Samples collected from ACPA and total serum IgG isolation procedures were reduced, alkylated, and digested in solution with trypsin and Trypsin/Lys-C Mix at 37 °C as previously described [48 (link)]. A nano LC-MS(MS) system was used for peptide separation and glycopeptide analysis. The chromatographic separation was carried out on an Ultimate 3000 nanoRSLC system (Dionex, Sunnyvale, CA, USA). Samples were desalted using an Acclaim PepMap100 C-18 trap column (Thermo Scientific, Sunnyvale, CA, USA) and peptide separation was achieved with an Acquity UPLC M-Class Peptide BEH C18 column (Waters, Milford, MA, USA). A Maxis II ETD Q-TOF (Bruker Daltonics, Bremen, Germany) equipped with a CaptiveSpray nanoBooster ion source was used for the mass spectrometry measurements. Glycopeptides were identified by scanning the MS spectra over the mass range of m/z 150−3000 at 2 Hz. CID analysis was performed on triply charged precursor ions at 0.5 Hz for low-abundance ones (>2500 cts/s) and 4 Hz for abundant precursors (>25,000 cts/s). Glycopeptide quantification was based on MS experiments performed over the mass range of m/z 300−3000 at 1 Hz.
Ultimate 3000 nano rslc system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Ultimate 3000 nano RSLC system is a high-performance liquid chromatography (HPLC) system designed for nano-scale separations. It features a low-volume, high-pressure pump capable of delivering precise flow rates in the nano- to micro-liter per minute range. The system is designed to handle small sample volumes and is suitable for a variety of applications, including proteomics, metabolomics, and other sensitive analyses.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (6 mentions)
Nanospray flex ion source, by Thermo Fisher Scientific (4 mentions)
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Acclaim pepmap 100 c18 trap column, by Thermo Fisher Scientific (2 mentions)
Maxis 2 etd q tof, by Bruker (2 mentions)
Acquity uplc m class peptide beh c18 column, by Waters Corporation (2 mentions)
Thermo qexactive plus mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Jupiter c18 particles, by Phenomenex (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Amazon speed etd, by Bruker (1 mentions)
Captivespray, by Bruker (1 mentions)
Xcalibur software, by Thermo Fisher Scientific (1 mentions)
Pierce gst protein interaction pull down kit, by Thermo Fisher Scientific (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Amazon etd, by Bruker (1 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (1 mentions)
Complete mini protease inhibitor cocktail, by Merck Group (1 mentions)
20 protocols using ultimate 3000 nano rslc system
1
Glycopeptide Analysis of Serum IgG
2
Peptide Separation and Analysis by Nano-HPLC-MS/MS
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Peptides were separated using an Ultimate 3000 nano RSLC system (Dionex) equipped with a trapping cartridge (Precolumn C18 PepMap100, 5 mm, 300 μm i.d., 5 μm, 100 Å) and an analytical column (Acclaim PepMap 100. 75 × 50 cm C18, 3 mm, 100 Å) connected to a nanospray-Flex ion source. The peptides were loaded onto the trap column at 30 μl per min using solvent A (0.1% formic acid) and eluted using a gradient from 2 to 80% Solvent B (0.1% formic acid in acetonitrile) over 90 min at 0.3 μl/min (all solvents were of LC–MS grade). The Orbitrap Fusion Lumos was operated in positive ion mode with a spray voltage of 2.4 kV and capillary temperature of 275°C. Full scan MS spectra with a mass range of 375–1500 m/z were acquired in profile mode using a resolution of 120 000 with a maximum injection time of 50 ms, AGC operated in standard mode and a RF lens setting of 30%. Fragmentation was triggered for 3 s cycle time for peptide like features with charge states of 2–7 on the MS scan (data-dependent acquisition). Precursors were isolated using the quadrupole with a window of 0.7 m/z and fragmented with a normalized collision energy of 34%. Fragment mass spectra were acquired in profile mode and a resolution of 30 000 in profile mode. Maximum injection time was set to 94 ms or an AGC target of 200%. The dynamic exclusion was set to 60 s.
3
Proteome Identification via Pull-Down
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Proteins eluted from the RNA pull-down assay were separated by 15 % SDS-PAGE gel. When the proteins went into the separating gel, the SDS-PAGE was stopped and the gel was then stained with the staining buffer (0.1 % Coomassie Blue R250 in 40 % ethanol supplemented with 10 % acetic acid). After destaining, the gels were washed and protein bands were excised followed by completely destaining. In-gel digestion was performed using DTT buffer and alkylated with the iodoacetamide. Afterwards, the protein digestion was done by incubating with trypsin at 37 °C overnight followed by extraction using 100 % Acetonitrile (CAN)/3 % Trifluoroacetic acid (TFA) and 40 % ACN/3 % TFA. Peptides were desalted using the C18 StageTips and analyzed using the Dionex Ultimate 3000 nanoRSLC system.
4
Optimized LC-MS Peptide Separation
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For the liquid chromatography-mass spectrometry measurements a Maxis II ETD Q-TOF (Bruker Daltonics, Bremen, Germany) equipped with a CaptiveSpray nanoBooster ionsource coupled to an Ultimate 3000 nanoRSLC system (Dionex, Sunnyvale, CA, USA) was used. Samples were dissolved in 2% AcN, 0,1% FA and injected onto an Acclaim PepMap100 C-18 trap column (100 µm x 20 mm, Thermo Scientific, Sunnyvale, CA, USA) for sample desalting. Peptides were separated on an ACQUITY UPLC M-Class Peptide BEH C18 column (130 Å, 1,7 µm, 75 µm x 250 mm, Waters, Milford, MA, USA) at 48 °C applying a flow rate of 300 nl/min using gradient elution (4% B from 0 to 11 min, followed by a 60 min or 90 min gradient to 50% B).
