Picofrit column

Manufactured by New Objective

Sourced in United States, Germany

The PicoFrit column is a laboratory equipment used for chromatographic separations. It is designed for high-performance liquid chromatography (HPLC) applications. The PicoFrit column features a fused silica capillary with an integrated spray tip for direct electrospray ionization (ESI) mass spectrometry (MS) detection.

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Lab products found in correlation

Ltq orbitrap xl, by Thermo Fisher Scientific (8 mentions) Sequencing grade modified trypsin, by Promega (7 mentions) Acquity m class nanolc system, by Waters Corporation (4 mentions) Magic c18aq resin, by Bruker (4 mentions) Q exactive plus mass spectrometer, by Thermo Fisher Scientific (4 mentions) Tempo nanolc system, by AB Sciex (4 mentions) Ltq orbitrap velos, by Thermo Fisher Scientific (3 mentions) Easy nlc 1200, by Thermo Fisher Scientific (3 mentions) Nanospray flex ion source es071, by Thermo Fisher Scientific (3 mentions) Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (3 mentions) Tripletof 6600 quadru pole time of flight mass spectrometer, by AB Sciex (3 mentions) As 1 autosampler, by AB Sciex (3 mentions) Qstar elite hybrid quadrupole time of flight mass spectrometer, by AB Sciex (3 mentions) Nanoease symmetry c18 trapping column, by Waters Corporation (2 mentions) Q exactive plus, by Thermo Fisher Scientific (2 mentions) C18 aq resin, by Dr. Maisch (2 mentions) Anti flag m2 affinity gel, by Merck Group (2 mentions) Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions) Nanoease symmetry c18 trapping column, by New Objective (2 mentions) Easy nlc 1000 hplc, by Thermo Fisher Scientific (2 mentions)

50 protocols using picofrit column

Nanoflow LC-MS/MS Proteomics Pipeline

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An Acquity M-class nanoLC system (Waters, Milford, MA, USA) was used, loading 5 µL of the sample (1 mg) at a rate of 15 mL/min for 3 min onto a nanoEase Symmetry C18 trapping column (180 mm × 20 mm). It was then washed onto a PicoFrit column (75 mm ID × 250 mm; New Objective, Woburn, MA, USA) packed with Magic C18AQ resin (Michrom Bioresources, Auburn, CA, USA). The column was then eluted of peptides into the Q Exactive Plus mass spectrometer (Thermofisher Scientific, NSW, Australia) using the following program: 5%–30% MS buffer B (98% acetonitrile + 0.2% formic acid) for 90 min, 30%–80% MS buffer B for 3 min, 80% MS buffer B for 2 min, 80%–5% for 3 min. The peptides that were eluted were ionised at 2000 V. A data-dependent MS/MS (dd-MS2) experiment was performed with a 350–1500 Da survey scan performed at a resolution of 70,000 m/z for peptides of charge state 2+ or higher with an automatic gain control (AGC) target of 3 × 106 and a 50 ms maximum injection time. The top 12 peptides were selectively fragmented in the higher-energy collisional dissociation (HCD) cell using a 1.4 m/z isolation window, an AGC target of 1 × 105 and a 100 ms maximum injection time. The fragments were scanned in the Orbitrap analyser at a resolution of 17,500 and the product ion fragment masses were measured over a 120–2000 Da mass range. The mass of the precursor peptide was then excluded for 30 s.

Fajardo J., Milthorpe B.K, & Santos J. (2020). Molecular Mechanisms Involved in Neural Substructure Development during Phosphodiesterase Inhibitor Treatment of Mesenchymal Stem Cells. International Journal of Molecular Sciences, 21(14), 4867.

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Quantitative Proteomics by UPLC-MS/MS

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LC-MS/MS was performed using an ACQUITY UPLC M-Class (Waters)
paired with a Q Exactive Plus (Thermo). Peptides were separated on a
65-cm-long, 75-µm-internal-diameter PicoFrit column (New
Objective) packed in-house to a length of 50 cm with 1.9 µm
ReproSil-Pur 120 Å C18-AQ (Dr. Maisch) using methanol as the
packing solvent. Peptide separation was achieved using a non-linear
90-min gradient from 1% ACN 0.1% formic acid to 90% ACN 0.1% formic acid
with a flowrate of 250 nl/min. Approximately 3–4 µg of
peptides were run for each of the six biological replicates with at
least one blank run between samples to avoid peptide carryover.

Rosa-Mercado N.A., Zimmer J.T., Apostolidi M., Rinehart J., Simon M.D, & Steitz J.A. (2021). Hyperosmotic stress alters the RNA Polymerase II interactome and induces readthrough transcription despite widespread transcriptional repression. Molecular cell, 81(3), 502-513.e4.

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HPLC-MS/MS Phosphopeptide Enrichment

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Samples were separated using a NanoAquity (waters) HPLC with a vented split on a 30mm 150 μM ID trap column with a Kasil frit packed with Magic C18AQ 3 mM 200Å resin (Bruker) and a 20cm 75 μM ID self-packed Pico-frit column (New Objective) packed with 1.9 μM 120Å reprosil-Pur C18-AQ resin (Dr. Maisch) on a non-linear 120min gradient from 5% ACN 0.1% formic acid to 95% ACN 0.1% formic acid and analyzed with a LTQ Orbitrap Velos (Thermo) using a Top 10 method. ERLIC samples were run in duplicate, with the first ERLIC fraction of each set of 3 run separately, while the 2^nd and 3^rd fraction of each set were pooled for a single run. In addition, for each sample 2 μg of the pre-enriched peptides and 2 μg of the total (unfractionated) enriched phosphopeptides were run using a non-linear 200min gradient from 5% ACN 0.1% formic acid to 95% ACN 0.1% formic acid.

