NOD islets were prepared for LC–MS/MS analyses as described elsewhere (5 (link)). In vitro reactions and prepared NOD islets were analyzed by LC–MS/MS using an Agilent 1200 series UHPLC system with a nanoflow adapter and an Agilent 6550 Q-TOF equipped with a nano-ESI source. Online separation was accomplished by reversed-phase liquid chromatography using a Thermo Acclaim PepMap 100 C18 trap column (75 μm × 2 cm; 3 μm particles; 100 Å pores) and Thermo Acclaim PepMap RSLC C18 analytical column (75 μm inner diameter; 2 μm particles; 100 Å pores) in a trap forward-elute configuration using a water/acetonitrile gradient (buffer A: 0.1% formic acid in water; buffer B: 0.1% formic acid and 90% acetonitrile in water). A detailed description of the MS data collection was previously published (12 (link)).
Pepmap rslc c18 analytical column
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The PepMap RSLC C18 analytical column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of peptides. It features a C18 stationary phase and is suitable for reversed-phase chromatography applications.
Automatically generated - may contain errors
Lab products found in correlation
Easy spray ion source, by Thermo Fisher Scientific (4 mentions)
Easy nlc 1200 system, by Thermo Fisher Scientific (3 mentions)
Pepmap 100 c18 trap column, by Thermo Fisher Scientific (2 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (2 mentions)
C18 acclaim pepmap100 precolumn, by Thermo Fisher Scientific (2 mentions)
Zorbax extend c18 column, by Agilent Technologies (2 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (2 mentions)
Pepmap 100 μ precolumn, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
1200 series uhplc system, by Agilent Technologies (1 mentions)
6550 q tof, by Agilent Technologies (1 mentions)
Janus automated liquid handling workstation, by PerkinElmer (1 mentions)
Proteome discoverer, by Thermo Fisher Scientific (1 mentions)
Mascot search engine, by Matrix Science (1 mentions)
Easynano lc 1000 hplc system, by Thermo Fisher Scientific (1 mentions)
Xcalibur software, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap trap column, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion lumos tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive high field mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap 100 column, by Thermo Fisher Scientific (1 mentions)
10 protocols using pepmap rslc c18 analytical column
1
NOD Islet LC-MS/MS Analysis
2
Proteomic Analysis of IL-1β Stimulated RTS-11 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Supernatant (20 μL) from RTS-11 cells stimulated with 25 ng/mL IL-1β for 36 h was size separated in a 4–12% Bis-tris SDS-PAGE gel as described above. Supernatant sample equivalent to ~11 kDa to ~14 kDa was cut out from the gel and stored in double-distilled water at 4 °C. Proteins were digested in gel using a JANUS automated liquid-handling workstation (PerkinElmer, Beaconsfield, UK) with a customised method adapted from that of Shevchenko and colleagues [29 (link)]. Tryptic peptides were dried by vacuum centrifugation and dissolved in 0.1% TFA for liquid chromatographic–tandem mass spectrometric (LC-MS/MS) analysis using a Q Exactive Plus/UltiMate 3000 RSLCnano system (Thermo Fisher Scientific, Hemel Hempstead, UK) equipped with an EASY-Spray source and configured for pre-concentration onto a 25-cm long PepMap RSLC C18 analytical column (Thermo Fisher Scientific). Tandem mass spectra were acquired using a data-dependent “Top 10” method as described before [30 (link)]. Peptides were identified by comparison with the O. mykiss proteome (UniProt proteome UP000193380, downloaded 17 January 2019) using Proteome Discoverer (version 2.2, Thermo Scientific) and a workflow incorporating the Mascot search engine (version 2.6, Matrix Science, London, UK) and decoy database validation with a strict target false discovery rate (FDR) of 0.01.
3
Peptide Separation and Proteomic Analysis
4
Quantitative Mass Spectrometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Digested samples were analyzed using a Q-Exactive™ HF-X quadrupole Orbitrap benchtop mass spectrometer (Thermo Fisher Scientific). Equal amounts of the protein digests were injected into an Easy nano-LC 1000 HPLC system (Thermo Fisher Scientific) equipped with an Acclaim PepMap® 100 nanoViper pre-column (Thermo Scientific, C18, 3 μm particles, 75 μm i.d. 2 cm long) and a PepMap® RSLC C18 analytical column (Thermo Scientific, C18, 2 μm particles, 75 μm i.d. 25 cm long) (RSLC: rapid separation liquid chromatography). On-line reversed-phase separation was performed using a flow rate of 300 nL/min. A binary gradient of 125 min was used, starting with a 5 min increase from 5% B to 7% B, then increasing to 20% B over 85 min, further increasing to 30% B over 20 min, and with a final 5 min increase to 90% B, after which it ended with 90% B isocratic for 10 min. The system was controlled by Xcalibur™ Software (Thermo Fisher Scientific), and blank runs were injected between every sample to avoid cross-contamination. A heated ion transfer setting of 280°C was used for desolvation, together with a spray voltage of +1850 V.
