Details regarding the in solution protein digestion are provided in Supplemental Methods- Protein Digestion . Nanoflow liquid chromatography (LC) was conducted using the Thermo Scientific Easy-nLC 1000 Liquid Chromatography system. Details regarding all LC methods can be found in Supplemental Method- Nanoflow LC (Supplemental Table 2 ). Methods for global tandem mass spectrometry (MS) data collection using a dependent acquisition mode (DDA), and targeted MS data collected using a selected reaction monitoring (SRM) are thoroughly described in Supplemental Method- Mass Spectrometry .
Easy nlc 1000 liquid chromatography system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EASY-nLC 1000 is a liquid chromatography system designed for high-performance nano-flow liquid chromatography. It features a compact design, precise flow control, and compatibility with a range of chromatographic columns for sensitive sample separation and analysis.
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Lab products found in correlation
Q exactive plus hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Nanoflex source, by Thermo Fisher Scientific (3 mentions)
Picofrit c18 2 μm medium analytical columns, by New Objective (3 mentions)
Orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Q exactive hf orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easy spray c18 column, by Thermo Fisher Scientific (1 mentions)
Easy spray ion source, by Thermo Fisher Scientific (1 mentions)
Easy spray column, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Sep pak c18 cartridge column, by Waters Corporation (1 mentions)
Sequencing grade endoproteinase lys c, by Promega (1 mentions)
Easy spray reverse phase column, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Rslc c18, by Thermo Fisher Scientific (1 mentions)
Picoview ion source, by New Objective (1 mentions)
Picofrit, by New Objective (1 mentions)
19 protocols using easy nlc 1000 liquid chromatography system
1
Comprehensive Proteomics Workflow
2
Auto-ADP-ribosylation of SCO5461ΔN34 Protein
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Auto-ADP-ribosylation of SCO5461ΔN34 protein (12.5 μm ) was performed in the assay buffer (50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 2 mm MgCl2) with NAD+ (50 μm ) at room temperature for 30 min. Proteins were digested with trypsin according to the FASP protocol (75 (link)) with a 10-kDa cut-off Vivacon 500 flat ultrafiltration filters (Sartorius Stedim). The resulting peptides were analyzed by UHPLC-MS/MS on an EASY-nLC 1000 liquid chromatography system (Thermo Scientific) coupled online to a Q Exactive HF orbitrap mass spectrometer (Thermo Scientific) essentially as described before (2 (link)). The data were processed with MaxQuant (76 (link)) using parameters optimized for the detection of ADP-ribosylated peptides (2 (link)).
3
Tryptic Peptide Analysis by LC-MS/MS
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The TMT-labelled tryptic peptides were dissolved in 20 μL of 2% AcN/0.1% formic acid. Five μL samples were injected into a EASY-nLC™1000 liquid chromatography system (ThermoFisher Scientific) on-line coupled to a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer (ThermoFisher Scientific). The chromatographic separation of the peptides was achieved using a 50 cm long EASY-Spray™ C18 column (ThermoFisher Scientific), with an organic gradient: 4–26% B (solvent B = 98% AcN/0.1% formic acid) in 180 min, 26–95% B in 5 min, and 95% B for 8 min at a flow rate of 300 nL/min. The mass spectrometric (MS) acquisition method was comprised of one survey full spectrum ranging from m/z 350 to 1600, acquired with a resolution of R = 140,000 (at m/z 200), followed by data-dependent higher energy collision dissociation (HCD) fragmentations of maximum 16 most intense precursor ions with a charge state 2+ and 3+, applying 60 s dynamic exclusion. The tandem mass scans were acquired with a resolution of R = 70,000, targeting 2x105 ions, setting isolation width to 2.0 Th and normalized collision energy to 33%.
4
Quantitative Proteomics by LC-MS/MS
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Peptide samples were analyzed by LC-MS/MS on a Q Exactive mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nLC 1000 liquid chromatography system (Thermo Scientific) via an EASY-Spray ion source (Thermo Fisher Scientific). Peptides were fractionated on a 75 μm × 500 mm EASY-Spray column (Thermo Scientific) over various gradient lengths from 90 min to 240 min. The following describes the typical analytical set-up, but further specific details of MS run conditions can be found within the raw data files. Precursor ion full scan spectra were acquired over (m/z 300 to 1,800) with a resolution of 70,000 at m/z 200 (target value of 1,000,000 ions, maximum injection time 20 ms). Up to ten data dependent MS2 spectra were acquired with a resolution of 17,500 at m/z 200 (target value of 500,000 ions, maximum injection time 60 ms). Ions with unassigned charge state, and singly or highly (>8) charged ions were rejected. Intensity threshold was set to 2.1 × 104 units. Peptide match was set to preferred, and dynamic exclusion option was enabled (exclusion duration 40 s). The mass spectrometry proteomics raw data files have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/ ) with the dataset identifier PXD003530 for the label-free site ID analysis, and PXD004995 for the occupancy and SILAC half-life analysis.
