SLE lymphadenopathy is lymph node enlargement associated with SLE. Reactive follicular hyperplasia is the most frequent finding [22 (link)]. Lymph nodes from patients diagnosed with SLE (n = 3) and controls (n = 3) were used for LC-MS analysis. The controls were lymph nodes dissected during thyroidectomy for papillary carcinoma with no metastasis found. Proteins from FFPE tissue were extracted using the Liquid Tissue MS Protein Prep Kit (Expression Pathology Inc., Rockville, MD, USA). The extracts were diluted in 0.1% formic acid and 1 μg aliquots for each sample were separated by nanoflow reversed-phase LC (NanoLC-Ultra 2D-Plus; Eksigent, Dublin, CA, USA) equipped with cHiPLC Nanoflex (Eksigent). Eluted peptides were analyzed by a quadrupole time-of-flight hybrid mass spectrometer (Triple TOF5600+ system; AB SCIEX, Framingham, MA, USA).
Chiplc nanoflex
Manufactured by AB Sciex
Sourced in United States
The CHiPLC Nanoflex is a liquid chromatography system designed for ultra-low flow rate separations. It features a nanoflow liquid chromatography (nano-LC) pump capable of delivering flow rates from 20 nL/min to 2 μL/min.
Automatically generated - may contain errors
Lab products found in correlation
Triple tof 5600 system, by AB Sciex (8 mentions)
Nano lc ultra 2d plus, by AB Sciex (6 mentions)
Nanolc ultra, by AB Sciex (3 mentions)
Tripletof 5600 system with nanospray 3 source and heated interface, by AB Sciex (2 mentions)
Proteinpilot 5, by AB Sciex (2 mentions)
Ziptip c18 column, by Merck Group (2 mentions)
Sequencing grade modified porcine trypsin, by Promega (2 mentions)
Proteinpilot descriptive statistics template pdst, by AB Sciex (2 mentions)
Nanolc system, by AB Sciex (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Chromxp c18 cl 3 μm 120, by AB Sciex (1 mentions)
Nanospray 3 source, by AB Sciex (1 mentions)
In gel tryptic digestion kit, by Thermo Fisher Scientific (1 mentions)
Pierce silver stain for mass spectrometry kit, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Fujifilm (1 mentions)
Trypsin, by Promega (1 mentions)
Nano chiplc column, by AB Sciex (1 mentions)
Chromxp c18 cl, by AB Sciex (1 mentions)
Pierce c18 spin column, by Thermo Fisher Scientific (1 mentions)
13 protocols using chiplc nanoflex
1
Proteomic Analysis of SLE Lymphadenopathy
2
Nano-scale Peptide Separation and Tandem Mass Spectrometry
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The 1st dimension of peptide separation was conducted using an Eksigent nanoLC Ultra and ChiPLC-nanoflex (USA) in TrapElute configuration. Subsequently, the samples were loaded on a 200 μm × 0.5 mm column and eluted on an analytical 75 μm × 15 cm column (ChromXP C18-CL, 3 μm). A gradient formed by mobile phase A (2% acetonitrile, 0.1% formic acid) and mobile phase B (98% acetonitrile, 0.1% formic acid) was used to separate 2 and 5 μl of the sample at a 0.3 μl/min flow rate. The following gradient elution was used for peptide separation: 0 to 5% of mobile phase B in 1 min, 5 to 12% of mobile phase B in 15 min, 12 to 30% of mobile phase B in 114 min, 30 to 90% of mobile phase B in 2 min, 90% for 7 min, 90 to 5% in 3 min and finally held at 5% of mobile phase B for 13 min. The tandem MS analysis was performed using a 5600 TripleTOF system (AB SCIEX, USA) under Information Dependent Acquisition (IDA) mode. The mass range of 400–1800 m/z and accumulation times of 250 millisec per spectrum were chosen for precursor ion selection. MS/MS analysis was performed on the 20 most abundant precursors (accumulation time: 100 millisec) per cycle with 15 s dynamic exclusion. Recording of MS/MS was acquired under high sensitivity mode with rolling collision energy and adjusted capillary electrophoresis (CE) when iTRAQ reagent use was selected.
