The Ettan Spot Handling Workstation was used to core, digest and extract protein spots of interest (User Manual, Amersham Biosciences). Gel spots were washed, dehydrated, digested using 50 μL of sequencing grade-modified trypsin (5 μg/mL in 50 mM ammonium bicarbonate) containing 0.01% ProteaseMax (Promega, Madison WI), and extracted according to standard protocols. Peptide sequences were determined by capillary-liquid chromatography tandem mass spectrometry (Cap-LC/MS/MS) using an UltiMate™ 3000 LC system and an LTQ mass spectrometer (both from Thermo Scientific) equipped with a CaptiveSpray source (Bruker Daltonics Billerica, MA) operated in positive ion mode according to standard methods in the OSU CCIC Mass Spectrometry and Proteomics Facility.32 (link) The MS/MS data acquired was converted into mascot generic files (.mgf) using MS Convert (ProteoWizard) and the resulting files were searched against all NCBInr other mammalia using Mascot Daemon by Matrix Science version 2.2.1 (Boston, MA) setting the mass tolerance of the precursor ions and fragment ions at 1.8 and 0.8 Da, respectively. Considered modifications were oxidation, deamidation and carbamidomethylation. Protein identifications were checked manually and only proteins with a Mascot score of 100 or higher with a minimum of two unique bold red peptides were accepted.
Ltq mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The LTQ mass spectrometer is a versatile instrument designed for a wide range of analytical applications. It utilizes linear ion trap technology to provide high-sensitivity detection and accurate mass measurements. The LTQ mass spectrometer is capable of performing MS and MS/MS experiments, enabling the analysis of complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc, by Thermo Fisher Scientific (18 mentions)
Trace dsq, by Thermo Fisher Scientific (5 mentions)
Ultimate 3000 lc system, by Thermo Fisher Scientific (3 mentions)
Mascot server, by Matrix Science (3 mentions)
Trypsin, by Promega (3 mentions)
Novex tris glycine gel, by Thermo Fisher Scientific (2 mentions)
Sepharaose cl4b protein a beads, by GE Healthcare (2 mentions)
Flag m2 antibody, by Merck Group (2 mentions)
C18 integrafrit nano precolumn, by New Objective (2 mentions)
C18 reversed phase capillary column, by New Objective (2 mentions)
Acquity uplc, by Waters Corporation (2 mentions)
Thermo finnigan trace dsq ms, by Thermo Fisher Scientific (2 mentions)
Prodigy 5μ c18 column, by Phenomenex (2 mentions)
Pepmap c18, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap discovery mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Microlab star system, by Hamilton Company (2 mentions)
Acquity ultra performance liquid chromatography, by Thermo Fisher Scientific (2 mentions)
Acquity uplc system, by Thermo Fisher Scientific (2 mentions)
Proteasemax, by Promega (1 mentions)
Captivespray source, by Bruker (1 mentions)
96 protocols using ltq mass spectrometer
1
Protein Identification by Mass Spectrometry
2
Contactless Electrospray Ionization Source
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All MS experiments were carried out on LTQ mass spectrometer (Thermo Scientific, San Jose, CA). The parameters of instrument were set as follows: capillary temperature = 275 °C, capillary voltage = 9 V, tube lens voltage = 100 V, maximum inject time = 100 ms, microscans = 1. The commercial ionization source was removed and replaced by a home-made ionization source35 (link). The ionization source was achieved by inserting a copper wire (as electrode) into the emitter and ensure the wire and emitter’s tip are contactless (the distance is usually 5 mm). The emitter was placed 5 mm in front of MS inlet. The electrode voltage was −1.4 kV for negative mode analysis. When the emitter’s tip was bedewed by the droplet, the electrospray would be triggered. The contactless way could make the pico-liter sample have spray time of 18 s, which is adequate for full scan and MS/MS scan. The photograph of the ionization source was shown in Fig. 1b . MS/MS analysis was performed with 30 eV collision energy.
