Easy spray analytical column

Manufactured by Thermo Fisher Scientific

The EASY-Spray analytical column is designed for high-performance liquid chromatography (HPLC) applications. It features a packed bed of porous particles that facilitates the separation and analysis of complex samples. The column is engineered to provide efficient and reproducible chromatographic separations.

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Easy Spray PepMap C18 analytical column Acclaim PepMap EASY-Spray analytical column Easy spray C18 analytical column EASY-SprayTM C18 LC Analytical Column EASY-Spray column EASY-Spray Analytical columns Easy Column

11 protocols using easy spray analytical column

Peptide Fractionation and Mass Spectrometry

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Peptide sample fractions were dried and resuspended in 1: 99 acetonitrile: water containing 0.5% (v/v) acetic acid and 0.06% TFA. 2 μg peptides per fraction were injected in Dionex UltiMate 3000 UPLC system coupled to a Q Exactive HF mass spectrometer (ThermoFisher Scientific). Separation was performed on a 50 cm × 75  μm EASY-Spray analytical column (ThermoFisher Scientific) with a pre-programmed gradient by mixing solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) over 70 min. MS data was acquired using a top 12 data-dependent acquisition protocol at 60,000 resolution in MS1 and 45,000 resolution in MS2.

Sun W., Dai L., Yu H., Puspita B., Zhao T., Li F., Tan J.L., Lim Y.T., Chen M.W., Sobota R.M., Tenen D.G., Prabhu N, & Nordlund P. (2019). Monitoring structural modulation of redox-sensitive proteins in cells with MS-CETSA. Redox Biology, 24, 101168.

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Targeted Proteomics for Biomarker Discovery

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Each sample was resuspended in 100 μl injection buffer. An equal volume of SIS mixture was added to the sample for relative quantitation. All samples were subjected to analysis on an Easy-nLC 1000 system coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). The samples were grouped into injection blocks that covered all conditions of comparison (time points, strains, genders). Each block containing 20 samples was injected in a randomized order and analyzed in targeted-MS² mode with retention time scheduling (4 min window). Triplicate injections of 8 biological replicates were analyzed over 480 runs. An E.coli digest standard (1 μg/μl) was analyzed at the start and end of each block to ensure stability of the LC-MS/MS system.
A 4-μl injection was loaded onto a 75 μm i.d. × 25 cm EASY-Spray analytical column (Thermo Fisher Scientific). Peptides were eluted in a 50 min gradient of mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) at a flowrate of 300 nl/min: 5% B at 0 min, 29% B at 32 min, and 80% B from 40.5–50 min. The spray voltage was 2 kV and the capillary temperature was 250 °C. The mass spectrometer was operated in a targeted-MS2 acquisition mode with a maximum IT of 130 ms, 1 microscan, 35 000 resolution, 2E5 AGC target, 1.6 m/z isolation window, and 27% normalized collision energy.

Liu K., Singer E., Cohn W., Micewicz E.D., McBride W.H., Whitelegge J.P, & Loo J.A. (2019). Time-Dependent Measurement of Nrf2-Regulated Antioxidant Response to Ionizing Radiation towards Identifying Potential Protein Biomarkers for Acute Radiation Injury. Proteomics. Clinical applications, 13(6), e1900035.

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Quantitative proteomic analysis by LC-MS/MS

