatlnlЬ/Control ТАОК\ ТАОК2 FMCA RP�OЕЮ POGFRB РТК2 РТК2В РТК7 SУК ТК1 ТК2 ПIК1 ПIК2 ТУК2 VESI ACVR1 мw BRAF ILК IRAl,______ RlmatiniЬ/Control ""'' ""' sтк;,,; '"'" !IЕК& t,E.l(g тдз "''"е-----�--""" SТЮ9 ,_-�--.....; RlmatlnlЫControl RtmatiniЬ/Control RlmatiniЬ/Control RlmatlnlЬ/Control Figure 2. Difierential protein expression (а) and ATP-binding affinities (Ь) ofkinase pl'oteins in K-S62 cells induced Ьу imatinib treatment. Tl1e kinase protein expression data represent the mean of !'esults obtained fl'Dm three foпvard and hvo !'everse SILAC labeling experiments, and the ATP-Ьinding affinity results reflect five foпva!'d and six reverse АТР р!'оЬе labeling experiments. (The !'atios obtained from individual measшements а,·е listed in ТаЫе S2.) Вlпе, red, and gray bars represent thosekinases ,vith l'atios (in imatinib-t1·eated/control cells) that are <0.67, > l.S, and behveen 0.67 and l.S, respectively. the excellent reprodпciЬility of the taгgeted quantitative many kinases (Figure 2., ТаЫе S2). For instance, substaпtially proteomic methods. Our quantification results showed that exposure of К-562 more kinases display diminished ATP-Ьinding capaЬilities than cells to imatinib led to pronounced diminutions in the ATP­ ,vhereas а much larger number of kinases exhiЬit elevated Ьinding affinities of а large пumber of kinases while protein expression than those with reduced expression upon concomitantly stimulating the protein expression levels of imatinib treatment (48 vs 9, Figure 2, ТаЫе S2). those ,vith heightened ATP-Ьinding affinities (65 vs 8 ), 4 а We also examined the accuracy of t\1e РRМ method Ьу monitoring the expression levels of tl1ree representative kinases (AKl, CCNDЗ, and SCYLЗ) in К -562 cells ,vith and witlюut imatinib treatment. The Western Ыоt data slюwed tЬat the expression levels of these kinases are elevated after imatiпib treatmeпt, wblch is consisteпt with the РRМ results (Figure 3). ь а К-562 lmatinib (11М) О АК1 - - CCNDЗ !3-Actin - - с �g "' ::Е ОС D �;о "'·&!g .:; с -'i, 14 10 с ; i SCYLЗ 12 i .., ф ос AK 1 1 о.5 о о / 10114 о 1 0_5 Lt 113 о 1 CYS LЗ UL о Иl о о о i ,: Fo�vard о 5 1 8 47 49 51 1 2 1 2 Reverse u 48 49 UL 47 48 49 UL з8 0 ,1 ABL2, РRМ VGEAPGLOQPQPR .5 -омsо ii о Retention Tlme (mln) OPRM 172 UL 38 .5 39.5 40.5 g � о 86 88 о � .;; 8 -�w 90 �•;�: � ni b -омsо Ll 86 88 90 0.4 �----т-----, 0.2 о � ::Е -0.2 135 6 .. •• .• -0.4 -0.6 -0.6 -0.4 -0.2 О 0.2 0.4 0.6 Log 10(1matiniЫDMSO, PRM) 4 2 о u_ Retention Тime (min) PRM 160 ��::t�nib O: 40 '2� о::� ■Western Ыоt ABL 112, MRM YSLТVАVКТLК : : с MRM 11 7 49 50 51 127 129 39. 5 .: ь - lmatlnlb o.s o: § -омsо !l g o: 1 - lmatinib 0.5 0.5 i 0_5 CCNDЗ 1 ABL 1, РRМ GSALGTPAAAEPVТPTSK АК1 CCN03 Kinases Figнre 4. РRМ and МRМ resнlts s\10,viпg the diffeгeпtial expressio11 a11d ATP-biпdiпg affinities of kinases iп К-562 cells indнced Ьу imatinib treatment. (а) Rep1·esentative РRМ a11d МRМ traces for the quantification of АВL kinases. (Ь) Venn diagram depicting tl1e overlap iп the numbers of k.inases that ,vere qнaпtified Ьу the РRМ- and МRМ-based kinome profili11g methods for lysates ofK-562 cells after а 24 h treatme11t ,vith 1 рМ of imati11ib or DMSO. (с) А