3
Affinity Purification of BUD31 Complexes
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Hela cells transduced with lentivirus encoding BUD31 cDNA and non-transduced Hela cells were harvested, and nuclear extracts as well as whole cell lysates were collected as described previously33 . Lysates were treated with RNase A (500 ug/mL) for 1 hour on ice. For IPs, nuclear and whole cell extracts were ultracentrifuged at 100,000 rcf, and incubated with 25 ug M2 Flag antibody (Sigma) for 1 hour, followed by ultracentrifugation and incubation with Sepharaose-CL4B Protein A beads (GE Healthcare). Beads were washed with NTN (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.5% NP-40), and immunocomplexes were resuspended in 1× Laemmli buffer and resolved on pre-cast 4–20% Novex Tris-Glycine gels (Life Technologies). Gels were minimally stained with Coomassie brilliant blue, cut into 8 molecular weight ranges, and digested with trypsin. Immunocomplexes were identified on a Thermo Fisher LTQ mass spectrometer, and data processing was performed as previously described33 .
4
LCMS Analysis of Synthetic Peptides
5
Electrospray and MALDI-TOF Mass Spectrometry of Chitosan
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Electrospray analyses were performed on a linear trap quadrupole (LTQ) mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) in positive ion mode using direct infusion of the samples in a mixture of water/methanol (4/1, v/v) (0.1 mg/mL). Electrospray source parameters were as follows: capillary voltage +20 V, tube lens voltage +90 V, capillary temperature 300 °C, sheath and auxiliary gas flow (N2) 8 and 5, sweep gas 0, spray voltage 3.6. Spectra were acquired by full range acquisition covering m/z 50–2000. Collision-induced dissociations were made with helium.
MALDI-TOF spectra were registered on a Voyager mass spectrometer (Applied Biosystems, Waltham, MA, USA) equipped with a pulsed nitrogen laser (337 nm) and a time-delayed extracted ion source. Spectra were recorded in positive ion mode using reflectron mode with an accelerating voltage of 20 kV. The chitosan samples were dissolved in a mixture made of H2O/MeOH (50/50 v/v) and acetic acid (0.1%, v/v) at 10 mg/mL. The 2,5-dihydroxybenzoic acid (DHB) matrix solution was prepared by dissolving DHB (10 mg) in MeOH (1 mL). A MeOH solution of cationization agent (NaI, 10 mg/mL) was also prepared. The solutions were combined in a 10:1:1 volume ratio of matrix to sample to cationization agent. One to two microliters of the obtained solution were deposited onto the sample target and vacuum dried.
MALDI-TOF spectra were registered on a Voyager mass spectrometer (Applied Biosystems, Waltham, MA, USA) equipped with a pulsed nitrogen laser (337 nm) and a time-delayed extracted ion source. Spectra were recorded in positive ion mode using reflectron mode with an accelerating voltage of 20 kV. The chitosan samples were dissolved in a mixture made of H2O/MeOH (50/50 v/v) and acetic acid (0.1%, v/v) at 10 mg/mL. The 2,5-dihydroxybenzoic acid (DHB) matrix solution was prepared by dissolving DHB (10 mg) in MeOH (1 mL). A MeOH solution of cationization agent (NaI, 10 mg/mL) was also prepared. The solutions were combined in a 10:1:1 volume ratio of matrix to sample to cationization agent. One to two microliters of the obtained solution were deposited onto the sample target and vacuum dried.
6
Proteomic Analysis of Human Proteoglycans
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Proteoglycan-enriched fractions in 25 mM ammonium bicarbonate were reduced (10 mM DTT, 10 min, 95 °C), alkylated (25 mM iodoacetamide, 20 min, room temperature), and digested (sequencing grade trypsin, 20 μg/ml, 16 h, 37 °C). Samples were subjected to peptide analysis by liquid chromatography–tandem mass spectrometry (LC-MS2) as previously described (74 (link)). Samples were analyzed by LC-MS2 using an LTQ mass spectrometer (Thermo Fisher Scientific). The data was analyzed using the peaklist-generating software Mascot Daemon/extract_msn (version 2.5.1; Matrix Sciences) and the Mascot search engine (version 2.6.2) together with the NCBI sequence database with the homo sapiens taxonomy (November 2016 with 97,105,869 total sequences/309,980 human sequences in the database) with the following parameters: no fixed modifications; variable modifications = carbamidomethyl (C), oxidation (M) and propionamide (C); peptide mass tolerance = 4 ppm, fragment mass tolerance = 0.4 Da, maximum missed cleavages = 1; threshold score = MOWSE score >70.
