Samples were analyzed by using a nanoLC-MS/MS setup. nanoLC was configured with a 75 μm ID × 17 cm Reprosil-Pur C18-AQ (3 μm; Dr. Maisch GmbH, Germany) nano-column using an EASY-nLC nanoHPLC (Thermo Scientific, Odense, Denmark). The HPLC gradient was 2-35 % solvent B (A = 0.1 % formic acid; B = 95 % MeCN, 0.1 % formic acid) over 30 min and from 34 % to 100 % solvent B in 30 minutes at a flow-rate of 300 nL/min. LC was coupled with an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Full scan MS spectrum (m/z 290 − 1400) was performed in the Orbitrap with a resolution of 60,000 (at 400 m/z) with an AGC target of 1x10e6. The acquisition method contained both data-dependent and targeted scans. The targeted signals were the histone H3 and H4 peptides in isobaric forms, if any. MS/MS was performed with collision induced dissociation (CID) with normalized collision energy of 35, an AGC target of 10e4 and a maximum injection time of 100 ms. MS/MS data were collected in centroid mode. Precursor ion charge state screening was enabled and all unassigned charge states as well as singly charged species were rejected. All proteomics data has been deposited in the Chorus database (https://chorusproject.org/pages/dashboard.html#/projects/all/1013/experiments ).
Easy nlc nanohplc
Manufactured by Thermo Fisher Scientific
Sourced in Germany, Denmark
The EASY-nLC nanoHPLC is a high-performance liquid chromatography (HPLC) system designed for nanoscale separations. It is a versatile and robust instrument that can be used for a variety of applications, including proteomics, metabolomics, and lipidomics. The EASY-nLC nanoHPLC features a high-pressure liquid chromatography (HPLC) pump, an autosampler, and a column compartment, all of which are integrated into a compact, modular design. The system is capable of delivering precise and consistent flow rates in the nanoliter per minute range, making it well-suited for analyzing small sample volumes and achieving high separation efficiencies.
Automatically generated - may contain errors
Lab products found in correlation
Reprosil pur c18 aq, by Dr. Maisch (4 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion, by Thermo Fisher Scientific (1 mentions)
Orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Epiquik total histone extraction kit, by Epigentek (1 mentions)
Ltq velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Reprosil pur c18 aq 3 μm, by Dr. Maisch (1 mentions)
8 protocols using easy nlc nanohplc
1
Nanoflow LC-MS/MS Histone Peptides Analysis
2
Nano-LC-MS/MS DIA Proteomics Workflow
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Samples were resuspended in 0.1% trifluoroacetic acid (TFA) and injected onto a 75 µm ID × 25 cm Reprosil-Pur C18-AQ (3 µm; Dr. Maisch GmbH, Germany) nano-column packed in-house using an EASY-nLC nanoHPLC (Thermo Scientific, San Jose, CA, USA). The nanoLC pumped a flow-rate of 300 nL/min with a programmed gradient from 5% to 28% solvent B (A = 0.1% formic acid; B = 80% acetonitrile, 0.1% formic acid) over 45 minutes, followed by a gradient from 28% to 80% solvent B in 5 minutes and 10 min isocratic at 80% B. The instrument was coupled online with a Q-Exactive (Thermo Scientific, Bremen, Germany) or an Orbitrap Fusion (Thermo Scientific, San Jose, CA, USA) mass spectrometer acquiring data in a data-independent acquisition (DIA) mode as previously optimized25 (link),26 (link). Briefly, DIA consisted on a full scan MS (m/z 300−1100) followed by 16 MS/MS with windows of 50 m/z using HCD fragmentation and detected all in high resolution.
3
Quantitative Histone Proteomic Analysis
4
Quantitative Analysis of Histone PTMs
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Approximately 20 μg of extracted histones was resuspended in 30 μl of 100 mM ammonium bicarbonate, at pH 8.0. Chemical propionylation derivatization, digestion and desalting of histones followed by analysis by LC–MS and MS/MS were performed as described previously [32 (link), 33 (link)]. In brief, purified peptides were loaded onto 75-μm-ID fused-silica capillary columns packed with 12 cm of C18 reversed-phase resin (Reprosil-pur 120 C18, aq-3 μm particles, Fisher Scientific). Peptides were separated using EASY-nLC nano-HPLC (Thermo Scientific, Odense, Denmark) and introduced into a hybrid linear quadrupole ion trap–Orbitrap mass spectrometer (ThermoElectron) and resolved with a gradient from 0 to 35% solvent B (A = 0.1% formic acid; B = 95% MeCN, 0.1% formic acid) over 30 min and from 34 to 100% solvent B in 20 min at a flow-rate of 250 nL/min. The Orbitrap was operated in data-dependent mode essentially as previously described [33 (link)]. Relative abundances of peptide species were calculated by chromatographic peak integration of full MS scans using EpiProfile [34 (link)]. Where necessary, peptide and PTM identity were verified by manual inspection of MS/MS spectra.