scatter plot depicting. the \ack of coпelatio11 behvee11 the ratios of k.i11ases i11 imatinib-treated cells over co11trol DMSO-treated cells obtained from the РRМ a11d МRМ methods (plotted in logarithm scale). SCYL3 Figure 3. Westem Ыоt a11alyses for validati11g the protei11 expressio11 \evels of kinases i11 К-562 cells ,vith 01· ,vithoнt imati11ib treatme11t. (а) Images from _Western Ыоt a11alyses or'the expressio11 levels of select kinases iп К-562 cells ,vith or ,vithoнt imati11ib treatme11t. (Ь) Represe11tative РRМ traces for mo11itoriпg the expгessio11 levels of the same kinases as sho,v11 i11 (а). (с) The qнa11tificatio11 resнlts for the �atios of k.i11ase proteins in imatinib-treated cells over t111treated cells. -Error ba1·s represent standard deviations. ь. ·ь . kiпases were previous1у shown to шd d'rrect1у to rmat·ш1 . 29,30 Непсе, our isotope-coded АТР affinity р!'ОЬе, together with LC-MRМ analysis, allows fO!' the revelatioп of candidate kiпases that are targeted Ьу imatiпib. Apart from previoпsly reported kinase targets of imatinib, we ideпtified а ппmЬеr of kiпases with reduced ATP-Ьinding affinities but поt witl1 diminisl1ed proteiп expression iп К-562 cells after imatinib ехроsше, inclпding YESl and CHKl. ln this coпtext, our resпlts sho,ved that the expression levels of YESl апd CHKl proteins were not changed in К-562 cells uрон imatinib treatment, whereas their ATP-Ьindiпg affinities were decreased Ьу approximately 2-fold (ТаЬ\е S2 and Figure S), sпpportiпg that imatinib сап also comp1·omise the АТР- It is worth notiпg that our LC-PRМ method, which quaпtifies proteiпs based оп uпique peptides, permits the iпdepeпdent measurements of difiereпt isoforms of kiпases. For iпstaпce, fo1· the two isoforms of АВL kiпases, the expressioп of AВLl p!'Oteiп was clearly elevated, whereas that of АВL2 proteiп kept uпchaпged upon а 24 h tr·eatmeпt with imatinib (Figure 4а, ТаЬ!е S2). In tЬis veiп, imatinib was showп to Ьiпd to the kiпase domains of AВLl апd АВL2; 25-27 however, imatiпib admiпistratioп leads to the differential reprogramming of the two isofO!'ms of АВL. The detailed mechanism(s) contributing to the elevated expression of AВLl, but not АВL2, is not clear and awaits further investigatioп. The two methods facilitated the quantification of approx­ imately 160 common kinases (Figure 4Ь ). А comparison of kinase ratios ( ,vith/without kiпase inhiЬitor treatment) obtained from РRМ and МRМ aпalyses revealed tЬе lack of appareпt correlation between the inhiЬitor-induced alterations in kiпase protein expressioп and ATP-Ьinding capability (Figure 4с), пnderscoring that the alteratioпs in overall ATP­ Ьiпdiпg capacities of most kinases are not attr·ibutaЬ!e to chaпges in pr·oteiп expression levels of these kinases. Despite the fact that the two isofO!'ms of АВL kinase exhiЬit differential reprogramming after imatinib treatmeпt, the ATP­ Ьinding affiпities of both isoforms were substaпtially atteпuated (Figure 4а), which is iп keepiпg with а previous findiпg.28 In addition, our resпlts showed the decreased ATP­ Ьinding capacities of several other kinases, includiпg FGR, GAK, IRAK, and МЕLК (Figures 2Ь апd S4, ТаЬ!е S2). These !