Mass Spectrometry Methods

2.2.7.1 In-Gel Digestion of Proteins

In-gel hydrolysis of proteins was performed according to the modified protocol of Shevchenko et al., 2007. Unless otherwise stated, all the incubation steps were carried out at 26 °C with continuous shaking at 1050 rpm for 15 min. The solutions were removed after each incubation step. Each SDS-PAGE gel lane was cut into 22 equal slices with the help of in-house designed gel cutting device (Schmidt & Urlaub, 2009). Each gel slice was cut into small pieces, washed with 150 µl of water and dehydrated with 150 µl of acetonitrile. The gel pieces were then dried by SpeedVac. The proteins were reduced by adding 100 µl of 100 mM DTT prepared in 50 mM ammonium bicarbonate pH 8 and incubating at 56 °C for

55 50 min at 1050 rpm. The gel pieces were then dehydrated with 150 µl of acetonitrile and the reduced cysteines were alkylated with 100 µl of 60 mM Iodoacetamide (IAA) prepared in 50 mM ammonium bicarbonate pH 8 for 20 min at 26 °C and 1050 rpm. The gel pieces were washed with 150 µl of 50 mM ammonium bicarbonate pH 8, dehydrated and dried again as described before.

The dried gel pieces were rehydrated with 20 µl of trypsin digestion buffer (15 µl of 0.1 µg/µl modified trypsin making volume upto 100 µl with 25 mM ammonium bicarbonate pH 8), for 30 min on ice. The gel pieces were overlaid with 25 mM ammonium bicarbonate pH 8, if needed. Then they were incubated at 37 °C for overnight with constant shaking at 1050 rpm in thermomixer.

2.2.7.2 Extraction of Peptides

The peptides from in-gel digestion were extracted according to the protocol described by Shevchenko et al., 2007. All incubation steps were carried out at 37°C for 15 min with a constant shaking at 1050 rpm by thermomixer. The gel pieces were processed by series of extraction steps comprised of incubation with 50 µl of water and 50 µl of acetonitrile then with 50 µl of 5% (v/v) formic acid followed by twice with 50 µl of acetonitrile. The supernatants from each step were collected and pooled together in new microcentrifuge tubes. The extracted peptides were dried in SpeedVac and stored at -20 °C until subjected to LC-ESI-MS/MS analysis. For MS analysis the samples were dissolved in 20 µl of 5% (v/v) acetonitrile and 1% (v/v) formic acid by extensive vortexing and sonication for 2min each. The samples were then analyzed by LTQ Orbitrap XL mass spectrometer (Thermo Scientific).

2.2.7.3 LC-ESI-MS/MS

The mass spectrometric analysis was carried out by administering the samples to nanoflow-liquid chromatography (nano-LC) system coupled to electrospray ionisation mass spectrometer (ESI-MS). During the course of Ph.D. studies three mass spectrometers were used. The samples from in-gel digestion were analyzed on LTQ Orbitrap XL mass spectrometer (Thermo Scientific) coupled to Agilent nano-LC system (Agilent Technologies) whereas the RNA-protein

cross-56 linking samples were analyzed on LTQ Orbitrap Velos (Thermo Scientific) and Q-Exactive (Thermo Scientific) mass spectrometers coupled to Agilent nano-LC system (Agilent Technologies) and EASY-nLC II system (Thermo Scientific) respectively. The details regarding LC separation and MS analysis are given as follows.

(A) Nanoflow-Liquid Chromatography Separation (Nano-LC) (i) Nano-LC Separation (Agilent nano-LC system)

The sample was applied onto trapping column (C18 AQ 120 Å material with particle size of 5 µm, 20 mm length, 0.150 mm inner diameter) at a flow rate of 10μl/min (60 min gradient) and 15 μl/min (118 min gradient) in 3% buffer B (buffer A: 0.1% (v/v) formic acid; buffer B: 95% (v/v) acetonitrile, 0.1% (v/v) formic acid) followed by elution and separation on an analytical column (C18 AQ 120 Å material with particle size of 5 µm, 150 mm length, 0.075 mm inner diameter) at a flow rate of 130 nl/min (60 min gradient) and 150 nl/min (118 min gradient) using a linear gradient of 4-37% buffer B (buffer A: 0.1% (v/v) formic acid; buffer B:

95% (v/v) acetonitrile, 0.1% (v/v) formic acid) over 37 min (60 min gradient) and 102 min (118 min gradient). The column was then washed with 90-95% buffer B and re-equilibrated with 3% buffer B. Both the columns were packed in-house by Uwe Pleßmann (Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany).

(ii) Nano-LC Separation (Thermo EASY-nLC II system)

The sample was injected into a trapping column (C18 AQ 120 Å material with particle size of 3 µm, 40 mm length, 0.1 mm inner diameter) in-line with the analytical column (C18 AQ 120 Å material with particle size of 3 µm, 10 cm length, 50 µm inner diameter), both packed in-house by Uwe Pleßmann (Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany). The sample was loaded onto trapping column at a flow rate of 15 μl/min in 3% buffer

57 B (buffer A: 0.1% (v/v) formic acid; buffer B: 95% (v/v) acetonitrile, 0.1% (v/v) formic acid) followed by elution and separation on an analytical column at a flow rate of 320 nl/min using a linear gradient of 4-36% buffer B (buffer A: 0.1% (v/v) formic acid; buffer B: 95% (v/v) acetonitrile, 0.1% (v/v) formic acid) over 42 min (50 min gradient) and 97 min (105 min gradient). The column was then washed with 95% buffer B and equilibrated automatically by the instrument.

(B) ESI-MS/MS Analysis

(i) LTQ Orbitrap XL Mass Spectrometer

The instrument was operated in data dependent acquisition mode with Top 8 method. The MS scans were recorded in the m/z range 350-1600 at a resolution setting of 30,000 FWHM at m/z 400 and automatic gain control (AGC) at 10⁶. Fragmentation was generated by CID activation for the precursor ions having the charge state 2 and above. The MS/MS scans were recorded at normalized collision energy of 35 and a dynamic exclusion of 60 sec with a repeat count of 1.

(ii) LTQ Orbitrap Velos Mass Spectrometer

The instrument was operated in data dependent acquisition mode with Top 10 method. The MS survey scans were recorded in the m/z range 350-1600 at a resolution setting of 30,000 FWHM. The automatic gain control was set to 10⁶. Fragmentation was generated by HCD activation for the precursor ions having the charge state 2, 3 and 4. The MS/MS scans were recorded at normalized collision energy of 35 and a dynamic exclusion of 20 sec at a resolution setting of 7500 FWHM and isolation width of 2 Th.

(iii) Q-Exactive Mass Spectrometer

The instrument was operated in data dependent acquisition mode with Top 12 method. The MS survey scans were recorded in the m/z range 350-1600 at a resolution setting of 70,000 FWHM. The automatic gain control was set to 10⁶. Fragmentation was generated by HCD activation for the precursor ions having the charge state 2, 3 and 4. The MS/MS scans were recorded at normalized

58 collision energy of 28 and a dynamic exclusion of 15 sec at a resolution setting of 17,500 FWHM with a fixed first mass of m/z 100.