Nano electrospray source

Manufactured by Thermo Fisher Scientific

Sourced in United States, France

The Nano-electrospray source is a specialized ionization source designed for mass spectrometry applications. It generates a fine mist of charged droplets from a liquid sample, enabling the efficient transfer of analytes into the gas phase for detection and analysis.

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10 protocols using nano electrospray source

LC-MS/MS Proteomics Analysis Protocol

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MS-based proteomic measurements were performed as in ref. 26 (link). Briefly, ~2 µg of desalted peptides were loaded and analyzed by linear 4 h gradients. The LC system was equipped with an in-house made 50-cm, 75-µm inner diameter column slurry-packed into the tip with 1.9 µm C₁₈ beads (Dr. Maisch GmbH, Product Nr. r119.aq). Reverse phase chromatography was performed at 50 °C with an EASY-nLC 1000 ultra-high-pressure system (Thermo Fisher Scientific) coupled to the Q Exactive mass spectrometer (Thermo Fisher Scientific) via a nano-electrospray source (Thermo Fisher Scientific). Peptides were separated by a linear gradient of buffer B up to 40% in 240 min for a 4-h gradient run with a flow rate of 250 nl/min. The Q Exactive was operated in the data-dependent mode with survey scans (MS resolution: 50,000 at m/z 400) followed by up to the top 10 MS2 method selecting ≥2 charges from the survey scan with an isolation window of 1.6 Th and fragmented by higher energy collisional dissociation with normalized collision energies of 25. Repeated sequencing was avoided using a dynamic exclusion list of the sequenced precursor masses for 40 s.

Yahya G., Menges P., Amponsah P.S., Ngandiri D.A., Schulz D., Wallek A., Kulak N., Mann M., Cramer P., Savage V., Räschle M, & Storchova Z. (2022). Sublinear scaling of the cellular proteome with ploidy. Nature Communications, 13, 6182.

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LC-MS/MS Protein Identification Workflow

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All samples/peptides were analyzed by LC-MS/MS using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) coupled online to a RSLC nano HPLC (Ultimate 3000, UHPLC Thermo Fisher Scientific). Samples were loaded onto a 100 µm, 2-cm nanoviper Pepmap100 trap column, eluted and separated on a RSLC nano column 75 µm × 50 cm, Pepmap100 C18 analytical column (Thermo Fisher Scientific). The detailed gradients used for protein identification are listed in Supplementary Table S11. The eluent was nebulized and ionized using a nanoelectrospray source (Thermo Fisher Scientific) with a distal coated fused silica emitter (New Objective). The capillary voltage was set at 1.7 kV. The Q Exactive mass spectrometer was operated in the data-dependent acquisition mode to automatically switch between full MS scans and subsequent MS/MS acquisitions. Survey full-scan MS spectra (m/z 375–1575) were acquired in the Orbitrap with 70,000 resolution (at m/z 200) after accumulation of ions to a 3 × 10⁶ target value with a maximum injection time of 54 ms. Dynamic exclusion was set to 15 s. The 12 most intense multiply charged ions (z ≥ 2) were sequentially isolated and fragmented in the collision cell by higher-energy collisional dissociation (HCD) with a fixed injection time of 54 ms, 17,500 resolution, and automatic gain control (AGC) target of 2 × 10⁵.

Montgomery M.K., Bayliss J., Nie S., De Nardo W., Keenan S.N., Miotto P.M., Karimkhanloo H., Huang C., Schittenhelm R.B., Don A.S., Ryan A., Williamson N.A., Ooi G.J., Brown W.A., Burton P.R., Parker B.L, & Watt M.J. (2022). Deep proteomic profiling unveils arylsulfatase A as a non-alcoholic steatohepatitis inducible hepatokine and regulator of glycemic control. Nature Communications, 13, 1259.

