Reprosil pur c18 aq beads

Manufactured by Dr. Maisch

Sourced in Germany

Reprosil-Pur C18-AQ beads are a type of chromatographic material used for liquid chromatography. They are silica-based beads modified with C18 alkyl chains and a polar group, providing both reversed-phase and hydrophilic interaction chromatography (HILIC) selectivity.

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ReproSil-Pur C18-AQ (72 citations) ReproSil-Pur C18 beads (32 citations) C18 beads (23 citations) Reprosil-Pur C18 (21 citations) ReproSil-Pur C18-AQ 1.9 μm beads (7 citations) ReproSil-Pur 120 C18-AQ 2.4 μm beads (5 citations) ReproSil-Pur 120 C18-AQ 1.9 μm beads (4 citations) Reprosil-AQ Pur (3 citations) ReproSil-Pur C18-AQ 3 µm beads (2 citations) C18- Reprosil-AQ Pur reversed-phase beads (2 citations)

21 protocols using reprosil pur c18 aq beads

DIA-based Quantitative Proteomics of ccRCC Samples

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Unlabeled, digested peptide material from individual tissue samples (ccRCC and NAT) was spiked with index Retention Time (iRT) peptides (Biognosys) and subjected to data-independent acquisition (DIA) analysis. Peptides (~0.8 μg) were separated on an Easy nLC 1200 UHPLC system (Thermo Scientific) on an in-house packed 20 cm x 75 μm diameter C18 column (1.9 μm Reprosil-Pur C18-AQ beads (Dr. Maisch GmbH); Picofrit 10 μm opening (New Objective)). The column was heated to 50°C using a column heater (Phoenix-ST). The flow rate was 0.200 μl/min with 0.1% formic acid and 3% acetonitrile in water (A) and 0.1% formic acid, 90% acetonitrile (B). The peptides were separated with a 7%–30% B gradient in 84 mins and analyzed using the Thermo Fusion Lumos mass spectrometer (Thermo Scientific). The DIA segment consisted of one MS1 scan (350-1650 m/z range, 120K resolution) followed by 30 MS2 scans (variable m/z range, 30K resolution). Additional parameters were as follows: MS1: RF Lens – 30%, AGC Target 4.0e⁵, Max IT – 50 ms, charge state include - 2-6; MS2: isolation width (m/z) – 0.7, AGC Target - 2.0e⁵, Max IT – 120 ms.

Clark D.J., Dhanasekaran S.M., Petralia F., Pan J., Song X., Hu Y., da Veiga Leprevost F., Reva B., Lih T.S., Chang H.Y., Ma W., Huang C., Ricketts C.J., Chen L., Krek A., Li Y., Rykunov D., Li Q.K., Chen L.S., Ozbek U., Vasaikar S., Wu Y., Yoo S., Chowdhury S., Wyczalkowski M.A., Ji J., Schnaubelt M., Kong A., Sethuraman S., Avtonomov D.M., Ao M., Colaprico A., Cao S., Cho K.C., Kalayci S., Ma S., Liu W., Ruggles K., Calinawan A., Gümüş Z.H., Geiszler D., Kawaler E., Teo G.C., Wen B., Zhang Y., Keegan S., Li K., Chen F., Edwards N., Pierorazio P.M., Chen X.S., Pavlovich C.P., Hakimi A.A., Brominski G., Hsieh J.J., Antczak A., Omelchenko T., Lubinski J., Wiznerowicz M., Linehan W.M., Kinsinger C.R., Thiagarajan M., Boja E.S., Mesri M., Hiltke T., Robles A.I., Rodriguez H., Qian J., Fenyö D., Zhang B., Ding L., Schadt E., Chinnaiyan A.M., Zhang Z., Omenn G.S., Cieslik M., Chan D.W., Nesvizhskii A.I., Wang P, & Zhang H. (2019). Integrated Proteogenomic Characterization of Clear Cell Renal Cell Carcinoma. Cell, 179(4), 964-983.e31.

