Nanospray ion source

Manufactured by Thermo Fisher Scientific

Sourced in United States, Austria

The Nanospray ion source is a compact, high-performance ion source designed for use in mass spectrometry applications. It generates a fine, stable spray of charged particles from a sample solution, which is then introduced into the mass spectrometer for analysis. The Nanospray ion source operates at low flow rates, typically in the nanoliter-per-minute range, making it suitable for analyzing small sample volumes.

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4 protocols using nanospray ion source

Peptide Quantification Protocol with Internal Standards

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Samples were reconstituted in 5 µl 30% formic acid containing 10 fmol each of 4 synthetic standard peptides and then immediately diluted with 40 µl mobile phase A (98% H₂O, 2% acetonitrile, and 0.1% formic acid). The synthetic peptides [Glu1-Fribrinopeptide B, EGVNDNEEGFFSAR; M28, TTPAVLDSDGSYFLYSK; HK0, VLETKSLYVR; HK1, VLETK(ε-AC)SLYVR] were spiked into each sample as an internal quality control for monitoring LC–MS instrument stability. Five microliters of the solution were injected into the nano HPLC-system (Dionex Ultimate 3000) loading peptides on a 2 cm × 75 µm C18 Pepmap100 pre-column (Thermo Fisher Scientific) at a flow rate of 10 µl/min using mobile phase A. Afterwards, peptides were eluted to a 50 cm × 75 µm Pepmap100 analytical column (Thermo Fisher Scientific) at a flow rate of 300 nl/min, using a gradient from 8 to 40% mobile phase B (80% acetonnitrile, 20% H2O, 0.1% formic acid) over 235 min. The nano-HPLC system was coupled to a QExactive orbitrap with a nanospray ion source (Thermo Fisher Scientific). MS scans were performed in the range from m/z 400 to 1400 at a resolution of 70,000 (at m/z = 200), MS/MS scans at a resolution of 17,500 (at m/z = 200), using a top 12 method and applying HCD fragmentation at 30% normalized collision energy.

Drev D., Bileck A., Erdem Z.N., Mohr T., Timelthaler G., Beer A., Gerner C, & Marian B. (2017). Proteomic profiling identifies markers for inflammation-related tumor–fibroblast interaction. Clinical Proteomics, 14, 33.

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Permethylated Glycan Analysis via NSI-MS^n

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NSI-MSⁿ was performed as described by Aoki et al (17 (link)). Briefly, permethylated glycans were dissolved in 1 mM NaOH in 50% methanol and infused directly into a linear ion trap mass spectrometer (LTQ Orbitrap Discovery; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using a Thermo Fisher Scientific^™ nanospray ion source (Thermo Fisher Scientific, Inc.). MS analysis was performed in a positive ion mode and MS/MS spectra (at 28% collision energy) were obtained using the total ion mapping function of the Xcalibur software (version 2; Thermo Fisher Scientific, Inc.). The fragmentation derived from the MS/MS spectra was identified using the nomenclature described by Domon and Costello (19 (link)).

Talabnin K., Talabnin C., Ishihara M, & Azadi P. (2017). Increased expression of the high-mannose M6N2 and NeuAc3H3N3M3N2F tri-antennary N-glycans in cholangiocarcinoma. Oncology Letters, 15(1), 1030-1036.

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Peptide Characterization by Nano-LC-MS/MS

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Dried peptides were reconstituted in 5 µl 30% formic acid, containing four synthetic peptides [Glu1-fribrinopeptide B, EGVNDNEEGFFSAR; M28, TTPAVLDSDGSYFLYSK; HK0, VLETKSLYVR; HK1, VLETK(ε-AC)SLYVR] for quality control. The samples were further diluted with 40 µl mobile phase A (97.9% H2O, 2% acetonitrile, 0.1% formic acid). Peptides were analyzed with a Dionex UltiMate 3000 Nano LC system coupled to a Q Exactive Orbitrap mass spectrometer, equipped with a NanoSpray Ion Source (Thermo Fisher Scientific, Austria). Preconcentration of the peptides was done on a C18 2 cm × 100 μm precolumn and LC separation was performed with a 50 cm × 75 μm PepMap100 analytical column (Thermo Fisher Scientific), at a flow rate of 300 nl/min and injection volume of 10 µl. Gradient elution of the peptides was achieved by increasing the mobile phase B (79.9% acetonitrile, 20% H2O, 0.1% formic acid) from 8% to 40%, with a total chromatographic run time of 135 min including washing and equilibration. Mass spectrometric resolution on the MS1 level was set to 70,000 (at m/z = 200) with a scan range from 400 to 1,400 m/z. The 12 most abundant peptide ions were selected for fragmentation at 30% normalized collision energy and analyzed in the Orbitrap at a resolution of 17,500 (at m/z = 200).

Gerner M.C., Niederstaetter L., Ziegler L., Bileck A., Slany A., Janker L., Schmidt R.L., Gerner C., Del Favero G, & Schmetterer K.G. (2019). Proteome Analysis Reveals Distinct Mitochondrial Functions Linked to Interferon Response Patterns in Activated CD4+ and CD8+ T Cells. Frontiers in Pharmacology, 10, 727.

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Nano-RPLC-MS/MS Analysis of Peptide Samples

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The separated and lyophilized peptide samples were redissolved in Buffer A (acetonitrile:water:formic acid = 2 : 98 : 0.1), dissolved on a vortex mixer, moved to a sample bottle, and subsequently subjected to MS analysis. Online Nano-RPLC was performed via the Easy-nLC 1000 system (Thermo Fisher Scientific). The dissolved sample was loaded onto a trap column (PepMap100, C18 3 μm 75 μm × 20 mm NanoViper, Thermo Fisher Dionex ion chromatography; Thermo Fisher Scientific) at a flow rate of 2 µL/min followed by rinse desalination for 10 min. The analytical column was a C18 reverse-phase chromatography column (PepMap100, C18 2 μm 75 μm × 150 mm NanoViper, Thermo Fisher Dionex ion chromatography). The gradient used in the experiment increased the mobile phase B from 5% to 35% within 70 min.
The Q-Exactive MS system (Thermo Fisher Scientific) combined with a nanospray ion source (Thermo Fisher Scientific) was used. The spray voltage was 1.6 kV, and the temperature of the capillary tube was 250°C. The MS scanning mode was set as the data-dependent acquisition mode (Data Dependent Analysis, DDA). Twenty fragment maps were collected after each full scan. The full-scan resolution was 70,000, the MS/MS resolution was 17,500, the precursor ion scan range was 300–1,800 m/z, and the collision energy was 27% higher energy C-trap dissociation.

Li F., Yang B., Liu Y., Tang T., Wang C., Li M., Lv S., Qi Q., Liu H., Shi Z., Wu H, & Wang X. (2021). Acupuncture Regulates Serum Differentially Expressed Proteins in Patients with Chronic Atrophic Gastritis: A Quantitative iTRAQ Proteomics Study. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 9962224.

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