Human ccRCC cell line 786-O with PHD3 knockdown LC-MS/MS
Appreciating the pseudohypoxic signalling in ccRCC cells we decided to look into the effects of an established hypoxia-related enzyme, namely PHD3, in ccRCC development. Interestingly, these cells express PHD3 in unusually high amounts, a phenomena yet unexplained. We were interested in determining how ccRCC cells expressing elevated levels of PHD3 would respond to its knockdown. It has been shown that PHD3 has many other functions in addition to its given role as regulating HIF-1 by targeting it for proteosomal degradation. Motivated by these varied roles of PHD3 we decided to look for indications of a systemic-level changes that might be governed by PHD3 in ccRCC. To this end we chose a discovery proteomics –approach to see how ccRCC cells would respond on proteome level to PHD3 depletion.
Sample Processing Protocol
Gel electrophoresis and in-gel digestion Proteins were separated on Criterion XT 12% Bis-Tris gel (BioRad) and silver stained. Bands were cut into 1 millimetre pieces and reduced and alkylated with 20mM dithiothreitol (DTT) in 100mM NH4HCO3 and with 55 mM iodoacetamide (IAA) in 100mM NH4HCO3, respectively. For reaching optimal efficacy of the reduction and alkylation, the samples were incubated in DTT for 30 min in +56 °C and in IAA for 20 min in RT, dark. In between the reagents gel pieces were dehydrated with 100 % acetonitrile (ACN). After reduction and alkylation 60 µl of trypsin in 40mM NH4HCO3 /10% ACN was added and incubated in +37 ° C for 18 hours. After in-gel digestion, the peptides were extracted in two steps; first using 100 % ACN for 15 min in +37 ° C, and secondly using 50 % ACN in 5 % CHOOH for 15 min in +37 ° C followed by a mixing of the supernatants. The extracted peptides were vacuum dried and stored at -20 ° C until analysis. LC-MS/MS Analysis Tryptic peptides were dissolved in 0.2 % formic acid (CHOOH) and 200-ng samples were submitted for LC-MS/MS system, where peptides were separated according to their hydrophobicity on a reversed-phase chromatography column. Each sample was analysed in three biological replicates using a QExactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientiﬁc). The QExactive was coupled to an EASY-nLC 1000 nano ﬂow LC instrument (Thermo Fisher Scientiﬁc). Sample loading, solvent delivery, and scan functions were controlled by Xcalibur software (version 2.1.0 SP1.1160, Thermo Fisher Scientiﬁc). An in-house-build trap column (2.5 cm long, 75μ m inner diameter) (Magic AQ C18 resin, 5μm/200 Å, Bruker-Michrom, Billerica, MA, USA) was used for desalting and concentrating the sample, and an analytical column (15 cm long, 75μ m inner diameter) (PicoFrit, 15 μm, NewObjective, Woburn, MA, USA) with the same C18 resin was used for peptide separation. A 45 min gradient from 95% solvent A (98% H2O, 2% ACN, and 0.2% HCOOH) to 90% solvent B (95% ACN, 5% H2O, and 0.2% HCOOH) with a ﬂow rate 0.3 μL/min was used for peptide elution. Data-dependent acquisition was performed in positive ion mode. The MS spectra were acquired from the range of 300 - 2000 m/z in the Orbitrap with resolution of 70 000, an AGC target value of 1 × 106 ions, and a maximal injection time of 120 ms. The MS/MS spectra were acquired in the Orbitrap with a resolution of 17 500, isolation window of 2.0 m/z, an AGC target value of 2 × 104 ions, maximal injection time of 250 ms, lowest mass ﬁxed at 100 m/z, and dynamic exclusion duration set to 15 s.
Data Processing Protocol
The database search for the raw spectrum ﬁles was performed in Proteome Discoverer (version 18.104.22.1689, Thermo Fisher Scientiﬁc) by using Mascot (Matrix Science, London, U.K.). The search was done for peptides formed with trypsin digestion, where one missed cleavage site was allowed, against UniProtKB/Swiss-Prot human database (2015-05-26). Search parameters were as follows: decoy database search was performed, accepted precursor mass tolerance was set to 5 ppm, and fragment mass tolerance to 0.02 Da, fixed modification of carbamidomethylation of cysteine and variable modification of methionine oxidation.
Petra Miikkulainen, University of Turku
Panu M. Jaakkola, Department of Medical Biochemistry, Faculty of Medicine, University of Turku, FI-20520 Turku, Finland and Department of Oncology and Radiotherapy, Turku University Hospital, FI-20520 Turku, Finland ( lab head )
Miikkulainen P, Högel H, Rantanen K, Suomi T, Kouvonen P, Elo LL, Jaakkola PM. HIF prolyl hydroxylase PHD3 regulates translational machinery and glucose metabolism in clear cell renal cell carcinoma. Cancer Metab. 2017 Jul 4;5:5. eCollection 2017 PubMed: 28680592