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LC-MS/MS analysis was performed using a QExactive HF mass spectrometer (ThermoFisher Scientific) coupled to an EASY-nLC 1000 system (ThermoFisher Scientific). Peptides were separated on a 75 μm by 50 cm EASY-Spray analytical column (Thermo Fisher Scientific) at 50 °C. The mass spectrometer was set to acquire in a data-dependent mode (Top10). Full scans were acquired at 60,000 resolution, with a target of 3 × 10⁶ ions with a maximum injection time of 20 ms. The top intense ions were fragmented by MS² scans were fragmented by HCD (NCE 33%) and acquired at 60,000 resolution, with a target of 1 × 10⁶ ions and a maximum injection time of 60 ms. MaxQuant version 1.5.3.30 was used to process MS data. The false discovery rate (FDR) was set at <0.01, enzyme was set to trypsin, and missed cleavages was set at <2. The Human UniProt FASTA database (March 2015) was used for peptide identification, with cysteine carbamidomethylation as a fixed modification and N-acetylation and oxidation of methionine as variable modifications. TMT quantification was performed by MaxQuant (Max Planck Institute of Biochemistry), using correction factors supplied with TMT reagents, reporter mass tolerance set to 0.01 Da, and a parent ion fraction (PIF) filter at 0.75.

Connor-Robson N., Booth H., Martin J.G., Gao B., Li K., Doig N., Vowles J., Browne C., Klinger L., Juhasz P., Klein C., Cowley S.A., Bolam P., Hirst W, & Wade-Martins R. (2019). An integrated transcriptomics and proteomics analysis reveals functional endocytic dysregulation caused by mutations in LRRK2. Neurobiology of Disease, 127, 512-526.

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Quantitative Proteomics by UPLC-MS/MS

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Online chromatography was performed using Dionex UltiMate 3000 UPLC system coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Inc.). Each sample was separated on a 50 cm ×75 µm EASY-Spray analytical column (Thermo Fisher Scientific, Inc.) using a 120-min gradient of a programmed mixture of solvents A (0.1% formic acid in water) and B (95% ACN and 5% water with 0.1% formic acid). MS data were acquired using a Top 12 data-dependent acquisition method. Full Scan MS spectra were acquired at 300-1,600 m/z at a resolution of 70,000 and AGC target of 3e6; Top12 ddMS2 35,000 and 1e5 with an isolation window of 1.6 m/z.
Proteome Discoverer 2.1 software (Thermo Fisher Scientific, Inc.) was used to analyze the Xcalibur^® raw files for subsequent protein identification and quantification. Both Mascot 2.6.0 (Matrix Science, Inc.) and Sequest HT (Thermo Fisher Scientific, Inc.) search engines were used to search against the human-reviewed UniProt database. MS precursor mass tolerance was set at 20 ppm, fragment mass tolerance at 0.05 Da and maximum missed cleavage sites at 3. Only the spectrum peaks with a signal-to-noise ratio of >4 were chosen for searches. The false discovery rate was set to 1% at both the peptide-spectrum match (PSM) and peptide levels. The Mascot score threshold for PSM was set at 10.

Yang C., Zhang Y., Segar N., Huang C., Zeng P., Tan X., Mao L., Chen Z., Haglund F., Larsson O., Chen Z, & Lin Y. (2021). Nuclear IGF1R interacts with NuMA and regulates 53BP1-dependent DNA double-strand break repair in colorectal cancer. Oncology Reports, 46(2), 168.

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Peptide Characterization by LC-MS/MS

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The dried peptide sample fractions were resuspended in 1% acetonitrile, 0.5% (v/v) acetic acid, and 0.06% TFA in water prior to analysis on LC/MS. Online chromatography was performed using the reverse phase liquid chromatography Dionex 3000 UHPLC system coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Each fraction was separated on a 50 cm x 75 µm Easy-Spray analytical column (Thermo Fisher Scientific) in 70 minutes gradient of programmed mixture of mobile phase A (0.1% formic acid) and mobile phase B (99.9% acetonitrile, 0.1% formic acid) to the following gradient ober time: 1-55 min (2-25%), 55-57 min (25-50%), 57-58 min (50-85%), 58-63 min (85%), 63-70 min (2%). MS data were acquired using data-dependent acquisition (DDA) with full scan MS spectra acquired in the range of 350-1550 m/z at a resolution of 70,000 and AGC target of 3e6; Top12 MS 2 (link) 35,000 and AGC target of 1e5, and 1e5 isolation window at 1.2 m/z.

