Histone peptides were resuspended with HPLC buffer A. Peptides were loaded on to 100 μm i.d. × 2-cm Reprosil-Pur C18-AQ (5 μm; Thermo Scientific, CA) trap column and separated by 75 μm i.d. × 25-cm Reprosil-Pur C18-AQ (2 μm; Thermo Scientific, CA) analytical column. The HPLC gradient was as follows: from 5 to 35% buffer B in 50 min, from 35 to 95% buffer B for 5 min, and 95% buffer B for 5 min at a flow rate of 250 nL/min. NanoLC was coupled to a Q Exactive HF mass spectrometer (Thermo Scientific). For data-independent acquisition (DIA), two full-scan MS spectra (m/z 300–1100) at a resolution of 60,000 were acquired in the orbitrap within a DIA duty cycle, and 16 MS/MS were performed at a resolution of 60,000 with an isolation window of 50 Da. Normalized collision energy (NCE) was set to 27.
Reprosil pur c18 aq
Manufactured by Thermo Fisher Scientific
Sourced in Japan
ReproSil-Pur C18-AQ is a reversed-phase HPLC column material designed for the separation of polar and hydrophilic compounds. It features a C18-modified silica stationary phase and is suitable for aqueous mobile phases.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
Nanospray 3 source, by AB Sciex (1 mentions)
Easy n lcii hplc system, by Thermo Fisher Scientific (1 mentions)
Reprosil pur c18 aq column, by Thermo Fisher Scientific (1 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Orbitrap fusion lumos, by Thermo Fisher Scientific (1 mentions)
Picofrit column, by New Objective (1 mentions)
Easy nano lc 2 system, by Thermo Fisher Scientific (1 mentions)
Nano bore stainless steel online emitter, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Q exactive hf, by Thermo Fisher Scientific (1 mentions)
Rslcnano system, by Thermo Fisher Scientific (1 mentions)
6 protocols using reprosil pur c18 aq
1
Histone Peptide Analysis by Nano-LC-MS/MS
2
Proteomic Analysis of Gel-digested Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gel band (from IP result) was eventually digested by the in-gel trypsin digestion process, following previous procedures36 (link). Peptide samples were analyzed on an LTQ-Orbitrap Velos (Thermo Fisher Scientific) connected to an Easy-nano LC II system (Thermo Fisher Scientific) incorporated with an autosampler. The dried peptide samples were resuspended in 70 μL of 0.1% formic acid, and an aliquot (7 μL) was injected into a reversed-phase peptide trap EASY-Column (L 2 cm, ID 100 μm, 5 μm, 120 Å, ReproSil-Pur C18-AQ, Thermo Fisher Scientific) and a reversed-phase analytical EASY-Column (L 10 cm, ID 75 μm, 3 μm, 120 Å, ReproSil-Pur C18-AQ, Thermo Fisher Scientific), and electrospray ionization was subsequently performed using a 30 μm (i.d.) nano-bore stainless steel online emitter (Thermo Fisher Scientific). The total duration of LC gradient analysis was 60 min. The peptides were eluted in a linear gradient of 10–40% buffer B over a period of 40 min, with buffer A (0.1% formic acid in H2O) and buffer B (0.1% formic acid in acetonitrile), and a flow rate of 0.3 μL/min. The temperature and voltage applied to the capillary was 275 °C and 1.9 V, respectively. All data were acquired with the mass spectrometer operating in automatic data-dependent switching mode. The MS survey was scanned from 350 to 2000 m/z with the resolution set to 100,000.
3
Peptide Separation and Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The peptides were separated on an Easy-nLC II HPLC system (Thermo Scientific) equipped with a trap column (ReproSil-Pur C18-AQ (5 μm, 2 cm x 100 μm I.D., Thermo Scientific) and an analytical column (ReproSil-Pur C18-AQ column, 3 μm, 10 cm x 75 μm I.D., Thermo Scientific) in-line to a NanoSpray III source (AB Sciex) connected to a TripleTOF 5600 mass spectrometer (AB Sciex) operated under Analyst TF 1.5.1 control. Peptides were eluted at a constant flow of 250 nl/min with a 50 min gradient from 5 to 35 % solvent B (90 % ACN, 0.1 % formic acid) followed by re-equilibration for 10 min back to the starting conditions. Information dependent acquisition was employed acquiring up to 25 MS/MS spectra per cycle using 1.6 s cycle time with an exclusion window of 6 s.
