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Nanoacquity

Manufactured by Thermo Fisher Scientific
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The NanoAcquity is a high-performance liquid chromatography (HPLC) system designed for nano-scale separations. It features precise flow control and sensitive detection capabilities for applications requiring low sample volumes.

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Lab products found in correlation

Fusion lumos mass spectrometer, by Thermo Fisher Scientific (2 mentions) Thermo pepmap c18 trap, by Thermo Fisher Scientific (2 mentions) Easy spray pepmap c18 analytical column, by Thermo Fisher Scientific (2 mentions) Mascot software, by Matrix Science (1 mentions) Ltq orbitrap velos pro, by Thermo Fisher Scientific (1 mentions) Q exactive, by Thermo Fisher Scientific (1 mentions) Symmetry c18 material, by Waters Corporation (1 mentions) Aeris c18 material, by Phenomenex (1 mentions)

8 protocols using nanoacquity

1

Quantifying ApoE Isoforms by LC-MS/MS

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Total apoE levels and the individual apoE isoform (apoE3 and apoE4) levels were previously quantified by use of a mass-spectrometry based method [31 (link)], and the results were reported in a previous pub-lication [21 (link)]. In brief, plasma samples where diluted 1:100, trypsin-digested and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a Waters NanoAcquity coupled to a Thermo Vantage mass spectrometer at the Mayo Cli-nic Proteomics Core, USA. For quantification of apoE3 and apoE4 isoforms two tryptic peptides, LGADMEDVCGR or LGADMEDVR, derived from the two single major APOE single nucleotide polymorphisms (SNP112 and SNP158) were used. Total apoE was quantified using a peptide present in both isoforms, LGPLVEQGR, which also served as a control to assess the accuracy of the total sum of the two isoforms within each sample.
Edlund A.K., Chen K., Lee W., Protas H., Su Y., Reiman E., Caselli R, & Nielsen H.M. (2021). Plasma Apolipoprotein E3 and Glucose Levels Are Associated in APOE ɛ3/ɛ4 Carriers. Journal of Alzheimer's Disease, 81(1), 339-354.
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2

Peptide Analysis by LC/MS/MS

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The peptide samples were analyzed by LC/MS/MS using a Waters nanoAcquity coupled to a Thermo Fusion Lumos mass spectrometer. Samples were injected onto a Thermo PepMap C18 trap column, washed, and then loaded onto an Easy Spray PepMap C18 analytical column (75 μM id × 25 cm, 2 μM particle size) (Thermo Scientific). The samples were separated over a 120 min method, where the gradient for separation consisted of 2%–25% mobile phase B at a 300 nl/min flow rate; mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 100% acetonitrile. MS1 orbitrap scans were collected at a resolution of 120,000 and 1e6 AGC target. The MS2 spectra were acquired either in the orbitrap or the linear ion trap depending on peak charge and intensity using a 3 s TopSpeed CHOPIN method (Davis et al., 2017 (link)). Orbitrap MS2 scans were acquired at 7500 resolution, with a 5e4 AGC, and 22ms maximum injection using HCD fragmentation with a normalized energy of 30%. Rapid linear ion trap MS2 scans were acquired using a 4e3 AGC, 250 ms maximum injection time, CID fragmentation set at 30%. Dynamic exclusion was set to 30 s and precursors with unknown charge or a charge state of 1 and ≥ 8 were excluded.
Waters A.M., Khatib T.O., Papke B., Goodwin C.M., Hobbs G.A., Diehl J.N., Yang R., Edwards A.C., Walsh K.H., Sulahian R., McFarland J.M., Kapner K.S., Gilbert T.S., Stalnecker C.A., Javaid S., Barkovskaya A., Grover K.R., Hibshman P.S., Blake D.R., Schaefer A., Nowak K.M., Klomp J.E., Hayes T.K., Kassner M., Tang N., Tanaseichuk O., Chen K., Zhou Y., Kalkat M., Herring L.E., Graves L.M., Penn L.Z., Yin H.H., Aguirre A.J., Hahn W.C., Cox A.D, & Der C.J. (2021). Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition. Cell reports, 35(13), 109291.
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3