3
Nanoflow LC-MS Peptide Separation
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The samples were analyzed using a nanoLC system (Eksigent, Dublin, CA) equipped with a LCchip system (cHiPLC nanoflex, Eksigent, CA, USA) coupled online to a Q Exactive Mass Spectrometer (Thermo Scientific, Bremen, Germany). From each sample, 0.2 µg peptide material was separated using a linear gradient from 93% solvent A (0.1% formic acid in water), 7% solvent B (0.1% formic acid in acetonitrile) which was increased to 32% solvent B over 60 min. The mass spectrometer was operated in data-dependent mode, selecting up to the 12 most intense precursors for fragmentation from each precursor scan.
4
Nano-LC-MS/MS Peptide Separation and Analysis
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Protein digests were separated using Nano-LC-Ultra 2D-plus equipped with cHiPLC Nanoflex (Eksigent, Dublin, CA, USA) in trap-and-elute mode, with trap column (200 μm x0.5 mm ChromXP C18-CL 3 μm 120 Å, Eksigent) and analytical column (75 μm x15 cm ChromXP C18-CL 3 μm 120 Å, Eksigent). The separation was carried out with a binary gradient, in which solvent A (0.1 % formic acid/water) and solvent B (0.1 % formic acid/acetonitrile) were used. The gradient program was 2%–33.2% solvent B for 250 min, 33.2%–98% solvent B in 2 min, 98 % solvent B for 5 min, 98 % to 2% solvent B in 0.1 min, and 2% solvent B for 17.9 min, at 300 nL/min. The eluates were infused on-line into the mass spectrometer (TripleTOF 5600+ System with NanoSpray III source and heated interface, SCIEX, Framingham, MA, USA) and ionized in an electrospray ionization-positive mode. Datasets were acquired using an information-dependent acquisition method.
5
Proteomic Analysis of Protein Samples
6
Peptide Separation and Tandem Mass Spectrometry
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Peptide separation was performed using an Eksigent nanoLC Ultra and ChiPLC-nanoflex (Eksigent, Dublin, CA) in TrapElute configuration. Samples were loaded on a 0.5 mm × 200 μm column and eluted on an analytical 15 cm × 75 μm column (ChromXP C18-CL, 3 μm). A gradient formed by mobile phase A (2% (v/v) ACN, 0.1% (v/v) formic acid) and mobile phase B (98% (v/v) ACN, 0.1% (v/v) formic acid) was used to separate 2 μL of the sample. The flow rate was set at 0.3 μL/min. The following gradient elution was used for peptide separation: 0 to 5% of mobile phase B in 1 min, 5 to 12% of mobile phase B in 19 min, 12 to 30% of mobile phase B in 40 min, 30 to 90% of mobile phase B in 2 min, 90 to 90% in 7 min, 90 to 5% in 3 min and finally held at 5% of mobile phase B for 13 min. The tandem MS analysis was performed using a TripleTOF 5600 system (SCIEX) under Information Dependent Mode. For precursor ions selection, the mass range of 400–1800 m/z and accumulation times of 250 ms per spectrum were chosen. MS/MS analysis was performed on the 20 most abundant precursors with accumulation time of 100 ms per cycle. The dynamic exclusion time was at 15 s. High sensitivity mode with rolling collision energy was used to acquire the MS/MS spectra.
7
Reversed-Phase Peptide Separation by nanoLC
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The separation of peptides was performed with an Eksigent nanoLC Ultra and ChiPLC-nanoflex (Eksigent, Dublin, CA, USA) in Trap-Elute configuration. The samples were desalted with a Sep-Pak tC 18 μL Elution Plate (Waters, Miltford, MA, USA), and reconstituted using 20 μL of 2% acetonitrile and 0.05% formic acid. Five microliters (μL) of each sample was loaded on a 200 μm × 0.5 mm trap column and eluted on a 75 μm × 15 cm analytical column (ChromXP C18-CL, 3 μm). A gradient formed by mobile phase A (2% acetonitrile, 0.1% formic acid) and mobile phase B (98% acetonitrile, 0.1% formic acid) was used to separate the sample content at a 0.3 μL/min flow rate. The following gradient elution was used for peptide separation: 0–5% of mobile phase B in 1 min, 5–12% of mobile phase B in 15 min, 12–30% of mobile phase B in 104 min, 30–90% of mobile phase B in 2 min, 90–90% in 7 min, 90–5% in 3 min and held at 5% of mobile phase B for 13 min (protocol modified from [33 (link)]).
