Q exactive hybrid quadrupole orbitrap mass spectrometer

Spectra were collected on a modified QExactive hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific) optimized for transmission of high-mass complexes (68 (link)). Protein concentration was 15 μm by monomer. Capillary voltage was 1.4 kV in positive ion mode with source temperature 200 °C and S-lens RF 200%. UHV pressure (argon) was between 1.4 × 10⁻⁹ and 1.7 × 10⁻⁹ mbar. In-source trapping fragmentation voltage ranged from −150 to −180 V. Ion transfer optics were as follows: injection flatapole 10 V, inter-flatapole lens 8 V, bent flatapole 6 V, transfer multipole 4 V, C-trap entrance lens 3 V. Nitrogen was used in the HCD cell and HCD energy was 0 V for intact spectra and tuned for optimal dissociation of each protein for CID spectra, ranging from 200 to 230 V. Resolution was kept at 17,500 at m/z = 200 for a transient time of 64 ms and the noise threshold was set to 3. For CID spectra, groupings of 30 microscans were combined to improve signal quality. Data were visualized using Xcalibur (ThermoFisher Scientific) and calibrated manually according to expected peak positions for WT αB-crystallin. Calibrated CID data were processed using UniDec software, which allowed for stoichiometric assignment and post-hoc correction for dissociated subunits (62 (link)).

For proteomic analysis, the colon mucosa tissue samples were lysed and labeled with tandem mass tag (TMT, Thermo Scientific, San Jose, CA, USA) according to the manufacturer’s instructions. The labeled peptide samples were pooled into a new vial and dried using SpeedVac (Thermo Scientific). The following processes, including strong cation exchange fraction and liquid chromatography (LC)-mass spectrometry (MS) and database searching, were performed by Poochon Scientific (Frederick, MD) as described previously^{22 (link)}. Briefly, TMT-multiplex labeled peptide mixture (100 µg protein/each plex) fractionation was performed using an Agilent AdvanceBio Column and Agilent UHPLC 1290 system (Agilent, Santa Clara, CA). LC/MS/MS analysis was performed using a Thermo Scientific Q-Exactive hybrid Quadrupole-Orbitrap Mass Spectrometer and Thermo Dionex UltiMate 3000 RSLCnano System (Thermo Scientific). Raw MS data files were searched against the human protein sequence databases obtained from the NCBI website using Proteome Discoverer 1.4 software (Thermo Scientific) based on the SEQUEST and percolator algorithms. The false positive discovery rate (FDR) was set at 5%. The resulting Proteome Discoverer Report from Poochon Scientific contained all assembled proteins with peptide sequences and peptide spectrum match counts (PSM#) and TMT-tag-based quantification ratios.

Proteins were isolated from adult feather skeleton and analyzed as previously described^{36 (link)}. All organic material was removed from the skeleton by extensive washing with sodium hypochlorite followed by guanidine isothiocyanate. The skeleton was demineralized with acetic acid followed by dialysis. Both soluble and insoluble protein fractions were analyzed. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Each lane was excised into 20 equal sized segments and processed. In-gel digestion with trypsin was performed on each fraction, followed by analysis by nano Liquid chromatography–mass spectrometry (LC-MS/MS) with a Waters nanoAcquity high-performance liquid chromatography system interfaced to a ThermoFisher Q Exactive hybrid quadrupole-orbitrap mass spectrometer. The mass spectrometer was operated in a data-dependent mode. Data were used to search predicted peptides from the A. japonica genome using Mascot and then parsed into the Scaffold algorithm for validation and filtering, using a 95% protein identification score with at least two peptides per protein.

Approximately 7.0 μg of original bNK2A-C10C20 peptide was aliquoted into four tubes (10 μL each) and 20 mM triethylammonium borocarbonate buffer, pH 8.0 (13 μL each) was added to each tube. Two microliters of tris(2-carboxyethyl)phosphine (TCEP; final concentration = 50 mM) were added to two tubes and PBS to the other two tubes and samples were incubated at room temperature for 10 min. An alkylating agent iodoacetamide (IAA) was added to one of the PBS and TCEP tubes (IAA; 110 mM final concentration) and incubated at room temperature for additional 60 min. Samples were diluted in PBS and matrix-assisted laser desorption/ionization (MALDI-TOP) mass spectrometry analysis was performed using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher Scientific) at the Protein Facility, Iowa State University.

The protein sequence analysis of protease from S. fusiformis was performed nano-Reverse Phase LC coupled to a QExactive Hybrid Quadrupole—Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) through a nanoelectrospray ion source (ThermoScientific, Bremen, Germany) as following: a protein band of protease was in gel-digested for 3 h at 37 °C by trypsin. The resulting peptide mixture was resolved and applied on LC–MS/MS. Raw MS files were analyzed by the MaxQuant v1.5.3.3 proteomics software package. Precursor and MS/MS mass tolerance was set to 20 ppm for the first search (for the identification of the maximum number of peptides for mass and retention time calibration) and 4.5 ppm for the main search (for the refinement of the identifications). Protein and peptide false discovery rate (FDR) were set to 1%. FDR was calculated based on the number of spectra matched to peptides of a random proteome database (reversed sequence database) in relation to the number of spectra matching to the reference proteome. Peptide features were aligned between different runs and masses were matched (“match between runs” feature), with a match time window of 3 min and a mass alignment window of 20 min. Protein quantification was performed using the iBAQ algorithm through MaxQuant software.

First, extracted peptides were loaded onto an in-house packed C18 ‘trapping’ column (0.15 mm x 20 mm, Reprosil-Pur 120 C18-AQ 5 μm, Dr. Maisch GmbH, Germany). Then a second C18 column was connected in tandem (an analytical C18 capillary column; 0.075 mm x 250 mm column self-packed with 3 μm Reprosil-Pur 120 C18-AQ). Peptides were then eluted using a 105 min linear gradient (5– 35% acetronitrile in 0.1% FA at 300 nl/min) on an EASY nLC-1000 system in-line coupled to a Q Exactive hybrid quadrupole/orbitrap mass spectrometer (Thermo Scientific, Dreieich). The instrument was operated in data-dependent acquisition mode with a survey scan resolution of 70,000 at m/z 200 and an AGC target value of 1 x 10⁶. Up to 15 of the most intense precursor ions with charge state 2 or higher were sequentially isolated at an isolation width of 2.0 m/z for higher collision dissociation (HCD) with a normalized collision energy of 25%. Dynamic exclusion was set to 30 s to avoid a repeating sequencing of the same precursor ion.