5
Peptide Profiling by Nano-HPLC-MS/MS
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Peptide fractions were analyzed by nanoflow HPLC-MS/MS using an Ultimate 3000 nano RSLC system (Thermo Fisher Scientific) coupled with a Q-Exactive HF mass spectrometer fitted with a nano spray Flex ion source (Thermo Fisher Scientific) and controlled using Xcalibur software (version 4.3). Approximately 1 µg of each fraction was injected and separated using a 90-minute segmented gradient by preconcentration onto a 20 mm x 75 µm PepMap 100 C18 trapping column then separation on a 250 mm x 75 µm PepMap 100 C18 analytical column at a flow rate of 300 nL/min and held at 45 °C. MS Tune software (version 2.9) parameters used for data acquisition were: 2.0 kV spray voltage, S-lens RF level of 60 and heated capillary set to 250 °C. MS1 spectra (390 -1500 m/z) were acquired at a scan resolution of 120,000 followed by MS2 scans using a Top15 DDA method, with 30-second dynamic exclusion of fragmented peptides. MS2 spectra were acquired at a resolution of 15,000 using an AGC target of 2e5, maximum IT of 28ms and normalized collision energy of 30.
6
Nano-RSLC-MS/MS Proteomic Analysis
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Peptide samples were dissolved in 12 µL loading buffer (0.05% (w/w) trifluoroacetic acid in water/acetonitrile (98:2)). Approximately 1 μg of each sample was analyzed using an Ultimate 3000 nano RSLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Q-Exactive HF mass spectrometer fitted with nanospray Flex ion source (Thermo Fisher Scientific, Waltham, MA, USA) and controlled using Xcalibur version 4.3. Peptides were preconcentrated onto a 2 cm PepMap 100 C18 trap column at a flow rate of 5 µL/min and then separated at 300 nL/min on a 25 cm PepMap 100 C18 analytical column held at 45 °C using a 120-min segmented gradient. Mobile phase A comprised 0.1% (w/w) formic acid in water and mobile phase B comprised 0.08% (w/w) formic acid in acetonitrile/water (80:20). Data acquisition parameters were: 2.0 kV spray voltage, S-lens RF level of 60 and heated capillary set to 250 °C. MS1 spectra (390–1240 m/z) were acquired at a scan resolution of 120,000 in profile mode with an AGC target of 3e6 and followed by sequential MS2 scans across 26 DIA × 25 amu windows over the range of 397.5–1027.5 m/z, with 1 amu overlap between sequential windows. MS2 spectra were acquired in centroid mode at a resolution of 30,000 using an AGC target of 1 × 106, maximum IT of 55 ms and normalized collision energy of 27.
7
Identifying circRTN4-Interacting Proteins
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CircRTN4-interacting proteins in PANC-1 cells were identified as described previously [9 ]. Briefly, in vitro transcribed circRTN4 and two negative controls (RTN4 and circGreen Fluorescent Protein (circGFP)) were used incubated with PANC-1 cell lysate at 4 °C overnight with rotation. The pulled-down proteins were identified by Dionex Ultimate3000 nanoRSLC system coupled to Thermo Fisher Orbitrap Fusion Tribid Lumos.
8
Orbitrap Fusion Lumos Mass Spectrometry
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Samples were measured using an Ultimate 3000 nano RSLC system coupled to an Orbitrap Fusion Lumos mass spectrometer (both Thermo Scientific). Peptides were preconcentrated on a 100 μm × 2 cm C18 trapping column for 10 min using 0.1% TFA (v/v) at a flow rate of 20 μl/min. Next, the separation of the peptides was performed on a 75 μm × 50 cm C18 main column (both Pepmap, Thermo Scientific 164567) with a 120 min LC gradient ranging from 3 to 35% of 84% ACN (Biosolve 12041), 0.1% FA (v/v) (Biosolve 69141) at a flow rate of 230 nl/min. MS1 spectra were acquired in the Orbitrap from 300 to 1,500 m/z at a resolution of 120,000 using the polysiloxane ion at m/z 445.12003 as lock mass(Olsen et al, 2005 ), with maximum injection times of 50 ms and ACG target was set at 2.0 × 105 ions. Top fifteen most intense signals were selected for fragmentation by HCD with a collision energy of 30%. MS2 spectra were acquired in the ion trap at a resolution of 120,000, with maximum injection times of 300 ms, a dynamic exclusion of 15 s. The ACG target was set at 2.0 × 103 for MS2.
9
Quantitative Secretagogin Interactome
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Filter-aided sample preparation (FASP), LC-MS/MS in-line analysis on an Ultimate 3000 nanoRSLC system (ThermoFisher) coupled to a Thermo Q-Exactive Plus mass spectrometer (ThermoFisher), database search via the open-source software MaxQuant 1.5.3.30 [32] and statistical processing using the Perseus statistical package (version 1.5.4.1) of His6-secretagogin (n = 3) and control pull-downs (n = 3) were carried out as described previously [23] (link) with minor modifications. Proteins were identified against the UniProt mouse (Mus musculus) reference proteome database (as of October 2017; 60,177 entries). Secretagogin pull-downs were compared to control samples via two-tailed Student's t-test applying permutation-based FDR equal to 0.01. Significant hits identified by at least two peptides and matching the criteria of the minimum fold-change (s0 = 2) in the target pull-down were considered as specific for secretagogin.
10
Nano-LC-MS/MS Peptide Sequencing Protocol
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