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Peptide Separation and Identification by HPLC-MS

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Peptide digests were injected onto a 1260 Infinity HPLC stack (Agilent, Santa Clara, CA, USA) and separated using a 75 micron I.D. x 15 cm pulled tip C-18 column (00G-4053-E0, Jupiter C-18 300 Å, 5 micron, Phenomenex, Torrance, CA, USA). This system runs in-line with a Thermo Orbitrap Velos Pro hybrid mass spectrometer, equipped with a nano-electrospray source (Thermo Fisher Scientific), and all data was collected in CID mode. The HPLC was configured with binary mobile phases that include solvent A (0.1%FA in ddH₂O), and solvent B (0.1%FA in 15% ddH₂O / 85% ACN), programmed as follows; 10min @ 0%B (2μL/ min, load), 120min @ 0%−40%B (0.5nL/ min, analyze), 15min @ 0%B (2μL/ min, equilibrate). Following each parent ion scan (350–1200m/z @60k resolution), fragmentation data (MS2) was collected on the topmost intense 15 ions. For data dependent scans, charge state screening and dynamic exclusion were enabled with a repeat count of 2, repeat duration of 15.0s, and exclusion duration of 60.0s.

Sanchez R.G., Parrish R.R., Rich M., Webb W.M., Lockhart R.M., Nakao K., Ianov L., Buckingham S.C., Broadwater D.R., Jenkins A., de Lanerolle N.C., Cunningham M., Eid T., Riley K, & Lubin F.D. (2019). HUMAN AND RODENT TEMPORAL LOBE EPILEPSY IS CHARACTERIZED BY CHANGES IN O-GLCNAC HOMEOSTASIS THAT CAN BE REVERSED TO DAMPEN EPILEPTIFORM ACTIVITY. Neurobiology of disease, 124, 531-543.

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Quantitative Mass Spectrometry of Peptides

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MS was carried out as previously described (12 (link),30 (link),31 (link)). Briefly, dried peptides were resuspended in trifluoroacetic acid (TFA) and 6 μl was injected and loaded onto a C18 trap column. The sample was subsequently separated by a C18 reverse-phase column. The mobile phases consisted of water with 0.1% formic acid (A) and 90% acetonitrile with 0.1% formic acid (B). A 65-min linear gradient from 5 to 60% B was used. Eluted peptides were introduced into the mass spectrometer via a 10-μm silica tip (New Objective, Ringoes, NJ) adapted to a nano-electrospray source (Thermo Scientific). The spray voltage was set at 1.2 kV and the heated capillary at 200 °C. The linear trap quadrupole (LTQ) mass spectrometer (ThermoScientific) was operated in data-dependent mode with dynamic exclusion in which one cycle of experiments consisted of a full-MS (300–2,000 m/z) survey scan and five subsequent MS/MS scans of the most intense peaks. Pathways enriched with the proteins were generated by Ingenuity Pathways Analysis (IPA, version 8.5, Ingenuity Systems, Redwood City, CA). Relative protein amounts for each sample compared to the blood control were done by using peptide counts as a measurement of “amount” for each identified protein.

Val S., Jeong S., Poley M., Krueger A., Nino G., Brown K, & Preciado D. (2017). Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology. Pediatric research, 81(6), 911-918.

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Mass Spectrometry-based Proteomics Analysis

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MS was carried out as previously described[14 (link)]. Briefly, dried peptides were resuspended in trifluoroacetic acid (TFA) and 6μL was injected and loaded onto a C18 trap column. The sample was subsequently separated by a C18 reverse-phase column. The mobile phases consisted of water with 0.1% formic acid (A) and 90% acetonitrile with 0.1% formic acid (B). A 65-min linear gradient from 5 to 60% B was used. Eluted peptides were introduced into the mass spectrometer via a 10-μm silica tip (New Objective Inc., Ringoes, NJ) adapted to a nano-electrospray source (Thermo Fisher Scientific). The spray voltage was set at 1.2 kV and the heated capillary at 200°C. The linear trap quadrupole (LTQ) mass spectrometer (ThermoFisher Scientific) was operated in data-dependent mode with dynamic exclusion in which one cycle of experiments consisted of a full-MS (300–2000 m/z) survey scan and five subsequent MS/MS scans of the most intense peaks. Pathways enriched with the proteins were generated by Ingenuity Pathways Analysis (IPA, version 8.5, Ingenuity Systems, Redwood City, CA).

