Computational protocol: Network of protein interactions within the Drosophila inner kinetochore

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Protocol publication

[…] Peptides mixtures were analysed by LC-MS-MS/MS (liquid chromatography coupled to tandem mass spectrometry) using the Nano-Acquity (Waters) LC system and Orbitrap Velos mass spectrometer (Thermo Electron Corp., San Jose, CA, USA). Prior to the analysis, proteins were subjected to standard ‘in-solution digestion’ procedure during which proteins were reduced with 100 mM DTT (for 30 min at 56°C), alkylated with 0.5 M iodoacetamide (45 min in darkroom at room temperature) and digested overnight with trypsin (sequencing Grade Modified Trypsin, Promega V5111). Peptide mixture was applied to an RP-18 precolumn (nanoACQUITY Symmetry® C18, Waters 186003514) using water containing 0.1% TFA as mobile phase and then transferred to a nano-HPLC RP-18 column (nanoACQUITY BEH C18, Waters 186003545) using an acetonitrile gradient (0–60% AcN in 120 min) in the presence of 0.05% formic acid with a flowrate of 150 nl min−1. The column outlet was directly coupled to the ion source of the spectrometer working in the regime of data-dependent MS to MS/MS switch. A blank run ensuring lack of cross contamination from previous samples preceded each analysis. Acquired raw data were processed by Mascot Distiller followed by Mascot Search (Matrix Science, London, on-site licence) against FlyBase (in the case of affinity purifications from D. melanogaster cell extracts) or NCBI (in the case of Duet co-purifications from E. coli cell cultures) databases. Search parameters for precursor and product ion mass tolerance were 20 ppm and 0.6 Da, respectively, with search parameters set as follows: one missed semitrypsin cleavage site allowed, fixed modification of cysteine by carbamidomethylation and variable modification of lysine carbamidomethylation and methionine oxidation. Peptides with Mascot score exceeding the threshold value corresponding to less than 5% false positive rate, calculated by Mascot, were considered to be positively identified. [...] The list of peptic CENP-C1–94, Nnf1a, Mis12, Nsl1 peptides was established using non-deuterated protein samples. For this aim, a 5 µl aliquot of protein sample (at least 25 µM in 300 mM NaCl, 20 mM Tris–HCl pH 8.0, 10% (vol/vol) glycerol and 2 mM 2-mercaptoethanol) was diluted 10 times by adding 45 µl of 20 mM Tris pH 8 and 150 mM NaCl (H2O Reaction buffer). Next, the sample was acidified by mixing with 10 µl of 2 M glycine buffer, pH 2.5 (H2O Stop buffer) and injected into the nanoAQUITY UPLC system (Waters, Milford, MA, USA). The sample was digested online using an immobilized pepsin column (Porozyme, ABI, Foster City, CA, USA) with 0.07% formic acid in water as mobile phase (flow rate 200 µl min−1). Digested peptides were next trapped on a C18 column (ACQUITY BEH C18 VanGuard Pre-column, Waters) and then were directed into a reverse phase column (Acquity UPLC BEH C18 column, Waters) with a 6–40% gradient of acetonitrile in 0.1% formic acid at 40 µl min−1 using nanoACQUITY Binary Solvent Manager. Total time of a single run was 13.5 min. All fluidics, valves and columns were kept at 0.5°C using HDX Manager (Waters). The pepsin column was kept at 13°C inside the temperature-controlled digestion compartment of the HDX manager. Leucine–enkephalin solution (Sigma) was used as a Lock mass. For protein identification, mass spectra were acquired in MSE mode over the m/z range of 50–2000. The spectrometer parameters were as follows: ESI positive mode, capillary voltage 3 kV, sampling cone voltage 35 V, extraction cone voltage 3 V, source temperature 80°C, desolvation temperature 175°C and desolvation gas flow 800 l h−1. Peptides were identified using ProteinLynx Global Server software (Waters). The list of identified peptides containing peptide m/z, charge and retention time was further processed with DynamX v. 2.0 program (Waters). Hydrogen–deuterium exchange experiments were carried out as described for non-deuterated samples with the Reaction buffer prepared using D2O (99.8% Armar Chemicals, Switzerland), in which the pHread (uncorrected meter reading) was adjusted using DCl or NaOD (Sigma). Five microlitres of protein stock was mixed with 45 µl D2O Reaction buffer and exchange reaction was carried out for a specific time period (either 10 s, 1 min or 20 min) at room temperature. The exchange was quenched by reducing the pHread to 2.5 by adding the reaction mixture into an Eppendorf tube containing ice-cold Stop buffer (2 M glycine buffer, pHread 2.5). Immediately after quenching, the sample was manually injected into the nanoACQUITY (Waters) UPLC system. Further pepsin digestion, LC and MS analysis were carried out exactly as described for the non-deuterated sample. To assess minimum exchange (in-exchange control), D2O Reaction buffer was added to Stop buffer cooled on ice prior to protein stock addition and the mixture immediately subjected to pepsin digestion and LC-MS analysis as described above. The deuteration level in the in-exchange experiment was calculated using DynamX and denoted as 0% exchange (). For out-exchange analysis, the 5 µl protein stock was mixed with 45 µl of D2O Reaction buffer, incubated overnight in 4°C to avoid protein degradation, mixed with Stop buffer and analysed as described above. The deuteration level in the out-exchange experiment was calculated and denoted as 100% exchange (). All HDX experiments and control experiments were repeated three times, and the final results represent a mean of these triplicates. […]

Pipeline specifications

Software tools Mascot Distiller, PLGS
Databases FlyBase
Application MS-based untargeted proteomics
Organisms Drosophila melanogaster
Chemicals Deuterium, Hydrogen