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Comprehensive Proteomic Profiling Workflow

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Global proteome and phosphoproteome fractions were analyzed using the same instrumentation and methodology. Peptides (~0.8 μg) were separated on an Easy nLC 1200 UHPLC system (Thermo Scientific) on an in-house packed 20 cm x 75 mm diameter C18 column (1.9 mm Reprosil-Pur C18-AQ beads (Dr. Maisch GmbH); Picofrit 10 mm opening (New Objective)). The column was heated to 50°C using a column heater (Phoenix-ST). The flow rate was 0.200 μl/min with 0.1% formic acid and 2% acetonitrile in water (A) and 0.1% formic acid, 90% acetonitrile (B). The peptides were separated with a 6%–30% B gradient in 84 min and analyzed using the Thermo Fusion Lumos mass spectrometer (Thermo Scientific). Parameters were as follows: MS1: resolution – 60,000, mass range – 350 to 1800 m/z, RF Lens – 30%, AGC Target 4.0e⁵, Max IT – 50 ms, charge state include - 2-6, dynamic exclusion – 45 s, top 20 ions selected for MS2; MS2: resolution-50,000, high-energy collision dissociation activation energy (HCD)-37, isolation width (m/z) – 0.7, AGC Target – 2.0e⁵, Max IT – 105 ms.

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Orbitrap-based Quantitative Proteomics and Glycoproteomics

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For data acquisition, peptide samples were analyzed using an Orbitrap Lumos Fusion system. Peptide (1 μg) was separated using an EASY-nLC 1200 UHPLC system (Thermo Scientific) on an in-house packed 20 cm × 75 μm diameter C18 column (1.9 μm ReproSil-Pur C18-AQ beads (Dr. Maisch GmbH); Picofrit 10 μm opening (New Objective)). The column was heated to 50 °C using a column heater (Phoenix-ST). The flow rate was 0.300 μL/min with 0.1% formic acid and 2% acetonitrile in water (A) and 0.1% formic acid and 90% acetonitrile (B). The peptides were separated with a 6–30% B gradient in 84 min and analyzed using a Thermo Fusion Lumos mass spectrometer (Thermo Scientific). Parameters were as follows: MS1: resolution, 60,000; mass range, 350–800 m/z, RF lens, 30%, AGC target, 4.0 × 10⁵; Max IT, 50 ms; charge state, 2–6; dynamic exclusion, 45 s; top 20 ions selected for MS2; MS2: resolution, 50,000; high-energy collision dissociation activation energy (HCD), 34 (global peptide) or 36 (intact glycopeptide); isolation width (m/z), 0.7; AGC target, 2.0 × 10⁵; Max IT, 105 ms. Three replicates were analyzed for the proteomic and glycoproteomic analyses.

Clark D.J., Schnaubelt M., Hoti N., Hu Y., Zhou Y., Gooya M, & Zhang H. (2020). Impact of Increased FUT8 Expression on the Extracellular Vesicle Proteome in Prostate Cancer Cells.. Journal of proteome research, 19(6), 2195-2205.

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Nanoscale Proteome Separation and Analysis

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Online separation was done with a nanoflow Proxeon EASY-nLC 1200 UHPLC system (Thermo Fisher Scientific). In this set up, the LC system, column, and platinum wire used to deliver electrospray source voltage were connected via a stainless-steel cross (360 μm, IDEX Health & Science, UH-906x). The column was heated to 50°C using a column heater sleeve (Phoenix-ST) to prevent over-pressuring of columns during UHPLC separation. Each peptide fraction containing ~1ug (based on protein-level BCA prior to digestion, with uniform distribution of fraction content presumed), the equivalent of 12% of each global proteome sample in a 2 ul injection volume or 50% of each phosphoproteome sample in a 4 ul injection volume, was injected onto an in-house packed 20cm x 75um diameter C18 silica picofrit capillary column (1.9 μm ReproSil-Pur C18-AQ beads, Dr. Maisch GmbH, r119.aq; Picofrit 10um tip opening, New Objective, PF360–75-10-N-5). Mobile phase flow rate was 200 nL/min, comprising 3% acetonitrile/0.1% formic acid (Solvent A) and 90% acetonitrile/0.1% formic acid (Solvent B). The 110-minute LC-MS/MS method consisted of a 10-min column-equilibration procedure, a 20-min sample-loading procedure, and the following gradient profile: (min:%B) 0:2; 1:6; 85:30; 94:60; 95;90; 100:90; 101:50; 110:50 (the last two steps at 500 nL/min flow rate).