Val S., Poley M., Brown K., Choi R., Jeong S., Colberg-Poley A., Rose M.C., Panchapakesan K.C., Devaney J.C., Perez-Losada M, & Preciado D. (2016). Proteomic Characterization of Middle Ear Fluid Confirms Neutrophil Extracellular Traps as a Predominant Innate Immune Response in Chronic Otitis Media. PLoS ONE, 11(4), e0152865.

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Peptide Separation and Mass Spectrometry

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Peptides were separated on an EASY-nLC 1000 HPLC system (Thermo Fisher Scientific) coupled online to the Q Exactive mass spectrometer via a nanoelectrospray source (Thermo Fisher Scientific). Peptides were loaded in buffer A (0.5% formic acid) on in house packed columns (75 μm inner diameter, 50 cm length and 1.9 μm C18 particles from Dr Maisch GmbH). Peptides were eluted with a nonlinear 240 min gradient of 5–60% buffer B (80% ACN, 0.5% formic acid) at a flow rate of 250 nl min⁻¹ and a column temperature of 50 °C. Operational parameters were real-time monitored by the SprayQC software. The Q Exactive was operated in a data-dependent acquisition mode with a survey scan range of 300–1,700 m/z and a resolution of 70,000 at m/z 200. Up to the five most abundant isotope patterns with a charge ≥2 were isolated with a 2.2 Thomson (Th) isolation window and subjected to higher-energy collisional dissociation fragmentation at a normalized collision energy of 25. Fragmentation spectra were acquired with a resolution of 17,500 at m/z 200. Dynamic exclusion of sequenced peptides was set to 45 s. Thresholds for ion injection time and ion target values were set to 20 ms and 3E6 for the survey scans and 120 ms and 1E5 for the MS/MS scans, respectively. Data were acquired using the Xcalibur software (Thermo Scientific).

Böttcher J.P., Beyer M., Meissner F., Abdullah Z., Sander J., Höchst B., Eickhoff S., Rieckmann J.C., Russo C., Bauer T., Flecken T., Giesen D., Engel D., Jung S., Busch D.H., Protzer U., Thimme R., Mann M., Kurts C., Schultze J.L., Kastenmüller W, & Knolle P.A. (2015). Functional classification of memory CD8+ T cells by CX3CR1 expression. Nature Communications, 6, 8306.

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Proteomic Analysis of Cell Surface Glycoproteins

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Lysates were prepared in M-Per in quadruplicates and peptide digests separated and analyzed with a Thermo Orbitrap Velos Pro hybrid mass spectrometer equipped with a nano-electrospray source (Thermo Fisher Scientific) similar to our prior report (76 (link)). The XCalibur RAW files were converted and mgf files searched using SEQUEST to generate peptide IDs that were filtered using Scaffold (Protein Sciences). Normalized spectral counts were used to calculate fold changes and proteins with greater than 5-fold changes among the nontargeting control and KD samples further analyzed to determine cell surface N-glycoproteins.

GC S., Tuy K., Rickenbacker L., Jones R., Chakraborty A., Miller C.R., Beierle E.A., Hanumanthu V.S., Tran A.N., Mobley J.A., Bellis S.L, & Hjelmeland A.B. (2022). α2,6 Sialylation mediated by ST6GAL1 promotes glioblastoma growth. JCI Insight, 7(21), e158799.

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Orbitrap-based Proteomic Profiling Protocol

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Mass spectrometry analysis and data evaluation were carried utilizing a Thermo Scientific EASY nLC 1000 HPLC system directly coupled to an Orbitrap Elite™ quadrupole Orbitrap mass analyzer via a nano-electro spray source (Thermo Fischer Scientific) (Michalski et al. 2012), with the software Xcalibur acquiring the data. Peptides were loaded onto in-house packed columns (75 µM inner diameter, 20-cm length, 1.8 µM C18 particles) in MS Buffer A and separated for 70 min within a linear gradient from 5% MS buffer B to 60% MS buffer B at a flow rate of 250 nl/min with a column temperature set to 40 °C. The mass analyzer was operated in a data-dependent top15 mode with a survey scan range set to 300 to 1650 m/z and a resolution of 240,000 at 400 m/z. Selected peptides were subjected to collision induced dissociation with a normalized collision energy of 35. Repeated sequencing was limited by dynamically excluding sequences features for 30 seconds.