4
LC-MS/MS Peptide Fractionation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The peptide fractions were resuspended in 20 uL 2% acetonitrile in 0.1% formic acid; approximately 0.5 ug (2 uL) was loaded onto a C18 trap (S-10 uM, 120 Å, 75 um × 2 cm; YMC Co., LTD., Kyoto, Japan) and then separated on an in-house packed PicoFrit column (75 um × 200 mm, 15 um, ±1 um tip, New Objective) with C18 phase (ReproSil-Pur C18-AQ, 3 um, 120 Å, www.dr-maisch.com ) using 2%–90% acetonitrile gradient at 300 nL/min over 120 min on a EasyLC nanoLC 1000 (Thermo Scientific). Eluting peptides were sprayed at 2.0 kV directly into an Orbitrap Fusion Lumos (Thermo Scientific) mass spectrometer. Survey scans (full ms) were acquired from 360–1,700 m/z with a cycle time of 3 s. Precursor ions isolated in a 0.7 Da window and fragmented using HCD activation collision energy 39 and 15 s dynamic exclusion, with a scan range of 116 m/z–2,000 m/z. Precursor and fragment ions were analyzed at resolutions 120,000 and 30,000, respectively, with automatic gain control (AGC) target values at 4 × 105 with 50 ms maximum injection time (IT) and 1 × 105 with 118 ms maximum IT, respectively.
5
Shotgun Proteomics using LC-MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For LC-MS/MS purposes, desalted peptides were injected in an Ultimate 3000 RSLCnano system (Thermo), separated in a 15-cm analytical column (75 mm ID home-packed with ReproSil-Pur C18-AQ 2.4 mm from Dr. Maisch) with a 50-min gradient from 5 to 60% acetonitrile in 0.1% formic acid. The effluent from the HPLC was directly electrosprayed into a Qexactive HF (Thermo) operated in data dependent mode to automatically switch between full scan MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 375-1600) were acquired with resolution R = 60,000 at m/z 400 (AGC target of 3x10 6 ). The 10 most intense peptide ions with charge states between 2 and 5 were sequentially isolated to a target value of 1x10 5 , and fragmented at 27% normalized collision energy. Typical mass spectrometric conditions were: spray voltage, 1.5 kV; no sheath and auxiliary gas flow; heated capillary temperature, 250 C; ion selection threshold, 33.000 counts. MaxQuant 1.5.2.8 was used to identify proteins and quantify by iBAQ with the following parameters: Database, UP000002311_559292_Scerevisiae_20171017; MS tol, 10ppm; MS/MS tol, 0.5 Da; Peptide FDR, 0.1; Protein FDR, 0.01 Min. peptide Length, 5; Variable modifications, Oxidation (M); Fixed modifications, Carbamidomethyl (C); Peptides for protein quantitation, razor and unique; Min. peptides, 1; Min. ratio count, 2.
6
Histone Peptides Analysis by Nano-LC-MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Desalted histone peptides in 0.1% TFA were injected in an RSLCnano System (Thermo Fisher Scientific) and separated in a 15 cm analytical column (75 μm ID home packed with ReproSil‐Pur C18‐AQ 2.4 μm from Dr. Maisch) with a 50 min gradient from 4 to 40% ACN in 0.1% formic acid at 300 nl/min flowrate. The effluent from the HPLC was electrosprayed into Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The MS instrument was programmed to target several ions except for the MS3 fragmentation (22). Survey full‐scan MS spectra (from m/z 270 to 730) were acquired with resolution R = 60,000 at m/z 400 (AGC target of 3 × 106). Targeted ions were isolated with an isolation window of 0.7 m/z to a target value of 2 × 105 and fragmented at 27% normalized collision energy. Typical mass spectrometric conditions were as follows: spray voltage, 1.5 kV; no sheath and auxiliary gas flow; and heated capillary temperature, 250°C.
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!