Plasma Proteomics Analysis Protocol

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Plasma proteomics experiments were performed by the Caprion Biosciences, Inc. All plasma samples went through standard sample preparation protocol including depletion of highly abundant proteins and trypsin digestion, followed by strong cation-exchange chromatography fractionation. Each fraction of a sample was analyzed by Q ExactiveTM mass spectrometer (Thermo Fisher) with separation through NanoAcquity UPLC-based liquid chromatography. For protein identification, the tandem mass spectra were searched against UniProt human protein database UP000005649 using Mascot software (Matrix Science, version 2.5.1) and peptides and associated protein IDs were identified at 6.4% false discovery rate. After cross-sample alignment by chromatographic retention time, peptides with 25% or higher frequency of missing data were removed, and the missing peptide intensities were imputed using the K-nearest neighbors method. The processed peptide intensity data was normalized so that the median intensity is the same across all the samples. Finally, protein intensity was derived by the sum of peptide-level intensities. See Supplementary Table S3.
Choi H., Koh H.W., Zhou L., Cheng H., Loh T.P., Parvaresh Rizi E., Toh S.A., Ronnett G.V., Huang B.E, & Khoo C.M. (2019). Plasma Protein and MicroRNA Biomarkers of Insulin Resistance: A Network-Based Integrative -Omics Analysis. Frontiers in Physiology, 10, 379.
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4

Quantification of Plasma ApoE Isoforms

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Plasma total apoE, apoE3 and apoE4 isoform concentrations were quantified using a previously described mass spectrometry-based multiplex selected reaction monitoring (SRM) assay [32 (link)] and the services provided by the Mayo Clinic Proteomics Core. Briefly, two tryptic peptides derived from the two major APOE single nucleotide polymorphisms (SNP112 and SNP158) were employed for the quantification of apoE3 and apoE4 isoforms (LAVYQAGAR and LGADMEDVR). As a control, total apoE concentrations were quantified in parallel using a tryptic peptide with a sequence present in all three known apoE isoforms (LGPLVEQGR). Plasma samples were diluted 1:100, digested as described previously [32 (link)] and analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) with a Waters NanoAcquity coupled to a Thermo Vantage mass spectrometer.
Nielsen H.M., Chen K., Lee W., Chen Y., Bauer RJ I.I.I., Reiman E., Caselli R, & Bu G. (2017). Peripheral apoE isoform levels in cognitively normal APOE ε3/ε4 individuals are associated with regional gray matter volume and cerebral glucose metabolism. Alzheimer's Research & Therapy, 9, 5.
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5

Crosslinking and Mass Spectrometry Analysis of Influenza Polymerase

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Crosslinking was performed using recombinant FluA or FluB polymerase or reconstituted FluA-vRNA polymerase complex by addition of isotope-labeled DSS or DSG as described previously (Leitner et al., 2014 (link)). Protein digestion was performed at 37°C using LysC for 4 hr and trypsin for 12 hr, and crosslinked peptides were enriched using gel filtration. Fractions were analyzed by liquid-chromatography-based mass spectrometry using a nanoAcquity ultraperformance liquid chromatography column connected to a LTQ Orbitrap Velos Pro instrument (Thermo Scientific). Mass spectrometry data were processed using the xQuest/xProphet. Identified crosslinks were mapped to known polymerase structures and analyzed using Xlink Analyzer (Kosinski et al., 2015 (link)).
Thierry E., Guilligay D., Kosinski J., Bock T., Gaudon S., Round A., Pflug A., Hengrung N., El Omari K., Baudin F., Hart D.J., Beck M, & Cusack S. (2016). Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains. Molecular Cell, 61(1), 125-137.
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6

