A targeted mass spectrometry (MS)‐based method, previously validated for cats,20 was applied to determine the concentrations of IGF‐I, IGF‐II, IGFBP‐3, and IGFBP‐5 in 3 cats with DM later achieving remission. Analyses were performed in serum at T0 and T1 exactly as described by Sundberg et al.20 Briefly, the serum proteins were digested with trypsin and isotopically labeled internal standards, 4 QPrESTs (Atlas antibodies, Stockholm, Sweden) and 1 synthetic peptide (New England Peptide, Gardner, Massachusetts), were added to the samples. The tryptic peptides were separated in reversed phase on an EASY‐nLC 1000 system (EASY‐nLC 1000 system, ThermoFisher Scientific, Waltham, Massachusetts) and electrosprayed on‐line to a QExactive Plus Orbitrap mass spectrometer (QExactive Plus Orbitrap mass spectrometer, ThermoFisher Scientific, Waltham, Massachusetts) operating in parallel reaction monitoring mode. The Skyline software (Skyline software, MacCoss Lab Software, University of Washington, Washington state) was applied for data analysis and quantification.
Easy nlc 1000 system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Denmark
The EASY-nLC 1000 system is a high-performance liquid chromatography (HPLC) instrument designed for nano-flow applications. It is capable of delivering flow rates from 50 nL/min to 2 μL/min with high precision and reproducibility. The system is equipped with a solvent delivery module, an autosampler, and a column compartment, making it suitable for a wide range of liquid chromatography applications.
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Lab products found in correlation
Q exactive mass spectrometer, by Thermo Fisher Scientific (51 mentions)
Q exactive plus mass spectrometer, by Thermo Fisher Scientific (23 mentions)
Q exactive, by Thermo Fisher Scientific (21 mentions)
Trypsin, by Promega (13 mentions)
Q exactive plus, by Thermo Fisher Scientific (12 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (11 mentions)
Orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (9 mentions)
Orbitrap fusion tribrid mass spectrometer, by Thermo Fisher Scientific (9 mentions)
Q exactive hf, by Thermo Fisher Scientific (8 mentions)
Ltq orbitrap elite mass spectrometer, by Thermo Fisher Scientific (8 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (8 mentions)
Easy spray c18 column, by Thermo Fisher Scientific (8 mentions)
Easy spray column, by Thermo Fisher Scientific (7 mentions)
Proteome discoverer 1, by Thermo Fisher Scientific (7 mentions)
Easy column, by Thermo Fisher Scientific (7 mentions)
Acclaim pepmap c18, by Thermo Fisher Scientific (6 mentions)
Reprosil pur c18 aq 1.9 μm resin, by Dr. Maisch (6 mentions)
Proteome discoverer software, by Thermo Fisher Scientific (6 mentions)
Omix c18 tips, by Agilent Technologies (6 mentions)
Mascot, by Matrix Science (5 mentions)
274 protocols using easy nlc 1000 system
1
Targeted MS-based Biomarker Quantification
2
Proteomic Profiling of Gel Spots
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SDS-PAGE gels were selected from the Coomassie-stained gels. Each was covered with 100 μL of 50 mM ammonium bicarbonate (ABC) buffer in 50% acetonitrile (ACN) with 50 mM dithiothreitol (DTT). Each gel spot was covered with 100 μL of 50 mM ammonium bicarbonate (ABC)/50% ACN containing 50 mM iodoacetamide (IAA) and sonicated for 5 min. After 15 min, the supernatant was discarded and replaced with 100 μL of 50 mM ABC/50% ACN containing 50 mM DTT. The supernatant was discarded, and the samples were again sonicated for 5 min in 100 μL of HPLC/MS-grade water. The water was discarded, and the samples were again sonicated for 5 min in 100 μL of ACN. The ACN was discarded, and the sample-containing microtubes were air-dried to remove the remaining ACN. Next, trypsin in 10 μL of 50 mM ABC was added to each sample, and the samples were incubated overnight at 37 °C followed by the addition of trifluoroacetic acid (TFA) and ACN to final concentrations of 1% and 30%, respectively. Each sample was assayed in triplicate. LC-MS/MS analysis was performed on a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nano-liquid chromatography system (Thermo Scientific EASY-nLC 1000 System, Waltham, MA, USA).
