# Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock
## 4 Materials and Methods
### Protein Identification Using Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS)
#### 4.7.2 LC-MS/MS Analysis  
The MS shotgun analysis was done with an UltiMate 3000 (Thermo Fisher Scientific, Bremen, Germany) nanoflow high-performance liquid chromatography (HPLC) system connected to a hybrid LTQ-Orbitrap XL (Thermo Fisher Scientific, Bremen, Germany) equipped with a nanoelectrospray ion source (New Objective). This HPLC system included a solvent degasser, a nanoflow pump, a thermostatted column compartment kept at 30 \({}^{\circ}\)C, and a thermostatted autosampler kept at 8 \({}^{\circ}\)C to reduce sample evaporation. This system uses two mobile phases: mobile A (99.9% Milli-Q water and 0.1% v/v formic acid); and mobile B (99.9% acetonitrile and 0.1% v/v formic acid). An aliquot of 10 \(\upmu\)L of prepared peptide mixture was loaded over 3 min at a flow rate of 25 \(\upmu\)L/min onto a PepMap C18 pre-column trap (5 mm \(\times\) 300 \(\upmu\)m i.d., 300 A pore size, 5 \(\upmu\)m, Thermo Fisher Scientific, Bremen, Germany) in 2% buffer B. This step was followed by reverse-phase separations (0.250 \(\upmu\)L/min) using a PepMap C18 analytical column (15 cm \(\times\) 300 \(\upmu\)m i.d., 300 A pore size, 5 \(\upmu\)m). We ran a gradient from 2-35% buffer B for the first 60 min, with 35-60% buffer B during minutes 60 to 85, and finally with 60-90% buffer B for minutes 85 through 105. The column was washed with 90% buffer B for 16 min and then with 2% buffer B for 19 min, after which the next sample was loaded. The peptides were detected by directly eluting them from the HPLC column into the electrospray ion source of the mass spectrometer. The HPLC buffer underwent an electrospray ionization (ESI) of 1.6 kV using the liquid junction provided by the nanoelectrospray ion source, with the ion transfer tube temperature set to 200 \({}^{\circ}\)C. The LTQ-Orbitrap XL was operated in data-dependent mode by automatically switching between full-scan MS and consecutive MS/MS acquisitions. The full survey scan spectra (mass range of 400-2000) were acquired in the Orbitrap with a resolution of \(r\) = 60,000 at m/z 400, and the ion injection times for each spectrum were calculated to allow for the accumulation of 106 ions. In each survey, the ten most intense peptide ions (each having an intensity above 2000 counts and a charge state \(\geq\) 2) were sequentially isolated at a target value of 10,000, then fragmented in the linear ion trap by collision-induced dissociation. Normalized collision energy was set to 35%, with an activation time of 30 milliseconds. To avoid selecting the same ion more than once, the peaks selected for fragmentation were automatically put on a dynamic exclusion list for 30 s, with a mass tolerance of \(\pm\)10 ppm. The following parameters were used: the repeat count was set to 1; the exclusion list was limited to 500; singly-charged precursors were rejected; and the maximum injection time was set to 500 milliseconds for full-scan MS, or 300 milliseconds for MS/MS. The fragment ion spectra were recorded in the LTQ mass spectrometer in parallel with the Orbitrap full-scan detection. The Orbitrap was externally calibrated before each injection series to ensure an overall error rate under 5 ppm for the detected peptides. MS data were saved in the RAW file format using Xcalibur 2.0.7 and Tune 2.4 (Thermo Fisher Scientific, Bremen, Germany). A data analysis was performed using Proline 1.4 software, Mascot Distiller, and Mascot Server 2.5.1 (Matrix Science, Boston, MA, USA). The identification of peptides and proteins was done using the Mascot search engine via its automatic decoy database search, calculating a false discovery rate of 1% at the peptide level. MS/MS spectra were compared to the _Escherichia coli_ Reference Proteome. For the MS and MS/MS experiments, mass tolerances were set to 10 ppm and 0.5 Da, respectively. The protein modifications used for this are as follows: fixed carbamidomethylation of cysteines, variable oxidation of methionine, variable acetylation of lysine, and variable acetylation of N-terminal residues.