5
Quantitative Analysis of Histone PTMs
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Histone samples were analyzed using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) essentially as described previously [27 (link)]. Briefly, nanoLC was configured with a 75 μm ID × 17 cm Reprosil-Pur C18-AQ (3 μm; Dr. Maisch GmbH, Germany) nano-column using an EASY-nLC nanoHPLC (Thermo Scientific, Odense, Denmark). Detection was performed by using an Orbitrap XL mass spectrometer (Thermo Scientific, Odense, Denmark). Peak area was extracted from raw files by using our in-house software EpiQuant. The relative abundance of a given PTM was calculated by dividing its intensity by the sum of all modified and unmodified peptides sharing the same sequence. All raw files are available at the Chorus database (https://chorusproject.org ).
6
Quantitative Histone Profiling of Malaria Parasites
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3D7 parasites
were tightly synchronized to a 4 h window as above and seeded at 4%
parasitemia and 2% hematocrit. Drug was added at 29 hpi. After 6 h,
infected RBCs were pelleted and immediately frozen in liquid nitrogen.
After thawing, parasites were isolated from RBCs using 0.05% saponin
in cold PBS and subsequently washed 3× in cold PBS. Histones
were extracted using the EpiQuik Total Histone Extraction kit (Epigentek,
Farmingdale, NY) followed by TCA precipitation. A portion of the histone
extracts were run on a 15% SDS-PAGE gel and visualized with Quick
Coomassie Stain (Protein Ark, Sheffield, United Kingdom) to determine
purity and concentration. Histones were prepared for mass spectrometry
by chemical derivatization using propionic anhydride and digested
to peptides with trypsin, followed by another round of derivatization.
Peptides were desalted using C18 stage tips, and about 1–2
μg of peptides were analyzed using an EASY-nLC nanoHPLC (Thermo
Scientific, Odense, Denmark) coupled with a Q-Exactive mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany). HPLC gradients and mass
spectrometry parameters were defined previously.80 (link) To facilitate MS/MS-based quantification, both data-dependent
acquisition and targeted acquisition for isobaric peptides were included.
The relative abundances of histone H3 and H4 peptides were calculated
using EpiProfile.81 (link)
were tightly synchronized to a 4 h window as above and seeded at 4%
parasitemia and 2% hematocrit. Drug was added at 29 hpi. After 6 h,
infected RBCs were pelleted and immediately frozen in liquid nitrogen.
After thawing, parasites were isolated from RBCs using 0.05% saponin
in cold PBS and subsequently washed 3× in cold PBS. Histones
were extracted using the EpiQuik Total Histone Extraction kit (Epigentek,
Farmingdale, NY) followed by TCA precipitation. A portion of the histone
extracts were run on a 15% SDS-PAGE gel and visualized with Quick
Coomassie Stain (Protein Ark, Sheffield, United Kingdom) to determine
purity and concentration. Histones were prepared for mass spectrometry
by chemical derivatization using propionic anhydride and digested
to peptides with trypsin, followed by another round of derivatization.
Peptides were desalted using C18 stage tips, and about 1–2
μg of peptides were analyzed using an EASY-nLC nanoHPLC (Thermo
Scientific, Odense, Denmark) coupled with a Q-Exactive mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany). HPLC gradients and mass
spectrometry parameters were defined previously.80 (link) To facilitate MS/MS-based quantification, both data-dependent
acquisition and targeted acquisition for isobaric peptides were included.
The relative abundances of histone H3 and H4 peptides were calculated
using EpiProfile.81 (link)
7
Nano-LC-MS/MS Proteomic Workflow
8
Nano-LC-MS/MS Proteomic Analysis Protocol
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