Krug K., Jaehnig E.J., Satpathy S., Blumenberg L., Karpova A., Anurag M., Miles G., Mertins P., Geffen Y., Tang L.C., Heiman D.I., Cao S., Maruvka Y.E., Lei J.T., Huang C., Kothadia R.B., Colaprico A., Birger C., Wang J., Dou Y., Wen B., Shi Z., Liao Y., Wiznerowicz M., Wyczalkowski M.A., Chen X.S., Kennedy J.J., Paulovich A.G., Thiagarajan M., Kinsinger C.R., Hiltke T., Boja E.S., Mesri M., Robles A.I., Rodriguez H., Westbrook T.F., Ding L., Getz G., Clauser K.R., Fenyö D., Ruggles K.V., Zhang B., Mani D.R., Carr S.A., Ellis M.J, & Gillette M.A. (2020). Proteogenomic Landscape of Breast Cancer Tumorigenesis and Targeted Therapy. Cell, 183(5), 1436-1456.e31.

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Peptide Separation and Mass Spectrometry

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One µg of peptide was separated using Easy nLC 1200 UHPLC system (Thermo Scientific) on an in-house packed 20 cm × 75 μm diameter C18 column (1.9 μm Reprosil-Pur C18-AQ beads (Dr. Maisch GmbH); Picofrit 10 μm opening (New Objective)). The column was heated to 50 °C using a column heater (Phoenix-ST). The flow rate was 0.200 μl/min with 0.1% formic acid and 2% acetonitrile in water (A) and 0.1% formic acid, 90% acetonitrile (B). The peptides were separated with a 6-30% B gradient in 84 min and analyzed using the Thermo Fusion Lumos mass spectrometer (Thermo Scientific). Parameters were as followed MS1: resolution – 60,000, mass range – 350 to 1800 m/z, RF Lens – 30%, AGC Target 4.0e⁵, Max IT – 50 ms, charge state include - 2-6, dynamic exclusion – 45 s, top 20 ions selected for MS2; MS2: resolution – 15,000, high-energy collision dissociation activation energy (HCD) – 34 (nonTMT-labeled samples) or 37 (TMT-labeled samples), isolation width (m/z) – 0.7, AGC Target – 5.0e⁴.

Theodros D., Murter B.M., Sidhom J.W., Nirschl T.R., Clark D.J., Chen L., Tam A.J., Blosser R.L., Schwen Z.R., Johnson M.H., Pierorazio P.M., Zhang H., Ganguly S., Pardoll D.M, & Zarif J.C. (2020). High-dimensional Cytometry (ExCYT) and Mass Spectrometry of Myeloid Infiltrate in Clinically Localized Clear Cell Renal Cell Carcinoma Identifies Novel Potential Myeloid Targets for Immunotherapy. Molecular & Cellular Proteomics : MCP, 19(11), 1850-1859.

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Proteomic Analysis of Cellular Signaling

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Control and TAP-CrkL samples were separated by SDS-PAGE and visualized by silver staining (LC6070; Invitrogen). Each lane was cut into 17 equal individual slices without regard to the staining pattern. The samples were destained, reduced with 20 mM dithiothreitol (DTT), and alkylated with 50 mM iodoacetamide. The samples were then digested overnight with 0.1 μg trypsin per gel slice. Tryptic peptides were extracted, dried under a vacuum, and then resuspended in 12 μl 0.1% formic acid. Eight microliters of each sample was loaded onto a 75-μm by 12-cm column self-packed with 3 μm ReproSil-Pur C₁₈-AQ beads (Dr. Maisch GmbH), eluted with a gradient of 2 to 40% acetonitrile in 0.1% formic acid over 50 min at 300 nl/min, and analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific). Proteins were identified using the Andromeda search engine (MaxQuant version 1.2.2.5) with cysteine carbamidomethylation specified as a fixed modification and methionine oxidation as a variable modification to search the Swiss-Prot database. Relative quantities of the proteins were determined using the iBAQ feature of MaxQuant. The entire data set is included as Data Set S1 in the supplemental material, and the raw data are hosted at ftp://massive.ucsd.edu/MSV000079311/.