8
Silver Staining and Mass Spectrometry Proteomics
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Proteins in the polyacrylamide gels underwent silver staining using a Pierce Silver Stain for Mass Spectrometry Kit (Thermo Scientific). The stained bands were excised and in-gel-digested with trypsin using an In-Gel Tryptic Digestion Kit (Thermo Scientific). The tryptic digests were separated using nanoflow liquid chromatography (Nano-LC-Ultra 2D-plus equipped with cHiPLC Nanoflex [Eksigent, Dublin, CA, USA]). The eluate was directly introduced into a mass spectrometer (TripleTOF 5600+ System coupled to a NanoSpray III source and heated interface [AB SCIEX, Framingham, MA]) and ionized in an electrospray ionization-positive mode. Data were acquired using an information-dependent acquisition method. The acquired datasets were analyzed by ProteinPilot software, version 4.5 beta (AB SCIEX), with the NCBI nr database (June 2016).
9
FFPE Tissue Proteome Analysis by LC-MS
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Proteins for LC-MS analysis were extracted from FFPE tissue samples (five IgG4-related pancreatitis and three normal pancreas tissue) using the Liquid Tissue MS Protein Prep kit (Expression Pathology Inc, Rockville, MD, USA) [15 (link)]. All cases analyzed for LC-MS were the partial resection for pancreas. For normal pancreas tissues, negative surgical margins taken at resection for pancreatic cancer were selected. Briefly, after deparaffinization, three 0.4 μm thick tissue sections (10 × 10 mm) were dissected using a needle and solubilized in 20 uL of Liquid Tissue buffer and protein digestion was performed with trypsin (Promega Corp, Madison, WI, USA) for 18 h at 37°C. Samples were dried and solubilized in 0.1% formic acid (Wako, Osaka, Japan) and 1 μg aliquots for each sample were separated by nanoflow reversed-phase LC (NanoLC-Ultra 2D-Plus, Eksigent, Dublin, CA, USA) equipped with cHiPLC Nanoflex (Eksigent). Eluted peptides were analyzed by a quadrupole-time-of-flight hybrid mass spectrometer (Triple TOF5600+ system, AB SCIEX, Framingham, MA, USA).
10
Proteomic Analysis of Protein Samples
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Protein concentrations were measured by BCA-protein assay. Approximately 30 μg of proteins were separated by SDS-PAGE using 10% Bis-Tris Nupage gels. Serial gel slices were excised and diced into smaller fragments. Samples were reduced with 10 mM dithiothreitol in 25 mM NH4HCO3 at 56 °C for 1 h and alkylated with 55 mM iodoacetamide for 45 min at RT. In-gel trypsin digestion was performed using 10 ng/μl of sequencing grade modified porcine trypsin (Promega) diluted in 505 mM NH4HCO3 at 37 °C overnight. Peptides were extracted with 0.5% formic acid and 50% acetonitrile. Following evaporation of acetonitrile, peptides were purified using a ZipTipC18 column (Millipore). The volume of each eluted sample was reduced in a Speedvac to 5 μl to evaporate acetonitrile and adjusted to 20 μl with 0.1% formic acid prior to LC–MS/MS analysis. An AB SCIEX TripleTOF 5600 System (Foster City) equipped with an Eksigent nanoLC Ultra and ChiPLC-nanoflex (Eksigent) in Trap Elute configuration was employed for LC-MS measurement. The acquired mass spectrometric raw data was processed using ProteinPilot 5.0 software (AB SCIEX) with the Paragon search mode. The ProteinPilot Descriptive Statistics Template (PDST, AB SCIEX) was used for alignment of multiple results and evaluation of the false discovery rate.
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