Wirjanata G., Lin J., Dziekan J.M., El Sahili A., Chung Z., Tjia S., Binte Zulkifli N.E., Boentoro J., Tham R., Jia L.S., Go K.D., Yu H., Partridge A., Olsen D., Prabhu N., Sobota R.M., Nordlund P., Lescar J, & Bozdech Z. (2024). Identification of an inhibitory pocket in falcilysin provides a new avenue for malaria drug development. Cell chemical biology, 31(4).

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Optimized Proteomics Workflow for EV Analysis

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LC–MS/MS experiments were performed using an Easy-nLC 1000 coupled to an Orbitrap Fusion mass spectrometer (both Thermo Fisher Scientific, Bremen, Germany). A detailed description of the nLC-MS/MS methods is available in the Supplementary Information (Supplementary Methods S4). Briefly, the peptide digest was diluted 1:1 with 10% formic acid prior to injection. The peptides were separated using an EASY-Spray analytical column (ES803: 75 µm × 50 cm, C18, 2 µm, 100 Å; Thermo Scientific) using a flow rate of 300 nl/min. A 145-min LC gradient (1 μg of peptide digest injected) was used for the comparison of PPT-EV and SEC-EV purification methods and a 75-min gradient (0.5 μg of peptide digest injected) was used for all the SEC-EV proteolysis optimizations and the SEC-EV GBM longitudinal study. The Orbitrap Fusion was operated in data dependent Top Speed mode, with a 3 s cycle-time. MS1 scans were performed in the Orbitrap (375 to 1500 m/z at 120 K resolution), ions with charge states from 2 + to 7 + and intensity greater than 5e3 were selected for HCD fragmentation and MS2 scans were acquired in the ion trap using a 1.6 m/z isolation window.

Anastasi F., Greco F., Dilillo M., Vannini E., Cappello V., Baroncelli L., Costa M., Gemmi M., Caleo M, & McDonnell L.A. (2020). Proteomics analysis of serum small extracellular vesicles for the longitudinal study of a glioblastoma multiforme mouse model. Scientific Reports, 10, 20498.

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Peptide Characterization by LC-MS

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The dried peptide sample fractions were resuspended in 1% acetonitrile, 0.5% (v/v) acetic acid, and 0.06% TFA in water prior to analysis on LC/MS. Online chromatography was performed using the reverse phase liquid chromatography Dionex 3000 UHPLC system coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Each fraction was separated on a 50 cm × 75 μm Easy-Spray analytical column (Thermo Fisher Scientific) in 70 min gradient of programmed mixture of mobile phase A (0.1% formic acid) and mobile phase B (99.9% acetonitrile, 0.1% formic acid) to the following gradient over time: 1 to 55 min (2–25%), 55 to 57 min (25–50%), 57 to 58 min (50–85%), 58 to 63 min (85%), 63 to 70 min (2%). MS data were acquired using data-dependent acquisition with full scan MS spectra acquired in the range of 350 to 1550 m/z at a resolution of 70,000 and AGC target of 3e6; Top12 MS2 35,000 and AGC target of 1e5, and 1e5 isolation window at 1.2 m/z.

Chung Z., Lin J., Wirjanata G., Dziekan J.M., El Sahili A., Preiser P.R., Bozdech Z, & Lescar J. (2023). Identification and structural validation of purine nucleoside phosphorylase from Plasmodium falciparum as a target of MMV000848. The Journal of Biological Chemistry, 300(1), 105586.