Hornburg D., Kruse T., Anderl F., Daschkin C., Semper R.P., Klar K., Guenther A., Mejías-Luque R., Schneiderhan-Marra N., Mann M., Meissner F, & Gerhard M. (2019). A mass spectrometry guided approach for the identification of novel vaccine candidates in gram-negative pathogens. Scientific Reports, 9, 17401.

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Peptide Analysis via Nano-HPLC-MS/MS

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Dried peptides were reconstituted in 16 µL of 0.1% formic acid (FA). 8 µL of each sample was injected onto a 1260 Infinity nHPLC stack (Agilent Technologies, Santa Clara, CA, USA), and separated using a 75 micron I.D. × 15 cm pulled tip C-18 column (Jupiter C-18 300 Å, 5 micron, Phenomenex). This system runs in-line with a Thermo Orbitrap Velos Pro hybrid mass spectrometer, equipped with a nano-electrospray source (Thermo Fisher Scientific, Waltham, MA, USA), and all data were collected in CID mode. The nHPLC was configured with binary mobile phases that included solvent A (0.1%FA in ddH2O), and solvent B (0.1% FA in 15% ddH₂O / 85% ACN), programmed as follows; 10 min at 2% solvent B; 90 min at 5-40% solvent B; 5 min at 70% solvent B; 10 min at 0% solvent B. Following each parent ion scan (300-1200m/z @ 60k resolution), fragmentation data (MS2) was collected on the top most intense 15 ions. For data dependent scans, charge state screening and dynamic exclusion were enabled with a repeat count of 2, repeat duration of 30 s, and exclusion duration of 90 s.

Han D., Yang J., Zhang E., Liu Y., Boriboun C., Qiao A., Yu Y., Sun J., Xu S., Yang L., Yan W., Luo B., Lu D., Zhang C., Jie C., Mobley J., Zhang J, & Qin G. (2020). Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart. Theranostics, 10(24), 11324-11338.

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Nano-LC-MS/MS Peptide Characterization

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The medium extracts were characterized by online nano-LC and electrospray tandem mass spectrometry. The analyses were performed on a U3000 Dionex nanoflow system connected to a LTQ Orbitrap mass spectrometer equipped with a nano-electrospray source (Thermo-Fischer, Les Ulis, France). Chromatographic separation took place in a C18 pepmap 100 column (75 µm ID, 15 cm length, 5 µm, 10 nm, Dionex). The extracts were injected on pre-concentration column with a flow rate of 20 µL.min -1 of water/TFA (0.1 %). After three minutes of wash with the same solvent, the compounds were eluted and separated in the analytical column with a flow of 200 nL/min and a gradient from 2 % to 60 % acetonitrile with 0.1 % formic acid in 30 minutes. The mass spectrometer was operated in the data dependent mode to automatically switch between Orbitrap MS and MS 2 in CID mode in the linear trap. Survey full scan MS spectra from m/z 200 to m/z 1500 were acquired in the Orbitrap with mass resolution of 30 000 at m/z 400, after accumulation of 500 000 charges in the linear ion trap. The most intense ions (up to four, depending on signal intensity) were sequentially isolated for fragmentation, in the linear ion trap using CID at a target value of 100 000 charges. The resulting fragments were recorded in the Orbitrap with a mass resolution of 7 500.

Vallet M., Vanbellingen Q.P., Fu T., Le Caer J.P., Della-Negra S., Touboul D., Duncan K.R., Nay B., Brunelle A, & Prado S. (2017). An Integrative Approach to Decipher the Chemical Antagonism between the Competing Endophytes Paraconiothyrium variabile and Bacillus subtilis. Journal of natural products, 80(11).

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