targeting the ubiquitin system
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1 DU activity based probe explorer panel (human, synthetic) UbiQ code : UbiQ-L02 Storage : powder at 20 C; solution at 80 C. void multiple freeze/thaw cycles. ackground. UbiQ-L02 is a panel of 10 deubiquitylating enzyme (DU) activity based probes 1-9 (Ps, 10 ug each) prepared by total chemical synthesis. These DU Ps are potent, irreversible and specific inhibitors of DUs which can be used for: o inhibiting hydrolysis of poly-ub chains on substrate proteins and thus enhancement of poly-ub chain accumulation. 1, 8,9 o structural biology studies of DU-Ub complexes o DU activity profiling experiments 1-9 1, 3, 4 o determine DU inhibitor specificity The panel consists of Ps with two types of C-terminal warheads: the vinyl methyl ester (VME) 3-9 and the recently developed propargylamide (P) warhead. 1,2 Furthermore various detection/affinity tags are present: the and biotin tag 3,4, 6-9 and the fluorescent dyes Cy and TMR 1,3 for in-gel fluorescence scanning as read-out. Ub-VME= UbiQ-00 -hx-hx-ub-vme= UbiQ-03 iotin-hx-ub-vme= UbiQ-04 TMR-Ub-VME= UbiQ-00 Cy-Ub-VME= UbiQ-071 Ub-P= UbiQ-07 -hx-hx-ub-p= UbiQ-078 iotin-hx-ub-p= UbiQ-076 TMR-Ub-P= UbiQ-08 Cy-Ub-P= UbiQ-072 Important: sample preparation. Dissolve the powder in as little DMS as possible (e.g. 20 mg/ml= 10 ug in 0. ul DMS) and add this DMS stock slowly to milliq (please note the order of addition). ext buffer with e.g. 1M EPES to 0 mm EPES. In general EPES and Tris buffers are standard for DU assays - please note that certain DUs react different to low or high acl concentrations. Furthermore, 2- mm TCEP or DTT can be used as reducing agent for the DU (see Wrigley et al. Cell iochem. iophys. 2011, 60, 99). final buffered volume of 20 ul yields a stock of 0. mg/ml (± um) which contains 2. vol% DMS. If required, total removal of DMS is accomplished by dialysis or spin-filtration (3 kda cut-off membrane). Literature. 1. Ekkebus et al. J. m. Chem. Soc. 2013, 13, Sommer et al. ioorg. Med. Chem. 2013, 21, de Jong et al. ChemioChem 2012, 13, ltun et al. Chem. iol. 2011, 18, El ualid et al. ngew. Chem. Int. Ed. 2010, 49, Misaghi et al. J. iol. Chem. 200, 280, Galardy et al. Methods in Enzymology 200, 399, orodovsky et al. Chemistry and iology 2002, 9, orodovsky et al. EM J. 2001, 20, 187. ote: higher molecular weight artefacts are observed sometimes during SDS-PGE analysis of monoub reagents (especially with reactive DU activity based probes). There is no proof for these higher mol. weight bands actually being present in the material as judged by LC-MS analysis. For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 1
2 Ub-VME (human, synthetic) UbiQ code : UbiQ-00 atch # : Purity : 9% by RP-PLC Mol. Weight : 8609 Da by MS (calc Mw 860 Da) ) vinylmethyl ester (VME) Sequence Ub-VME MQIFVKTLTGKTITLEVEPSDTIEVKKIQDKEGIPPDQQRLIFGKQLEDGRTLSDYIQKESTLLVLRLRG-VME UC-L3 Ub-VME UC-L3 Ub-VME : LC-MS analysis. Mobile phase = 1% C 3C, 0.1% formic acid in milliq and = 1% milliq and 0.1% formic acid in C 3C. Phenomenex Kinetex C18, (2.1 0 mm, 2.6 μm); flow rate = 0.8 ml/min, column T = 40 C. Gradient: % 9% over 3. min. : SDS-PGE analysis (12% is-tris, MES buffer) of reaction between UC-L3 and UbiQ-00 (excess). For exp. details, see ref. 1 & 2 For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 2