3
Nano-LC-MS/MS Proteomics Workflow
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LC-MS/MS was performed as described previously 39 (link). Nano-high-performance liquid chromatography (nano-LC) analyses were performed using an Easy n-LC 1000 system (Thermo Fisher Scientific, San Jose, CA). The column (15 cm x 75 μm) was packed in-house with Jupiter 2 μm, 100 Å pore size C18 beads (Phenomenex, Torrance, CA). Mobile phase A for LC separation consisted of 0.1% formic acid in deionized water and the mobile phase B consisted of 0.1% formic acid in acetonitrile. For analysis of fractionated samples, the mobile phase was programmed from 5% B over 10 min, 5% B to 30% B over 35 min, 30% B to 90% B over 12 min, and finally to 90% B to 5% B over 13 min at a flow rate of 300 nL/min. A Q-ExactiveTM mass spectrometer (Thermo Fisher) was used for MS analyses and was operated with Xcalibur (version 2.1, Thermo Fisher) to generate peak lists. For peptide ionization, 2400 V was applied and a 250 °C capillary temperature was used. The full scan event was collected using a m/z 350 - 2000 mass selection, an Q-Exactive MS resolution of 70,000, a target automatic gain control (AGC) value of 1 X 106, and a maximum injection time of 80 ms. Fragmentation was performed with a normalized collision energy of 25.
4
Proteomics Analysis of Extracellular Vesicles
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Proteomics analysis of EV samples was processed on a liquid chromatography electrospray ionisation tandem mass spectrometer (LC-ESI MS/MS, Shanghai Applied Protein Technology Co., Ltd., Shanghai, China) as previously reported [37 (link)]. After EVs (equivalent to 30 μg total proteins) were treated with trypsin digestion, the peptides were collected and loaded onto the Easy-nLC1000 system equipped with the orbitrap Q Exactive MS (Thermo). The LC-ESI MS/MS full-scan signals were acquired within the range of the precursor ion from 300 to 1800 m/z, and original files were imported into Mascot software (version 2.2, Matrix Science, London, UK) for protein candidate identification and characterisation. The reference database was uniprot_Homo_sapiens_173343_20191014.fasta (173,343 protein sequences, download date of 14 October 2019).
5
Phosphoproteome Profiling by LC-MS/MS
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Samples were loaded onto 50-cm columns packed in-house with C18 1.9 μM ReproSil particles (Dr. Maisch GmbH), with an EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to the MS (Q Exactive HFX; Thermo Fisher Scientific). A homemade column oven maintained the column temperature at 60°C. Phosphopeptides were eluted with a 140 min gradient starting at 5% buffer B (80% ACN, 0.1% Formic acid) followed by a stepwise increase to 20% in 85 min, 40% in 35 min, 65% in 10 min and 80% in 2 × 5 min at a flow rate of 300 nl/min. Samples were measured in data-dependent acquisition with a (TopN) MS method in which one full scan (300–1,650 m/z, R = 60,000 at 200 m/z, maximum injection time 120 ms) at a target of 3 × 106 ions was first performed, followed by 10 data-dependent MS/MS scans with higher energy collisional dissociation (AGC target 105 ions, maximum injection time at 120 ms, isolation window 1.6 m/z, normalized collision energy 27%, R = 15,000 at 200 m/z). Dynamic exclusion of 40 s and the Apex trigger from 4 to 7 s was enabled.
6
Peptide Separation and Mass Spectrometry
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Fractions were re-suspended in 30 μL solvent C and 30 μL solvent D (C: water with 0.1% formic acid; D: ACN with 0.1% formic acid), separated by nano-liquid chromatography (nanoLC) and analyzed by on-line electrospray tandem mass spectrometry. The experiments were performed using an Easy-nLC 1000 system (Thermo Fisher Scientific, MA, USA) connected to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA) that was equipped with an online nano-electrospray ion source. For analysis, 10 μL peptide sample was loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100 μm × 2 cm) with a flow of 10 μL/min for 3 min, separated on an analytical column (Acclaim PepMap C18, 75 μm × 15 cm) with a linear gradient from 3% D to 32% D in 120 min. The column was re-equilibrated at the initial conditions for 10 min. The column flow rate was maintained at 300 L/min. An electrospray voltage of 2 kV (versus the inlet) of the mass spectrometer was used.
7
High-Throughput Proteomics Workflow
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Two independent replicates were dissolved in loading buffer (0.1% formic acid) and loaded onto a 20-cm capillary column packed in-house with 3-μm Reprosil-Pur C18 beads (Dr. Maisch; Ammerbuch, Germany) using an EASY-nLC 1000 system (Thermo Scientific; San Jose, CA, USA). Running buffer A was 0.1% formic acid in water; running buffer B was 0.1% formic acid in ACN. Total gradient was 120 min, with flow rate started at 300 nL/min. Detailed gradient was 6% ACN with linear increase to 30% ACN over 105 min, followed by 4 min linear increase to 90% ACN. MS data were acquired using data-dependent top-20 method on Q Exactive HF (Thermo Scientific). Analytical parameters were: spray voltage 2 kV; S-lens RF level 60; capillary temperature 275 °C; full-scan resolutions 60,000@m/z 200 with AGC 3e6, maximum fill time 20 ms, and mass range of full mass 350–1500; MS2 scan resolutions 15,000@m/z 200 with AGC 5e4 and maximum fill time 100 ms for proteomics analysis; isolation width 1.6 Th; fixed first mass 110; normalized collision energy 27; peptide match set to “preferred”; isotope exclusion on. Precursor ions with single, unassigned charge states were eliminated from fragmentation selection.