Hallock P.T., Chin S., Blais S., Neubert T.A, & Glass D.J. (2016). Sorbs1 and -2 Interact with CrkL and Are Required for Acetylcholine Receptor Cluster Formation. Molecular and Cellular Biology, 36(2), 262-270.

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Optimized DDA Proteomic Workflow

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All fractions were resuspended in 20 µl buffer A (2% acetonitrile, 0.1% formic acid) and peptide concentration was measured using the 280 nm NanoDrop assay. One microgram of total peptide was analysed in DDA mode on an Agilent 1260 HPLC coupled to a TripleTOF 5600+ mass spectrometer equipped with NanoSource III. Each fraction was spiked with 0.1 µl of iRT calibration mix (Biognosys, AG) and loaded onto a 0.3×5 mm ZORBAX C18 (Agilent Technologies) trap column. Peptides were separated on a 75 µm×15 cm analytical column packed with Reprosil Pur C18AQ beads, 3 µm, 120 Å (Dr. Maisch, GmbH) with a manually pulled integrated spraying tip. A linear gradient of 2-40% buffer B (98% acetonitrile, 0.1% formic acid) in 90 min and flow rate of 250 nl/min was used for peptide separation. All data were measured in positive mode. Full profile MS scans were acquired in the m/z mass range of 340-1500 with 250 ms filling time; MS/MS scans for the 20 most intense ions with charge state from 2+ to 5+ were acquired in m/z mass range of 280-1500 with 100 ms filling time. Dynamic exclusion of fragmented ions was set to 12 s.

Krasny L., Bland P., Burns J., Lima N.C., Harrison P.T., Pacini L., Elms M.L., Ning J., Martinez V.G., Yu Y.R., Acton S.E., Ho P.C., Calvo F., Swain A., Howard B.A., Natrajan R.C, & Huang P.H. (2020). A mouse SWATH-mass spectrometry reference spectral library enables deconvolution of species-specific proteomic alterations in human tumour xenografts. Disease Models & Mechanisms, 13(7), dmm044586.

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DIA Analysis of Tryptic Peptides and Glycopeptides

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Unlabeled tryptic peptides and glycopeptides were spiked with index retention time peptides (Biognosys) and subjected to DIA analysis. Approximately 1 ug of peptides was separated on an in-house packed 28 cm x 75 mm diameter C18 column (1.9 mm Reprosil-Pur C18-AQ beads (Dr Maisch GmbH); Picofrit 10 mm opening (New Objective)) lined up with an Easy nLC 1200 UHPLC system (Thermo Fisher Scientific). The column was heated to 50 °C using a column heater (Phoenix-ST). The flow rate was set at 200 nl/min. Buffer A and B were 3% ACN (0.1% FA) and 90% ACN (0.1% FA), respectively. The peptides were separated from 0% B to 30% B gradient in 121 min. Peptides were eluted from the column and nanosprayed directly into Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in a data-independent mode. The DIA segment consisted of one MS1 scan followed by 30 MS2 scans (i.e., 30 windows with overlaps). Additional parameters were as follows: MS1: m/z range – 350 to 1650, Resolution – 120K, RF Lens – 30%, AGC Target 1.0e6, Max IT – 60 ms, Cycle time – 4.62; MS2: m/z range – 300 to 1600, Resolution – 30K, AGC Target – 1.0e6, Max IT – 120 ms, Cycle time – 4.62.

Lih T.M., Cao L., Minoo P., Omenn G.S., Hruban R.H., Chan D.W., Bathe O.F, & Zhang H. (2023). Detection of Pancreatic Ductal Adenocarcinoma-Associated Proteins in Serum. Molecular & Cellular Proteomics : MCP, 23(1), 100687.