3 -hx-hx-ub-vme (human, synthetic) UbiQ code : UbiQ-03 atch # : Purity : 9% by RP-PLC Mol. Weight : 9913 Da by MS (calc Mw 9912 Da) YPYDVPDY hx-hx Sequence -hx-hx-ub-vme YPYDVPDY-(hx) 2-MQIFVKTLTG KTITLEVEPS DTIEVKKI QDKEGIPPDQ QRLIFGKQL EDGRTLSDY IQKESTLLV LRLRG-VME UC-L3 Ub-VME UC-L3 Ub-VME : LC-MS analysis. Mobile phase = 1% C3C, 0.1% formic acid in water (milliq) and = 1% water (milliq) and 0.1% formic acid in C 3C. Phenomenex Kinetex C18, (2.1 0 mm), 2.6 μm); flow rate= 0.8 ml/min, runtime= 6 min, column T= 40 C. Gradient: 0 0. min: % ; 0. 4 min: % 9% ; 4. min: 9%. : SDS-PGE analysis. 12% is-tris, MES buffer. Comparison DU labeling efficiency between conventional -Ub-VME (obtained from bacterial expressed Ub precursor) and synthetic -hx-hx-ub-vme (UbiQ-03). EL4 cell lysate was incubated at ambient temperature for 1 min. with indicated concentrations of probe; both probes (i.e. expressed and synthetic UbiQ-03) showed comparable DU labeling. = ackground bands due to cross-reactivity of anti- antibody. * For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 3
4 iotin-hx-ub-vme (human, synthetic) UbiQ code : UbiQ-04 atch # : Purity : 9% by RP-PLC Mol. Weight : found 8944 Da, calc 894 Da Sequence iotin-hx-ub-vme iotin-hx-mqifvktltgktitlevepsdtievkkiqdkegippdqqrlifgkqledgrtlsdyiqkestllvlrlrgg-vme LC-MS analysis. Mobile phase = 1% C 3C, 0.1% formic acid in water (milliq) and = 1% water (milliq) and 0.1% formic acid in C 3C. Phenomenex Kinetex C18, (2.1 0 mm, 2.6 μm); flow rate = 0. ml/min, runtime = 6 min, column T = 40 C. Gradient: % 9% over 3. min. For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 4
5 TMR-Ub-VME (human, synthetic) UbiQ code : UbiQ-00 atch # : Purity : 9% by RP-PLC Mol. Weight : 9017 Da by MS (calc Mw 9017 Da) C 2 Sequence TMR-Ub-VME TMR-MQIFVKTLTGKTITLEVEPSDTIEVKKIQDKEGIPPDQQRLIFGKQLEDGRTLSDYIQKESTLLVLRLRG-VME : LC-MS analysis. Mobile phase = 1% C 3C, 0.1% formic acid in milliq and = 1% milliq and 0.1% formic acid in C 3C. Phenomenex Kinetex C18, (2.1 0 mm, 2.6 μm); flow rate = 0. ml/min, column T = 40 C. Gradient: % 9% over 3. min. : SDS-PGE analysis. 12% is-tris gel, MES buffer. Fluorescence scan exc 0 nm, emi 90 nm. UbiQ-00 (μm) 0 ⅛ ¼ ½ 1 1 Labeling in lysate. EL4 lysate was incubated with indicated concentrations of UbiQ-00 at ambient temperature for 1 min. Compound is a pan-du inhibitor that is included to show how TMR-Ub-VME can be used to monitor DU inhibitor specificity. In-gel fluorescence scans were obtained with a ProXPRESS 2D Proteomic imaging system (Perkin Elmer) (resolution= 100 μm and exposure time of 60s, λ ex/λ em= 0/90 nm). For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands
6 Cy-Ub-VME (human, synthetic) UbiQ code : UbiQ-071 atch # : Purity : 9% by RP-PLC Mol. Weight : 9082 Da by MS (calc Mw 908 Da) + Cy-Ub-VME Cy-MQIFVKTLTGKTITLEVEPSDTIEVKKIQDKEGIPPDQQRLIFGKQLEDGRTLSDYIQKESTLLVLRLRG-VME M Ub UbiQ -071 : LC-MS analysis. Mobile phase = 1% C 3C, 0.1% formic acid in milliq and = 1% milliq and 0.1% formic acid in C 3C. Phenomenex Kinetex C18, (2.1 0 mm, 2.6 μm); flow rate = 0. ml/min, column T = 40 C. Gradient: % 9% over 3. min. : SDS-PGE analysis. 12% gel, MES buffer. Left: fluorescence scanning (60/690 nm), right: C staining. For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 6