Peptide Analysis by LC/MS/MS

Cited 1 time
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The peptide samples were analyzed by LC/MS/MS using a Waters nanoAcquity coupled to a Thermo Fusion Lumos mass spectrometer. Samples were injected onto a Thermo PepMap C18 trap column, washed, and then loaded onto an Easy Spray PepMap C18 analytical column (75 μM id × 25 cm, 2 μM particle size) (Thermo Scientific). The samples were separated over a 120 min method, where the gradient for separation consisted of 2%–25% mobile phase B at a 300 nl/min flow rate; mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 100% acetonitrile. MS1 orbitrap scans were collected at a resolution of 120,000 and 1e6 AGC target. The MS2 spectra were acquired either in the orbitrap or the linear ion trap depending on peak charge and intensity using a 3 s TopSpeed CHOPIN method (Davis et al., 2017 (link)). Orbitrap MS2 scans were acquired at 7500 resolution, with a 5e4 AGC, and 22ms maximum injection using HCD fragmentation with a normalized energy of 30%. Rapid linear ion trap MS2 scans were acquired using a 4e3 AGC, 250 ms maximum injection time, CID fragmentation set at 30%. Dynamic exclusion was set to 30 s and precursors with unknown charge or a charge state of 1 and ≥ 8 were excluded.
Waters A.M., Khatib T.O., Papke B., Goodwin C.M., Hobbs G.A., Diehl J.N., Yang R., Edwards A.C., Walsh K.H., Sulahian R., McFarland J.M., Kapner K.S., Gilbert T.S., Stalnecker C.A., Javaid S., Barkovskaya A., Grover K.R., Hibshman P.S., Blake D.R., Schaefer A., Nowak K.M., Klomp J.E., Hayes T.K., Kassner M., Tang N., Tanaseichuk O., Chen K., Zhou Y., Kalkat M., Herring L.E., Graves L.M., Penn L.Z., Yin H.H., Aguirre A.J., Hahn W.C., Cox A.D, & Der C.J. (2021). Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition. Cell reports, 35(13), 109291.
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7

Proteomics Analysis of Cardiac Remodeling

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All experiments on the Sham, MI, and CM+EC+SMC groups were performed with 3, 4, and 3 biological replicates, respectively. Proteins were extracted using MaSDeS [29 (link)], a mass spectrometry compatible surfactant, from 30–50 mg tissue by homogenization in HEPES buffer, followed by in-solution digestion. The protein samples were analyzed using a nanoACQUITY ultra high pressure liquid chromatography coupled to a Q Exactive (Thermo Scientific) mass spectrometer. Protein identification and quantification were performed using the SEQUEST-based Proteome Discoverer. All of the given protein intensities are presented in Log10 scale. After conversion, the intensity of each protein in the sample was divided by the median protein intensity for the entire sample for normalization. Protein changes between groups were considered significant if they: (1) passed the Kruskal-Wallis test (p < 0.05) and (2) had a greater than 1.3-fold change.
Chang Y.H., Ye L., Cai W., Lee Y., Guner H., Lee Y., Kamp T.J., Zhang J, & Ge Y. (2015). Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation. Proteomics, 15(15), 2560-2567.
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8

Shotgun Proteomics Workflow by HPLC-MS

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The digestion extracts were analyzed by reverse-phase high-performance liquid chromatography (HPLC) using Waters NanoAcquity pumps and autosampler and a ThermoFisher Orbitrap Elite mass spectrometer using a nano flow configuration. A 20 mm × 180 μm column packed with 5 μm Symmetry C18 material (Waters) using a flow rate of 15 μl per min for 3 min was used to trap and wash peptides. These were then eluted onto the analytical column which was self-packed with 3.6 μm Aeris C18 material (Phenomenex) in a fritted 20 cm × 75 μm fused silica tubing pulled to a 5 μm tip. Elution was carried out with a gradient of isocratic 1% Buffer A (1% formic acid in H2O) for 1 min (250 nL min−1), followed by increasing Buffer B (1% formic acid in acetonitrile) concentrations to 15% B at 20.5 min, 27% B at 31 min and 40% B at 36 min. The column was washed with high percent B and re-equilibrated between analytical runs for a total cycle time of approximately 53 min.
DeBenedictis E.A., Carver G.D., Chung C.Z., Söll D, & Badran A.H. (2021). Multiplex suppression of four quadruplet codons via tRNA directed evolution. Nature Communications, 12, 5706.
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