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Label-Free DIA Mass Spectrometry

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All samples (label-free for DIA
analysis, or fractioned pools for TMT experiment or DDA library generation)
were solubilized in 2% MeCN with 0.1% TFA to 0.2 μg/μL
concentration before being injected in volumes equivalent to 1 μg
on an UltiMate 3000 RSLC nano System (Thermo Fisher Scientific). Peptides
were trapped for 5 min in A (0.1% FA in water) at a flow rate of 10
μL/min on a PepMap 100 C18 LC trap column (300 μm ID ×
5 mm, 5 μm, 100 Å) and then separated using an EASY-Spray
analytical column (50 cm × 75 μm ID, PepMap C18, 2 μm,
100 Å) (Thermo Fisher Scientific) flowing at 250 nL/min. The
column oven temperature was set at 45 °C. All peptides were separated
using an identical linear gradient of 3–35% B (80% MeCN containing
0.1% FA) over 120 min.

George A.L., Sidgwick F.R., Watt J.E., Martin M.P., Trost M., Marín-Rubio J.L, & Dueñas M.E. (2023). Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling. Journal of Proteome Research, 22(8), 2629-2640.

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Proteomic Profiling of Salivary Glands in IgG4-Related Sialadenitis

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The SMG tissues from three IgG4-RS patients and three age-matched controls were detected by proteomics analysis. The tissue processing was performed as described in our previous study.^{43 (link)} Briefly, we homogenized the SMG tissues on ice and used 100 μg of homogenates per sample for proteomics screening. After lysis, the concentration of protein was quantified by the Bradford assay. Then, the samples were digested overnight in trypsin at 37 °C and labeled with iTRAQ reagents according to the manufacturer’s instructions (AB Sciex). The labeled samples were analyzed using an EASY-Spray analytical column (120 mm × 75 μm, 3 μm) on an EASY-nLC1000 connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific) and liquid chromatography–tandem mass spectrometry.
Analysis on the DEPs between IgG4-RS and control tissues was conducted with the edgeR package in R software. Data were standardized by log2 conversion. Specific screening conditions were followed by |fold change| >1.3 and P < 0.05. To dig into the potential function of these DEPs in SMGs, we precisely used the weighted enrichment analysis tools (WEAT) for the annotation of weighted gene function and pathway analysis through selecting “salivary gland” gene essentially score according to the instructions (http://www.cuilab.cn/weat/).^{44 (link)} GO-BP analysis were further used to assess the weighted DEPs.

Zhu M., Min S., Mao X., Zhou Y., Zhang Y., Li W., Li L., Wu L., Cong X, & Yu G. (2022). Interleukin-13 promotes cellular senescence through inducing mitochondrial dysfunction in IgG4-related sialadenitis. International Journal of Oral Science, 14, 29.

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Quantitative Analysis of Tau Peptides

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20 μL of sample was loaded onto a PepMap 300 C18 HPLC Column (300 um x 5 cm, 5 μm, Thermo) using an Ultimate 3000 UHPLC autosampler at a flow rate of 5 μl/min. Mobile phase consisted of 0.1% Formic Acid in water (Solvent A) and 0.1% Formic Acid in acetonitrile (Solvent B). Analyte was loaded on stationary phase for 3 minutes with 2% B, followed by a mobile phase gradient from 2–40% B over 70 minutes to elute surrogate peptides from a C18 stationary phase EASY-spray analytical column (75 um ID, 15 cm, Thermo) at a flow rate of 0.3 ul/min at 45 ⁰C. The analytical column was washed with 70% B for five minutes and re-equilibrated with 2% B for 15 minutes.
Mass spectrometry analysis was performed on a Q Exactive Quadrupole Orbitrap instrument (Thermo) operating in positive electrospray ionization mode. A parallel reaction monitoring (PRM) method with 17,500 resolution (m/z = 200), 0.7 m/z mass tolerance, automatic gain control of 2e5, and HCD fragmentation was used to analyze 5 surrogate tau peptides and their co-eluting N15-labeled internal standards with an error of < 5 ppm (S2 Table).

Self W., Awwad K., Savaryn J.P, & Schulz M. (2022). An immuno-enrichment free, validated quantification of tau protein in human CSF by LC-MS/MS. PLoS ONE, 17(6), e0269157.

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