7 Ub-P (human, synthetic) UbiQ code : UbiQ-07 atch # : Purity : 9% by RP-PLC Mol. Weight : 846 Da by MS (calc Mw 84 Da) Propargylamide (P) Ub Sequence Ub-P MQIFVKTLTGKTITLEVEPSDTIEVKKIQDKEGIPPDQQRLIFGKQLEDGRTLSDYIQKESTLLVLRLRG-P.. C. UbiQ-07 UbiQ-07 : verview of Click-on/Click-off pull down. Ub-Prg can be directly immobilized onto Sepharose resin. The immobilized probe is incubated with a mixture of DUs and non-dus (i.e. lysate). Cysteine DUs will selectively react with immobilized UbiQ-07, resulting in their covalent attachment. Stringent washing removes unbound non-dus. fter purification, the DUs can be cleaved under radical conditions for retrieval of active DUs or by treatment with % aq. trifluoroacetic acid for MS-profiling. : SDS-PGE analysis. In vitro reaction of three different classes of DUs with UbiQ-07. C: GFP fusions of DUs from the USP and TU-classes were transfected in MelJuSo cells and their reaction with UbiQ-07 visualized using anti-gfp western blot. DUs annotated with CS are catalytic Cys to Ser mutants. For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 7
8 -hx-hx-ub-p (human, synthetic) UbiQ code : UbiQ-078 atch # : Purity : 9% by RP-PLC Mol. Weight : 982 Da by MS (calc Mw 982 Da) YPYDVPDY hx-hx Sequence -hx-hx-ub-p YPYDVPDY-hx-hx-MQIFVKTLTGKTITLEVEPSDTIEVKKIQDKEGIPPDQQRLIFGKQLEDGRTLSDYIQKESTLLVLRLRG-P LC-MS analysis. Mobile phase = 1% C 3C, 0.1% formic acid in water (milliq) and = 1% water (milliq) and 0.1% formic acid in C 3C. Xridge E300 C18 μm 4.6x100mm; column T = 40 C, flow= 0.8 ml/min. Gradient: 30 9% over 3. min. For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 8
9 iotin-hx-ub-p (human, synthetic) UbiQ code : UbiQ-076 atch # : Purity : 9% by RP-PLC Mol. Weight : 888 Da by MS (calc Mw 888 Da) S iotin hx Sequence iotin-hx-ub-p iotin-hx-mqifvktltgktitlevepsdtievkkiqdkegippdqqrlifgkqledgrtlsdyiqkestllvlrlrgg-p : LC-MS analysis. Mobile phase = 1% C 3C, 0.1% formic acid in milliq, = 1% milliq and 0.1% formic acid in C 3C. Phenomenex Kinetex C18, (2.1 0 mm, 2.6 μm); flow rate= 0. ml/min, column T= 40 C. Gradient: % 9% over 3. min. : SDS-PGE analysis. Coommassie blue staining, 12% SDS-PGE gel. For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 9
10 TMR-Ub-P (human, synthetic) UbiQ code : UbiQ-08 atch # : Purity : 9% by RP-PLC Mol. Weight : 897 Da by MS (calc Mw 897 Da) C 2 Sequence TMR-Ub-P TMR-MQIFVKTLTGKTITLEVEPSDTIEVKKIQDKEGIPPDQQRLIFGKQLEDGRTLSDYIQKESTLLVLRLRG-P C EL4 cell lysate + MM UbiQ -08 I: a nti-β-actin : LC-MS analysis. Mobile phase = 1% C 3C, 0.1% formic acid in milliq and = 1% milliq and 0.1% formic acid in C 3C. Phenomenex Kinetex C18, (2.1 0 mm, 2.6 μm); flow rate = 0. ml/min, column T = 40 C. Gradient: % 9% over 3. min. : SDS-PGE analysis. 12% gel, MES buffer. Fluorescence scan λ ex/λ em= 0/90 nm. C: Cell lysate labeling in lysate. Labeling of increasing amount of EL4 lysate with UbiQ-08. For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 10
11 Cy-Ub-P (human, synthetic) UbiQ code : UbiQ-072 atch # : Purity : 9% by RP-PLC Mol. Weight : 8994 Da by MS (calc Mw 902 Da) + Cy-Ub-P Cy-MQIFVKTLTGKTITLEVEPSDTIEVKKIQDKEGIPPDQQRLIFGKQLEDGRTLSDYIQKESTLLVLRLRG-P : LC-MS analysis. Mobile phase = 1% C 3C, 0.1% formic acid in milliq and = 1% milliq and 0.1% formic acid in C 3C. Phenomenex Kinetex C18, (2.1 0 mm, 2.6 μm); flow rate = 0. ml/min, column T = 40 C. Gradient: % 9% over 3. min. : SDS-PGE analysis, 12% gel, MES buffer. Left: fluorescence scanning λ ex/λ em= 60/690 nm, right: C staining. For Laboratory Research Use nly, ot For Use in umans msterdam, The etherlands 11
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