C 18 spin columns

All reagents were purchased from Sigma unless otherwise indicated. Yeast nitrogen base with ammonium sulfate was obtained from MP Biomedicals. Dulbecco’s Phosphate Buffered Saline (DPBS) was obtained from Invitrogen. Bortezomib and epoxomicin were purchased from LC Laboratories and Millipore, respectively. Acetonitrile was obtained from Fisher. Hydrochloric acid, trifluoroacetic acid (TFA) mass spectroscopy grade and C-18 spin columns were purchased from Pierce Thermo Scientific. Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-Leu-Leu-Val-Tyr-AMC) was obtained from Bachem. The isotopic labeling reagents 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) containing either 0, 3, 6, or 9 atoms of deuterium (D0-, D3-, D6-, and D9-TMAB-NHS, respectively) were synthesized as described [34 (link)]. The blm10Δ::NatMX3 strain yMS63 [35 (link)] and its isogenic wild-type BY4741 parent MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 [36 (link),37 (link)] were obtained from Marion Schmidt (Department of Biochemistry, Albert Einstein College of Medicine). The deletion strains pdr5Δ::KanMX4 and snq2Δ::KanMX4 in BY4741 were generated in the yeast deletion project [38 (link)].

Q7Q7, Q7Q111 and Q111Q111 cells were obtained from Dr. Erik Snapp (Albert Einstein College of Medicine) and cultured as described [36 (link)]. High glucose Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s Phosphate Buffered Saline (DPBS), penicillin-streptomycin, and M-MLV Reverse Transcriptase were purchased from Invitrogen. Fluorescent substrates containing the 7-amino-4-methylcoumarin (AMC) group were procured from Bachem. Hydroxylamine, G-418, glycine, sodium hydroxide, dibasic sodium phosphate, dimethyl sulfoxide (DMSO), and bestatin were purchased from Sigma. Hydrochloric acid, mass spectroscopy grade trifluoroacetic acid (TFA), and C-18 spin columns were purchased from Pierce Thermo Scientific. Other reagents and their sources were puromycin (Tocris Bioscience), TriPure Reagent (Roche), Power SYBR Green Master Mix (Applied Biosystems), acetonitrile (Fisher), fetal bovine serum (Seradigm), epoxomicin (Calbiochem), and bortezomib (LC Laboratories). The isotopic labeling reagents 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) containing either 0, 3, 6, or 9 atoms of deuterium (D0, D3-, D6-,and D9-, respectively) or 9 atoms of deuterium and three ¹³C atoms (D12-) were synthesized as described [37 (link),38 (link)].

High glucose Dulbecco’s Modified Eagle’s Medium (DMEM), L-glutamine enriched Roswell Park Memorial Institute medium (RPMI) and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from Invitrogen. Hydroxylamine, glycine, sodium hydroxide, dibasic sodium phosphate and dimethyl sulfoxide (DMSO) were obtained from Sigma. Acetonitrile was obtained from Fisher. Hydrochloric acid, trifluoroacetic acid (TFA) mass spectroscopy grade and C-18 spin columns were purchased from Pierce Thermo Scientific. MG132, MG262 and clasto-Lactacystin β-lactone were purchased from Boston Biochem. AM114, butabindide and puromycin were purchased from Tocris Bioscience. Other inhibitors and their commercial sources were bortezomib (LC Laboratories), MLN2238 (Selleckchem), carfilzomib (ChemieTek), bestatin (Sigma), and bestatin methylester (Calbiochem). Recombinant human puromycin-sensitive aminopeptidase was purchased from R&D Systems. Suc-Leu-Leu-Val-Tyr-AMC, Ala-Ala-Phe-AMC, Leu-AMC and Ala-AMC were procured from Bachem. The isotopic labeling reagents 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) containing either 0, 3, 6, or 9 atoms of deuterium (D0-, D3-, D6-, and D9-TMAB-NHS, respectively) or 9 atoms of deuterium and three ¹³C atoms (D12-TMAB-NHS) were synthesized as described [30] .