7
Proteomic analysis of oral epithelial cells
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2-DE and LC-MS/MS in human immortalized oral epithelial cells (HIOECs) and in HB96 cells were described thoroughly in our previous study [22 (link)]. Briefly, HIOECs and HB96 cells were lysed, sonicated and protein was quantified. First-dimensional IEF was completed with an IPGphor IEF System (Amersham Biosciences, Uppsala, Sweden) and second-dimensional SDS–PAGE was performed with a Hoefer SE 600 Ruby System (Amersham). Differentially expressed protein spots were excised and digested for mass spectroscopy. The peptide mixtures were isolated and identified by a Finnigan LTQ mass spectrometer coupled with the Surveyor HPLC system (Thermo, Sunnyvale, CA). Differentially expressed protein identification in MS/MS raw data was determined using the SEQUEST program in the BioWorks 3.1 software suite (University of Washington, licensed to Thermo Finnigan) based on the International Protein Index human database version 3.15.1.
8
Proteomic Analysis by LC-MS/MS
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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on a multi-dimensional LC system (Ettan MDLC, GE Healthcare Life Sciences) coupled to a LTQ mass spectrometer (Thermo Scientific, Braunschweig, Germany). Further settings were the same as described in the study by Otte et al.13
9
In-Gel Trypsin Digestion and LC-MS/MS
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Protein bands on SDS-PAGE gels were de-stained and in-gel digested with sequencing grade trypsin (10 ng/μL trypsin, 50 mM ammonium bicarbonate, pH 8.0) overnight at 37 °C. Peptides were extracted with 5% formic acid/50% acetonitrile and 0.1% formic acid/75% acetonitrile sequentially and then concentrated to ∼20 μl. The extracted peptides were separated by an analytical capillary column (50 μm × 10 cm) packed with 5 μm spherical C18 reversed phase material (YMC, Kyoyo, Japan). An Agilent 1100 series binary pump system (Agilent Technologies, Santa Clara, CA, USA) was used to generate the following HPLC gradient: 0%-5% B in 5 min, 5%-40% B in 70 min, 40%-100% B in 10 min (A = 0.2 M acetic acid in water, B = 0.2 M acetic acid/70% acetonitrile). The eluted peptides were sprayed into a LTQ mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a nano-ESI ion source. The mass spectrometer was operated in data-dependent mode with one MS scan followed by five MS/MS scans for each cycle. Database searches were performed on an in-house Mascot server (Matrix Science, London, UK) against IPI (International Protein Index) rat protein database. Methionine oxidation was set as variable modification.
10
Proteomic Analysis of Oral Lesions
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Intact proteins were isolated from the FFPE tissues of OLP (n=6) and OLP-OSCC (n=6) with the Qproteome FFPE Tissue kit (Qiagen, Valencia, CA, USA). In total, 5 μg of proteins from pooled OLP or OLP-OSCC sample were digested with 50 ng enzyme-grade trypsin (Promega, Madison, WI, USA) for overnight. LC-MS/MS of the resulting peptides was performed using a NanoLC system (Eksigent Technologies, Dublin, CA, USA) and a LTQ mass spectrometer (Thermo Fisher, Waltham, MA, USA). The peptides were concentrated and desalted on a C18 IntegraFrit Nano-Precolumn (New Objective, Woburn, MA, USA) for 10 min, then eluted and resolved using a C18 reversed-phase capillary column (New Objective). LC separation was performed at 400 nL/min with the following mobile phases: A, 5% acetonitrile/0.1%formic acid (v/v); B, 95% acetonitrile/0.1% formic acid (v/v). The chosen LC gradient was: from 5% to 15% B in 1 min, from 15% to 100% B in 40 min, and then maintained at 100%B for 15 min. Database searches were performed using the X! Tandem search engine against the SwissProt protein sequence database. The search criteria were set with a mass accuracy of 0.4 Da and semi-style cleavage by trypsin. Proteins with two unique peptides are considered as positively identified.
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