5
Dermal Fibroblast-Derived ECM Proteomic Analysis
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Dermal fibroblast-derived ECMs were generated using MMC and decellularized using PLA2. To solubilize the acellular ECM, 8 M Urea/50 mM Tris-HCl pH 8.0 was added before scraping the matrix off the surface and transferring it to a microtube. The matrix mixture was reduced with 10 mM DTT (Sigma), alkylated with 55 mM Iodoacetamide (Sigma) and then diluted with 100 mM TEAB buffer to reach a Urea concentration of < 1 M. The matrix proteins were digested with sequencing grade endoproteinase Lys-C (Promega, WI, USA) and sequencing grade-modified trypsin (Promega) at a ratio of 1:100 at 25 °C for 4 h and 18 h respectively, samples were subsequently acidified with 1% TFA (trifluoroacetic acid) and desalted. Following desalting with a Sep-Pak C18 column cartridge (Waters, Milford MA), the samples were analysed using an Easy nLC 1000 liquid chromatography system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Mass Spectrometer (Thermo Fisher Scientific). Each sample was analyzed in a 60 min gradient using an Easy Spray Reverse Phase Column (50 cm × 75 µm internal diameter, C-18, 2 µm particles, Thermo Fisher Scientific). Data were acquired in −3 s cycle with the following parameters: MS in Orbitrap and MS/MS in ion trap with ion targets and resolutions (OT-MS 4 × E5 ions, resolution 120 K, IT-MS/MS 1000 ions/turbo scan, “Universal Method”).
6
Peptide Analysis by LC-MS/MS
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Peptides extracted after in-gel digestion were analyzed by LC-MS/MS on a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, San Jose, CA, USA) with pre–separation on an EASY-Spray column, 50 cm × 75 µm ID, PepMap RSLC C18, 2 µm ES803 mounted on an EASY-nLC 1000 Liquid Chromatography system (Thermo Scientific, San Jose, CA, USA). The separation was run as a 70 min gradient of solvents A (0.1% formic acid in dH2O) and B (80% acetonitrile, 0.1% formic acid) at a constant flow of 250 nL/min, starting at 6% B, increasing to 23% over 43 min, followed by increasing to 38% over 12 min and completed by reaching 60% in 5 min. The column was finally washed with 95% solvent B for 10 min.
MS spectra were obtained in data-dependent acquisition mode with the following top 10 HCD method: Scan range was 300–1750 m/z and full scan resolution was 70,000 m/z with an AGC target of 3 ∙ 106 and a maximum injection time of 20 ms. Normalized collision energy was set to 35 with a dynamic exclusion time at 30 s. Ions in charge state 1 and unassigned, were excluded. For the MS/MS resolution was set to 17,500 m/z with AGC target of 1 ∙ 106 and a maximum injection time of 60 ms.
MS spectra were obtained in data-dependent acquisition mode with the following top 10 HCD method: Scan range was 300–1750 m/z and full scan resolution was 70,000 m/z with an AGC target of 3 ∙ 106 and a maximum injection time of 20 ms. Normalized collision energy was set to 35 with a dynamic exclusion time at 30 s. Ions in charge state 1 and unassigned, were excluded. For the MS/MS resolution was set to 17,500 m/z with AGC target of 1 ∙ 106 and a maximum injection time of 60 ms.
7
Peptide Separation and Identification by UHPLC-MS
8
Optimized LC-MS/MS Peptide Analysis
9
Peptide Separation and Analysis by NanoLC-MS/MS
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NanoLC-MS/MS analysis was performed as described previously (Hör et al. 2020a (link)) at Rudolf-Virchow-Center Würzburg for Integrative and Translational Bioimaging. The peptides were loaded on capillary columns (PicoFrit, 30 cm × 150 µm ID, New Objective) filled with ReproSil-Pur 120 C18-AQ, 1.9 µm (Dr. Maisch), which is connected with an Orbitrap Fusion (Thermo Scientific) equipped with a PicoView Ion Source (New Objective) and an EASY-nLC 1000 liquid chromatography system (Thermo Scientific). Peptides were then separated with a 140 min linear gradient from 3% to 40% acetonitrile and 0.1% formic acid at a flow rate of 500 nL/min. Both MS and MS/MS scans were acquired in the Orbitrap analyzer with a resolution of 60,000 for MS scans and 15,000 for MS/MS scans. HCD fragmentation with 35% normalized collision energy was applied. A Top Speed data-dependent MS/MS method with a fixed cycle time of 3 sec was used. Dynamic exclusion was applied with a repeat count of 1 and an exclusion duration of 60 sec; singly charged precursors were excluded from selection. The minimum signal threshold for precursor selection was set to 50,000. Predictive AGC was used with a target value of 2 × 105 for MS scans and 5 × 104 for MS/MS scans. EASY-IC was used for internal calibration.
10
Quantitative Proteomics of HCT-116 Cells
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HCT-116
cell lysis was proteolyzed and labeled with TMT 10-plex (Thermo Fisher
Scientific) following the manufacturer’s protocol. For the
proteome analysis, HPLC-MS/MS analysis was performed using an Orbitrap
Fusion Lumos mass spectrometer equipped with an EASY-nLC 1000 liquid
chromatography system and a nanoelectrospray ionization source (Thermo
Scientific, U.S.A.). The specific method of the experiment was the
same as that reported in the literature.35 (link) Compared with the DMSO group, proteins quantified with a p value ≤ 0.05 and a fold change of >1.50 or <0.66
were considered to be significantly expressed and used for further
proteomic analysis. Differentially expressed protein interaction data
was identified by the STRING online tool (version 9.0).
cell lysis was proteolyzed and labeled with TMT 10-plex (Thermo Fisher
Scientific) following the manufacturer’s protocol. For the
proteome analysis, HPLC-MS/MS analysis was performed using an Orbitrap
Fusion Lumos mass spectrometer equipped with an EASY-nLC 1000 liquid
chromatography system and a nanoelectrospray ionization source (Thermo
Scientific, U.S.A.). The specific method of the experiment was the
same as that reported in the literature.35 (link) Compared with the DMSO group, proteins quantified with a p value ≤ 0.05 and a fold change of >1.50 or <0.66
were considered to be significantly expressed and used for further
proteomic analysis. Differentially expressed protein interaction data
was identified by the STRING online tool (version 9.0).
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