Л .:11�= �olдlo 75 76 77 17 18 19 � Relenlion Тime {mln) ь lmllfk\.'Ь(µM) О ci•· • ::, • -с,,\$0 _..,,.,,. • ;о.в u . � о., • о cs1 о: О 10 20 $0 NCS (пglПIL) 100 Figure S. Imatinib inhiblts the activity of CHKl. (а) Represe11tative МRМ a11d РRМ traces for the quantificatio11s of CHKl. (Ъ) Western Ыоt for the validation of the protein expression a11d activity of CHKl i11 К-562 cells ,vith a11d ,vithout imatinib treatme11t. (с) C\011oge11ic sпrvival assay resнlts sho,ving the efiect of imatinib 011 sensitiziпg МDА-МВ-231 cells to,va1·d NCS. Etтor bars represent sta11dard deviatio11. *, 0.01 < р < 0.05; **, 0.001 < р < 0.01; #, 110 sig11ifica11t difference. The p-values ,vere calculated agaiпst the co11trol t1si11g the hvo-tailed, t111paired Stнdent's t-test. . 5 Ьinding capaЬilities ofYESl and CHKl. Along this line, it ,vas previously obseгved tЬat imatinib-resistant cells displayed elevated expt'ession of YESl.31 In addition, our Western Ыоt data showed that imatinib tl'eatment led to а marked diminution in the kinase activity of CHKl ( as manifested Ьу гeduced autophosphorylation at Ser296), albeit ,vith no appreciaЬ\e cl1ange in its protein exp1·ession level (Figure Sа,Ъ). In l'esponse to DNA douЬ\e-st1·and break (DSB) formation in cells, CHKl coordinates DNA damage response signaling and cell сус\е checkpoint control.32 Therefore, we also asked ,vhether t\1e cellular sensitivity towal'd neocarzinostatin (NCS), а radiomimetic agent that induces DNA DSBs, could Ье modнlated Ьу cotl'eatment ,vitЬ imatinib. The l'esult fi·om the clonogenic survival assay indeed showed that imatinib 1·endered МDА-МВ-231 cells more sensitive toward NCS (Figure Sc). ln sнmma1y, we made signilicant advances in qнantitative analysis of kinome i11 lшman cells. Ву developing а PRМ-based method for qнantifying the protein expression of kinases a11d comЬi11i11g it with our pt'evioнsly reported MRМ-based 1 targeted proteomic approacl1, 4 ,ve are аЬ\е to assess indepe11dently the protein expt'ession and ATP-Ьi11ding affinities ofki11ases. ln paгticнlar, our РRМ and МRМkinome libгaries each encompass appюximately 80% of the lшma11 kinome, with 350 kinases bei11g commonly i11clнded i11 both libl'aгies (Figure lc). Previously puЬ\ished methods for profiling the kinase i11hiЬitor-i11duced alterations of the human ki11ome in cells rely on the use of affinity resin immo\Jilized with multiple ki11ase iпhiЬitors.6'33 However, the Ьindi11g of ki11ases to inhiЬitor-immoЬilized affinity resin, similar to the АТР affinity probe method employed here,1 3, 1 4, 16 can Ье influenced Ъу tЬе protein expl'ession level and sometimes Ъу activity-induced conformational changes df pгotein kinases.6 То the best of our kno,vledge, our PRМ­ based ta1·geted pгoteomic approach facilitated, for the first time, the assessme11t of the alterations in protein expression levels of kinases upon small-molecule inhiЬitor treatme11t. Our results unveiletl pronuunced increases iн expressioп levels of а large numbel' of ki11ases in cells upon treatment with imatinib. Analysis of kinase protei11 expression a11d ATP-Ьi11ding affinity data together led to the discove1y of а number of ki11ases