8
Peiminine-Induced Proteomic Changes in HCT-116 Cells
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A DIA-based proteomics approach was used to characterize the protein changes in peiminine-treated HCT-116 cells. Briefly, extracted proteins were quantified using a BCA protein assay kit (Bi Yuntian, Shanghai, China) and digested using the FASP method. The peptide concentrations were determined by measuring absorbance at 280 nm. The digested peptides were also analyzed in DDA mode as reported previously (Zheng et al., 2022 (link)). For DIA analysis, an Orbitrap Fusion Lumos mass spectrometer equipped with an EASY-nLC 1000 system (Thermo Fisher Scientific) were used. The full scan was performed at a resolution of 60,000 over an m/z range of 350–1500, and DIA scans were set at a resolution of 30,000. There were 45 variable DIA windows ranging from 350 to 1500 m/z. Spectronaut pulsar X 12.0 (Biognosys) software was used for protein identification and quantification.
9
Tryptic Peptide MS Analysis Protocol
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Con A-enriched proteins were reduced with 10 mM dithiothreitol (DTT) for 150 min at 37°C and then alkylated with 50 mM iodoacetamide (IAA) for 40 min in the dark. After washing twice with 50 mM NH4HCO3, proteins were incubated with trypsin (1 μg/50 μg protein) overnight at 37°C in 150 µl of 50 mM NH4HCO3. Tryptic peptides were concentrated and resuspended in 0.1% formic acid.
Tryptic peptides were separated using the Thermo Fisher Scientific EASY-nLC 1000 system and EASY-Spray column (C18, 2 μm, 100 Å, 75 μm × 50 cm) with a 2–100% acetonitrile gradient in 0.1% formic acid over 180 min at a flow rate of 250 nl/min. The separated peptides were analyzed using a Thermo Scientific Q Exactive mass spectrometer operating in data-dependent mode. Up to 10 of the most abundant isotope patterns with charge ≥2 from an initial survey scan were automatically selected for fragmentation by higher energy collisional dissociation with normalized collision energies of 27%. The maximum ion injection times for the survey scan and the MS/MS scans were 20 and 60 ms, respectively, and the ion target value for both scan modes was set to 1E6. An 18-s dynamic exclusion of previously sequenced ions was applied.
Tryptic peptides were separated using the Thermo Fisher Scientific EASY-nLC 1000 system and EASY-Spray column (C18, 2 μm, 100 Å, 75 μm × 50 cm) with a 2–100% acetonitrile gradient in 0.1% formic acid over 180 min at a flow rate of 250 nl/min. The separated peptides were analyzed using a Thermo Scientific Q Exactive mass spectrometer operating in data-dependent mode. Up to 10 of the most abundant isotope patterns with charge ≥2 from an initial survey scan were automatically selected for fragmentation by higher energy collisional dissociation with normalized collision energies of 27%. The maximum ion injection times for the survey scan and the MS/MS scans were 20 and 60 ms, respectively, and the ion target value for both scan modes was set to 1E6. An 18-s dynamic exclusion of previously sequenced ions was applied.
10
Nano-UHPLC-MS/MS Proteomics Pipeline
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Samples were dissolved in 0.1% formic acid and loaded into a nano‐flow UHPLC (Easy‐nLC 1000 system; Thermo Fisher Scientific) online‐coupled to an Orbitrap mass spectrometer equipped with a nanospray ion source (Q‐Exactive Plus; Thermo Fisher Scientific). Samples were separated by using a 75 μm × 20 cm capillary column with a particle size of 3 μm (NTCC‐360; Nikkyo Technos) by applying a linear gradient ranging from 5% to 35% buffer B (100% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min for 120 minutes. In mass spectrometry analysis, survey scan spectra were acquired at a resolution of 70 000 at 200 m/z with a target value of 3e6 ions, ranging from 350 to 2000 m/z with charge states between 1+ and 4+. We applied a data‐dependent top 10 method that generates high‐energy collision dissociation fragments for the 10 most intense precursor ions per survey scan. The tandem mass spectrometry (MS/MS) resolution was 17 500 at 200 m/z with a target value of 1e5 ions.
For MS/MS data analysis, we used the Sequest HT (Thermo Fisher Scientific) and Mascot version 2.5 (Matrix Science) algorithms embedded in the Proteome Discoverer 2.2 platform (Thermo Fisher Scientific), and the peak lists were searched against the UniProt human databases. The tolerance of precursor ions and fragment ions were set to 10 ppm and 0.02 Da, respectively.
For MS/MS data analysis, we used the Sequest HT (Thermo Fisher Scientific) and Mascot version 2.5 (Matrix Science) algorithms embedded in the Proteome Discoverer 2.2 platform (Thermo Fisher Scientific), and the peak lists were searched against the UniProt human databases. The tolerance of precursor ions and fragment ions were set to 10 ppm and 0.02 Da, respectively.
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