Ltq orbitrap elite mass spectrometer

In total, 0.15–2 μg of depolymerized GAGs was used for each analytical run. The nLC-MS/MS setup has previously been described in detail (14 (link)). Briefly, GAGs were trapped on a 2 cm × 100 μm Acclaim PepMap C18 precolumn, separated on a 30 cm × 75 μm C4 column or C18 column using a stepwise elution gradient from 0% to 70% methanol with 5 mM DBA and 8 mM acetic acid at 300 nl/min over 60 min, and analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) operated in the negative electrospray ionization mode. For the MS¹ only analysis, the spectra were acquired in the m/z range 220–2000 at 120,000 resolution, and for the MS² analysis, precursor ions were scanned in the m/z range 220–2000 at 60,000 resolution in MS¹, followed by the HCD-MS² spectra of the five most abundant precursor ions, each with normalized collision energies (NCEs) at 60%, 70%, and 80%. The MS² spectra were acquired in centroid mode in the m/z range 100–2000 at 15,000 resolution. Dynamic exclusion was disabled and precursor ions with unassigned charge states were rejected.

U87 cells (1.5 × 10⁶), stably expressing GFP or GFP-DIAPH3, were lysed in RIPA buffer and immunoprecipitated with anti-GFP antibodies at 4 ^oC, as above. DU145 cells (1.25 × 10⁶), stably expressing GFP or GFP-DIAPH3, were immuno-precipitated at 4 ^oC or 25 ^oC. Proteins eluted from the beads were separated in a 10% SDS-PAGE gel, and in-gel reduced, alkylated, and tryptically digested56 (link). Tryptic peptides were extracted, concentrated, reconstituted in 0.1% formic acid, separated on a 25 cm EASY-Spray C18 column, and analyzed by an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific). After each survey scan, up to 20 collision-induced dissociation (CID) spectra were acquired in the rapid CID scan mode. For protein identification and quantitation, raw mass spectrometric data were searched against the Uniprot_Human database (released on 02/20/14, including 88647 sequences) with MaxQuant (v 1.3.0.5)57 (link) and Andromeda58 (link). False discovery rates for protein and peptide identifications were set at 0.01. Identified proteins were quantitated based on their summed ion intensities. All data have been deposited into ProteomeXchange59 (link). The DIAPH3 interactome was analyzed using the DAVID bioinformatics database60 (link).

The dried peptide mixtures were reconstituted with 30% acetonitrile/0.1% formic acid for analysis using a LTQ-Orbitrap ELITE mass spectrometer (Thermo Fisher Scientific) as previously described [31 (link)]. The MS raw data files were analyzed by Proteome Discoverer software (version 1.4; Thermo Fisher Scientific) including the reporter ions quantifier node for iTRAQ quantification. The MS/MS spectra was searched against the TrichDB-33_TvaginalisG3 sequence database (97,471 entries) using the Mascot search engine (version 2.5; Matrix Science, London, UK). For peptide identification, 10 ppm mass tolerance was permitted for intact peptide masses, and 0.05 Da for HCD fragment ions with an allowance for two missed cleavages made from the trypsin or semi-trypsin digestion: oxidized methionine, acetyl (protein N-terminal), iTRAQ (N-terminal), and iTRAQ (lysine) as variable modifications; and Methylthio (cysteine) as the fixed modifications. The peptide-spectrum match (PSM) was then filtered based on high confidence and Mascot search engine rank 1 of peptide identification to ensure an overall false discovery rate below 0.01. Proteins with a single peptide hit were removed. The identified proteins with fold changes higher than 2-fold or lower than 0.5-fold in the MTZ-R proteome upon MTZ treatment were considered differentially expressed in this study.

Samples were loaded on a NuPAGE Bis-Tris 4%–12% gradient gel (Invitrogen) and the Coomassie stained bands were pooled and in gel digested with trypsin and GluC, respectively, as described elsewhere [62 (link)]. LC-MS analyses of the peptides were done on an EasyLC nano-HPLC (Proxeon Biosystems) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific) as described elsewhere [63 (link)]. MS data were processed using the software suite MaxQuant, version 1.2.2.9 [64 (link)] and searched using Andromeda search engine [65 (link)] against a target-decoy E. coli database containing 4,311 forward protein sequences, the sequences of the tagged and overexpressed proteins and 248 frequently observed protein contaminants. Trypsin or GluC, were set as proteases in which two missed cleavage sites were allowed. Carbamidomethylation of cysteine was set as fixed modification; N-terminal acetylation, methionine oxidation and serine/threonine/tyrosine phosphorylation were set as variable modifications. Initial precursor mass tolerance was set to 6 parts per million (ppm) at the precursor ion and 20 ppm at the fragment ion level. False discovery rates were set to 1% at peptide, phosphorylation site, and protein group level.

To analyse the in vivo crosslinked protein complexes, the corresponding protein bands were excised and divided into six samples and, respectively, subjected to the in-gel cleavage and digestion procedure mentioned above. The extracted peptides were reconstituted in 0.2% formic acid, loaded onto a 100 μm × 2 cm pre-column and separated on a 75 μm × 20 cm capillary column both of which were packed in-house with 4 μm C18 bulk materials (InnosepBio, China). An Easy nLC 1000 system (Thermo Scientific, USA) was used to generate the following HPLC gradient: 7–35% B in 40 min, 35–75% B in 4 min, then held at 75% B for 20 min (A=0.1% formic acid in water, B=0.1% formic acid in acetonitrile). The eluted peptides were sprayed into an LTQ-Orbitrap-Elite mass spectrometer (Thermo Scientific, USA) equipped with a nano-ESI source. The mass spectrometer was operated in data-dependent mode with one MS scan in FT mode at a resolution of 30,000 followed by 10 CID (Collision Induced Dissociation) MS/MS scans in the ion trap for each cycle.