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DIA-based Tissue Proteome Profiling

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Unlabeled, digested peptide material from individual tissue samples (PDAC and NAT) was spiked with index Retention Time (iRT) peptides (Biognosys) and subjected to DIA analysis. Approximately 1 μg of peptides were separated on an in-house packed 28 cm x 75 mm diameter C18 column (1.9 mm Reprosil-Pur C18-AQ beads (Dr. Maisch GmbH); Picofrit 10 mm opening (New Objective)) lined up with an Easy nLC 1200 UHPLC system (Thermo Scientific). The column was heated to 50°C using a column heater (Phoenix-ST). The flow rate was set at 200 nl/min. Buffer A and B were 3% ACN (0.1% FA) and 90% ACN (0.1% FA), respectively. The peptides were separated with a 7–30% B gradient in 118 min. Peptides were eluted from the column and nanosprayed directly into Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). The mass spectrometer was operated in a data-independent mode. The DIA segment consisted of one MS1 scan (350-1650 m/z range, 120K resolution) followed by 30 MS2 scans (variable m/z range, 30K resolution). Additional parameters were as follows: MS1: RF Lens – 30%, AGC Target 3.0e6, Max IT – 60 ms, charge state include – 2-6; MS2: isolation width (m/z) – 0.7, AGC Target – 3.0e6, Max IT – 120 ms.

Cao L., Huang C., Zhou D.C., Hu Y., Lih T.M., Savage S.R., Krug K., Clark D.J., Schnaubelt M., Chen L., Leprevost F.D., Eguez R.V., Yang W., Pan J., Wen B., Dou Y., Jiang W., Liao Y., Shi Z., Terekhanova N.V., Cao S., Lu R.J., Li Y., Liu R., Zhu H., Ronning P., Wu Y., Wyczalkowski M.A., Easwaran H., Danilova L., Mer A.S., Yoo S., Wang J.M., Liu W., Haibe-Kains B., Thiagarajan M., Jewell S.D., Hostetter G., Newton C.J., Li Q.K., Roehrl M.H., Fenyö D., Wang P., Nesvizhskii A.I., Mani D.R., Omenn G.S., Boja E.S., Mesri M., Robles A.I., Rodriguez H., Bathe O.F., Chan D.W., Hruban R.H., Ding L., Zhang B, & Zhang H. (2021). Proteogenomic Characterization of Pancreatic Ductal Adenocarcinoma. Cell, 184(19), 5031-5052.e26.

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Optimized nanoLC-MS Peptide Separation

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The general nanoLC–MS setup was similar to that previously described^{66 (link)}, with minor modifications. A Q Exactive Plus mass spectrometer (Thermo Fisher) and an Easy nanoLC-1200 (Thermo Fisher) were used for both DDA and DIA experiments. For the chromatographic separation of peptides, 4 μg peptide digest was analysed at 50 °C (controlled by Sonation column oven) on a 50-cm in-house-packed fused-silica emitter microcolumn (75 μm inner diameter × 360 μm outer diameter SilicaTip PicoTip; New Objective) packed with 1.9-μm reverse-phase ReproSilPur C18-AQ beads (Dr Maisch). Peptides were separated by a 4-h linear gradient of 5–80% (80% acetonitrile, 0.1% formic acid) at a constant flow rate of 300 nl min⁻¹.

Baixauli F., Piletic K., Puleston D.J., Villa M., Field C.S., Flachsmann L.J., Quintana A., Rana N., Edwards-Hicks J., Matsushita M., Stanczak M.A., Grzes K.M., Kabat A.M., Fabri M., Caputa G., Kelly B., Corrado M., Musa Y., Duda K.J., Mittler G., O’Sullivan D., Sesaki H., Jenuwein T., Buescher J.M., Pearce E.J., Sanin D.E, & Pearce E.L. (2022). An LKB1–mitochondria axis controls TH17 effector function. Nature, 610(7932), 555-561.

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