5
Mass Spectrometry-Based Peptide Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peptides generated by trypsin treatment of the excised gel slice were resuspended in 5% formic acid in water and injected on an UltiMate 3000 RSLCnano System coupled to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific). Peptides were loaded on an Acclaim Pepmap trap column (Thermo Fisher Scientific #164750) with prior analysis on a PepMap RSLC C18 analytical column (Thermo Fisher Scientific #ES903) and eluted on a 120-min linear gradient from 3% to 35% Buffer B (Buffer A: 0.1% formic acid in water, Buffer B: 0.08% formic acid in 80:20 acetonitrile:water (v:v)). Eluted peptides were then analyzed by the mass spectrometer operating in data-dependent acquisition mode. Peptides were searched against a reduced database containing only the four proteins used in this assay (Ub, His–UBA1, UBE2A and UBR4) using MaxQuant (v2.1.3.1)70 (link). All parameters were left as default except for the addition of Deamidation (N, Q) and GlyGly (Protein N-term, K, C, S, T, Y) as variable modifications and with the PSM, Protein and Site FDR increase to 1.00. Tandem mass spectrometry spectra of interesting GlyGly peptides were manually inspected.
6
Proteomic Analysis of Candida albicans by LC-MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LC-MS/MS analyses were performed on an EASY nLC 1200 system coupled to a Q Exactive HF mass spectrometer equipped with an EASY-Spray ion source (all from Thermo Scientific, Mississauga, ON, Canada). A C18 Acclaim PepMap 100 pre-column (3 μm, 100 Å, 75 μm × 2 cm) connected to a PepMap RSLC C18 analytical column (2 μm, 100 Å, 75 μm × 50 cm) (all from Thermo Scientific) was used to separate peptide mixtures prior to injection into the mass spectrometer. Sixty-minute gradients were used to elute peptides from columns. The quality of LC-MS/MS analysis was repeatedly controlled for by running trypsin-digested BSA MS Standard (BioLabs) between sample runs. The acquired MS data were searched using MaxQuant version 1.6.0.16 [71 (link)] against the UniProt reviewed C. albicans proteome protein sequence database.
7
TMT-labeled Proteome Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were reduced and alkylated with TCEP and iodoacetamide and precipitated in 10% trichloroacetic acid, then digested overnight with Trypsin/ LysC (protein : protease ratio = 80:1). Resulting peptides were purified and labeled with TMT reagents. High pH reverse phase chromatography was performed using a Thermo Ultimate 3000 HPLC system (Thermo) with a 250 mm Zorbax extend C18 column (Agilent, ID 4.6 mm, particle size 5 μm) and one hour gradient from 97% ammonium bicarbonate, pH 8, 3% to 40% acetonitrile. 96 fractions were collected and combined into 24 fractions. These fractions were analyzed on an Orbitrap Fusion Lumos mass spectrometer with a 5 mm C18 PepMap 100 μ-precolumn (ID 300 μm), a 75 μm by 50 cm PepMap RSLC C18 analytical column (Thermo), and an Easy-Spray ion source (Thermo Scientific). Peptides were eluted. MS3 quant spectra were acquired in the Orbitrap, while MS2 fragment spectra were acquired in the linear trap.
8
TMT-labeled Proteome Profiling
9
Reversed-Phase LC-MS/MS Analysis of Cross-Linked Peptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reversed phase chromatographic separation of cross-linked peptide samples was performed using an EASY-nLC 1200 system coupled with a Q-Exactive High Field mass spectrometer (Thermo Scientific). The nano-flow LC system was configured with a 20 mm, 75 micron ID PepMap100 C18 trap column (Thermo Scientific), and a 15 cm, 50 micron ID PepMap RSLC C18 analytical column (Thermo Scientific) with an steel emitter. Mobile phase A consisted of 2% acetonitrile/0.1% formic acid and mobile phase B consisted of 80% acetonitrile/ 0.1% formic acid. Samples containing cross-linked peptide pairs were injected through the autosampler onto the trap column. Peptides were then separated using the following linear gradient steps at a flow rate of 300 nL/min: 2% B for 1 min, 2% B to 35% B over 100 min, 35% B to 40% B over 5 min, 40% B to 80% B over 1 min, held at 80% B for 4 min, 80% B to 2% B over 1 min and held at 2% B for 8 min. Separation was accomplished with a 40 min linear gradient from 2% to 40% solvent B at 300 nL/min. The mass spectrometer was operated with a PRM method with the following settings: a resolving power of 15,000 @ 200 m/z, ACG target of 2E5 ions, maximum ion time of 10 ms, isolation window of 1 m/z and a normalized collision energy of 27.
10
Proteomic Analysis of Protein Bands
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For liquid chromatography with tandem mass spectrometry, the gel was destained, and bands were cut out and processed as follows. Briefly, the protein bands were divided into 10 mm sections and subjected to in-gel digestion with trypsin. The tryptic digests were separated by online reversed-phase chromatography on a Thermo Scientific Eazy nano LC 1200 UHPLC system equipped with an autosampler using a reversed-phase peptide trap Acclaim PepMap™ 100 column (75 μm inner diameter, 2 cm length) and a reversed-phase PepMap™ RSLC C18 analytical column (75 μm inner diameter, 15 cm length, 3 μm particle size), both from Thermo Scientific, followed by electrospray ionization at a flow rate of 300 nl/min. The chromatography system was coupled in line with an Orbitrap Fusion Lumos mass spectrometer. Spectra were searched against the UniProt-human DB in Proteome Discoverer 2.1 software via the Sequest-based search algorithm, and comparative analysis of proteins identified in this study was performed in Scaffold 4 Q+S.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!