exhiЬiting diminutions i11 ATP-Ьi11di11g affinity but not i11 protei11 exp1·essio11 in К 5 - 62 cells upon imati11ib admin­ istration, undeгscoгi11g that imati11ib disrupts the ATP-Ьi11ding activities of many kinases. Amo11g these kinases, we validated that the activity of CHKl kinase could Ъе inhiЬited upon imatinib tгeatment, whicЬ is further substantiated Ьу tЬе observatio11 that imatinib could sensitize саnсег cells to,vard NCS, а гadiomimetic agent сараЬ\е of i11ducing DNA doнЬ\e­ stra11d breaks. We envisio11 tl1at our metЬods will Ье ge11erally applicaЬ\e for i11vestigati11g Ьоw the protei11 exp1·ession levels and ATP-Ьi11di11g affinities of tЬе human ki11ome are modulated Ьу other ki11ase i11!1iЬitors iп live cells. SucЬ k11owledge will Ье valuaЬ\e for tЬе appropгiate нsе of ki11ase inhiЬitors i11 cell signaling гesearch a11d in therapeutic inteгve11tions of hнman diseases. D etailed expeгimental pюcedures, repl'esentative РRМ qua11tification data, and ki11ome map for tЬе qнantified kinases (PDF) List of ki11ases included i11 the Skyli11e huma11 РRМ kinome libra1y (XLSX) E xpl'ession levels a11d АТР probe labeli11g efficiency of kinases after imatiпib treatment ( XLSX) ■ . REFERENCES (1) ,Lemmon, М. А; Scblessinger,J. Cell 2010, 141, 1117-1134. (2) Wu, Р.; Nielsen, Т. Е.; Claнsen, М. Н. Tm1ds P/1amiacol. Sci. 2015, 36, 422-439. (3) Druker, В. J. Са11се1· Cell 2002, 1, 31-36. (4) Cui, У.; Steagall, W. К.; Lamattina, А. М.; Pacheco-Rodrigнez, G.; Stylianou, М.; КidamЬi, Р.; Stнmp, В.; Golzarri, F.; Rosas, I. О.; P1·iolo, С.; Henske, Е. Р.; Moss, ].; El-Chemaly, S. Са11се1· Res. 2017, 77, 1492. (5) Dancey, ].; Sausville, Е. А Nat. Rev. Dmg Discove,y 2003, 2, 296-313. (6) Duncan, J. S.; Whittle, М. С.; Nakamшa, К.; Abell, А. N.; Мidland, А. А.; Za,visto,vski,]. S.; Johnson, N. L.; Granger, D. А.; Jordan, N. V.; Darr, D. В.; Usary, ].; Kuan, Р. F.; Smalley, D. М.; Маjог, В.; Не, Х.; Hoadley, К. А.; Zhoн, В.; Shaгpless, N. Е.; Perou, С. М.; Кim, W. У.; Gomez, S. М.; Chen, Х.; Jin, ].; Frye, S. V.; Еагр, Н. S.; Graves, L. М.; Jolшson, G. L. Cell 2012, 149, 307-21. (7) Clюng, С. R.; Sнllivan, D. ]., JI. Nat111·e 2007, 448, 645-6. (8) Manning, G.; Whyte, D. В.; Ma,·tinez, R.; Hнnter, Т.; Sнdarsanam, S. Scie11ce 2002, 298, 1912-34. (9) Peteгson, А. С.; Rнssell,]. D.; Bailey, D. ].; WestpЬall, М. S.; Coon,].]. Mol. Cell. P,·oteomics 2012, 11, 1475-88. (10) Miao, W.; Li, L.; Wang, У. A11al. С/1е111. 2018, 90, 6835-6842. (11) Мiао, W.; Li, L.; Wang, У. A11al. С/1е111. 2018, 90, 1175111755. (12) Ong, S.-E.; Blagoev, В.; l(i-atcl1ma1·ova, I.; l(i-istensen, D. В.; Steen, Н.; Pandey, А.; Mann, М. Mol. Cell. P,·oteomics 2002, 1, 376386. (13) Patricelli, М. Р.; Nomanbhoy, Т. К.; Wн,J.; Bro,vn, Н.; Zhoн, D.; Zhang, J.; JagannatЬan, S.; Aban, А.; Okerberg, Е.; Herring, С.; Nordin, В.; Weissig, Н.; Yang, Q; Lee,J. D.; Gray, N. S.; КоzагiсЬ,J. W. С/1е111. Biol. 2011, 18, 699-710. (14) Мiао, W.; Xiao, У.; Gно, L.; Jiang, Х.; Huang, М.; Wang, У. Анаl. С/1е111. 2016, 88, 9773-9779. (15) Patricelli, М. Р.; Szardenings, А. К.; Liyanage, М.; NomanЬЬoy, Т. К.; Wu, М.; Weissig, Н.; Aban, А; Chнn, D.; Tanner, S.; Kozarich, ]. W. Bioc/1e111ist1y 2007, 46, 350-8. (16) Xiao, У.; Gно, L.; Wang, У. Mol. Cell. Pгoteomics 2014, 13, 1065-75. ■ 6 (17) Milani, G.; Lana, Т.; Bresolin, S.; Aveic, S.; Pasto, А.; Frasson, С.; Те Kronnie, G. Mol. Са11се1· Res. 2017, 15, 683-695. (18) Reiter, L.; Rinner, О.; Picotti, Р.; Hiittenhain, R.; Beck, М.; B1·usniak, М.-У.; Hengartner, М. О.; Aebersold, R. Nat. Met/1ods 2011, 8, 430. (19) Dоеп, А. Nat. Met/1ods 2012, 9, 950. (20) Rauniyal', N. I11t. ]. Mol. Sci. 2015, 16, 28566_:_28581. (21) MacLean, В.; Tomazela, D. М.; Shulman, N.; Chambe1·s, М.; Finney, G. L.; Fre,ven, В.; Kern, R.; ТаЬЬ, D. L.; LieЬ\er, D. С.; MacCoss, М.]. Bioi11fom1atics 2010, 26, 966-8. (22) Manning, G.; Whyte, D. В.; Martinez, R.; Hunter, Т.; Sudat'sanam, S. Scie11ce 2002, 298, 1912. (23) Escl1er, С.; Reiter, L.; MacLean, В.; Ossola, R.; Непоg, F.; Chilton, J.; MacCoss, М. J.; Rinner, О. P1·oteo111ics 2012, 12, 11111121. (24) de Graaf, Е. L.; Altelaar, А. F.; van Breukelen, В.; Mohammed, S.; Heck, А. J. ]. P/'oteome Res. 2011, 10, 4334-4341. (25) Уо1шg, М. А.; Shah, N. Р.; Chao, L. Н.; Seeliger, М.; Milanov, Z. V.; Biggs, W. Н.; Treiber, D. К.; Patel, Н. К.; Zarrinkar, Р. Р.; Lockhart, D.J.; Sa,vyers, С. L.; Kшiyan,J. Cmica Res. 2006, 66, 1007. (26) Procaccia, V.; Nakayama, Н.; Shimizu, А.; Кlagsbrнn, М. Bioc/1e111. Biopl1ys. Res. Со1111111111. 2014, 448, 134-138. (27) Virgili, А.; Koptyra, М.; Dasgпpta, У.; Glodko,vska-Mro,vka, Е.; Stoklosa, Т.; Nacheva, Е. Р.; Skorski, Т. Cmice1· Res. 2011, 71, 5381. (28) Giles, F. J.; Cortes, J. Е.; Kantarjian, Н. М. Сшт. Mol. Med. 2005, 5, 615-623. (29) Davis, М. I.; Hнnt, J. Р.; Herrgard, S.; Ciceri, Р.; Wodicka, L. М.; Pallares, G.; Hocker, М.; Treiber, D. К.; Zarrinkar, Р. Р. Nat. Biotec/1110/. 2011, 29, 1046-1051. (30) FaЬian, М. А.; Biggs, W. Н., 3rd; Treiber, D. К.; Atteridge, С. Е.; Azimioara, М. D.; Benedetti, М. G.; Carter, Т. А.; Ciceri, Р.; Edeen, Р. Т.; Floyd, М.; Ford, J. М.; Galvin, М.; Gerlach, J. L.; Grotzfeld, R. М.; Herrgard, S.; Insko, D. Е.; Insko, М. А.; Lai, А. G.; Lelias,J. М.; Mehta, S. А.; Мilanov, Z. V.; Velasco, А. М.; Wodicka, L. М.; Patel, Н. К.; Zarrinkar, Р. Р.; Lockl1art, D.J. Nat. Biotec/1110/. 2005, 23, 329-36. (31) Кim, Т. М.; На, S. А.; Кim, Н. К.; Уоо, J.; Кim, S.; Yim, S. Н.; Jнng, S. Н.; Кim, D. W.; Clшng, Y.J.; Кim,J. W. Blood Cmica]. 2011, 1, No. е32. (32) Li11, Q; Gнntuku, S.; Снi, Х. S.; Matsuoka, S.; Cortez, D.; Tamai, К.; Lно, G.; Carattini-Rivera, S.; DeMayo, F.; Bradley, А.; Doneho,ver, L. А.; Elledge, S. J. Ge11es Dev. 2000, 14, 1448-14S9. (33) Кlaege1·, S.; Heiпzlmeir, S.; Wilhelm, М.; Polze1·, Н.; Vick, В.; Koenig, Р.-А.; Reinecke, М.; Rнprecht, В.; Petzoldt, S.; Meng, С.; Zecha, J.; Reitel', К.; Qjao, Н.; Helm, D.; Koch, Н.; Schoof, М.; Canevari, G.; Casale, Е.; Depaolini, S. R.; Feнchtinger, А.; Wн, Z.; Schmidt, Т.; Rнeckert, L.; Becker, W.; Hнenges, J.; Garz, А.-К.; Goblke, В.-0.; Zolg, D. Р.; Kayse1·, G.; Vooder, Т.; Preissner, R.; Наlше, Н.; Tonisson, N.; Krame1·, К.; Gotze, К.; Bassermaпn, F.; Scblegl,J.; Eh1·1ich, Н.-С.; Aiche, S.; Walch, А.; Greif, Р. А.; Sclшeider, S.; Felder, Е. R.; Rнland,J.; Medard, G.; Je1·emias, I.; Spiekermaпп, К.; Kнster, В. Scie11ce 2017, 358, No. eaan4368. 7