Gassaway B.M., Cardone R.L., Padyana A.K., Petersen M.C., Judd E.T., Hayes S., Tong S., Barber K.W., Apostolidi M., Abulizi A., Sheetz J.B., K.s.h.i.t.i.z., Aerni H.R., Gross S., Kung C., Samuel V.T., Shulman G.I., Kibbey R.G, & Rinehart J. (2019). Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production. Cell reports, 29(11), 3394-3404.e9.

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Peptide Separation and Mass Spectrometry

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The protein bands were reduced, alkylated and digested with trypsin overnight at 37°C. An aliquot of the peptide mixture was separated using a 2-40% acetonitrile gradient in 0.1% formic acid using an analytical PicoFrit Column (20 cm x ID75 µm; 5-µm particle size; New Objective, Inc.), at a flow rate of 300 nl/min over 35 min. Peptides were analysed using the EASY-nLC II (Proxeon Biosystems) coupled to LTQ Orbitrap Velos (Thermo Fisher Scientific, Inc.), with electrospray ionization in positive mode and set up in the data-dependent acquisition mode. Full scan MS spectra (m/z 300-1,600) were acquired in the Orbitrap analyzer after accumulation to a target value of 1×10⁶. Resolution in the Orbitrap was set to r=60,000, and the 20 most intense peptide ions with charge states ≥2 were sequentially isolated to a target value of 5,000 and fragmented in the linear ion trap by low-energy collision-induced dissociation (normalized collision energy of 35%). Three independent experiments were performed.

Morelli A.P., Tortelli TC J.r., Pavan I.C., Silva F.R., Granato D.C., Peruca G.F., Pauletti B.A., Domingues R.R., Bezerra R.M., De Moura L.P., Leme A.F., Chammas R, & Simabuco F.M. (2021). Metformin impairs cisplatin resistance effects in A549 lung cancer cells through mTOR signaling and other metabolic pathways. International Journal of Oncology, 58(6), 28.

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LC-MS/MS Analysis of Trichinella Proteome

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LC-MS/MS analysis was performed at the Proteomics Platform of the Quebec Genomics Centre (Quebec City, QC, Canada). Proteins were solubilized in 25 µL 50 mM NH₄HCO₃, 1% sodium deoxycholate, 0.2 mM DTT, and 0.9 mM iodoacetamide, and digested with sequencing-grade trypsin (Promega, Madison, WI) for 16 h at 37 °C. Peptides were concentrated using a Stage tip (C18), vacuum-dried, and resuspended in 5 µL 0.1% formic acid, then resolved by reverse-phase (RP) on self-packed PicoFrit column (New Objective, Woburn, MA) packed with Jupiter (5.0 u, 300 Å C₁₈, 15 cm × 0.075 mm internal diameter) (Phenomenex, Torrance, CA) with a linear gradient from 2 to 50% Solvent B (ACN, 0.1% formic acid) over 90 min, at 300 nL/min. Full survey spectra were collected (400 to 2,000 m/z) and analyzed on a 5600 mass spectrometer using Analyst (version 1.6) (AB Sciex, Framingham, MA), and the seven most intense ions were submitted to fragmentation. Spectra were searched against a database of predicted tryptic peptides derived from the predicted protein sequences translated from the T. suis genome annotation using MASCOT version 2.3.02 (Matrix Science, London, UK). MS data was analyzed with Scaffold (version 4.0.1, Proteome Software Inc., Portland, OR) with a peptide confidence of 95.0% with a minimum of 1 peptide in each sample for a given life-stage.

Leroux L.P., Nasr M., Valanparambil R., Tam M., Rosa B.A., Siciliani E., Hill D.E., Zarlenga D.S., Jaramillo M., Weinstock J.V., Geary T.G., Stevenson M.M., Urban JF J.r., Mitreva M, & Jardim A. (2018). Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties. Scientific Reports, 8, 15921.

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TRIP13 Protein Purification and Identification

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UM-SCC-1-FLAG-TRIP13 cells were used for TRIP13 protein purification by anti-Flag-M2 affinity gel (Sigma, #A2220) and Flag-tagged protein was eluted with flag peptide. Lanes corresponding to control and TRIP13-flag were cut into 15 slices and destained with 30% methanol for 4hrs. Upon reduction (10mM DTT) and alkylation (65mM 2-chloroacetamide) of the cysteines, proteins were digested overnight with sequencing grade modified trypsin (Promega, Madison, WI). Resulting peptides were resolved on a nano-capillary reverse phase column (Picofrit Column, New Objective, Woburn, MA) using 1% acetic acid/acetonitrile gradient at 300nl/min and directly introduced into a linear ion-trap mass spectrometer (LTQ Orbitrap XL, ThermoFisher). Data-dependent MS/MS spectra on the five most intense ions from each full MS scan were collected (relative CE ~35%). Proteins were identified by searching the data against UniProt Human database appended with decoy (reverse) sequences using X!Tandem/Trans-Proteomic Pipeline (TPP) software suite. All peptides and proteins with a peotideProphet and ProteinProphet probability score of >0.9 (fdr<2%) were considered positive identifications. Proteins found in the control lane were subtracted using in-house subtraction program and the remaining were considered as potential interactors.

Banerjee R., Russo N., Liu M., Basrur V., Bellile E., Palanisamy N., Scanlon C.S., van Tubergen E., Inglehart R.C., Metwally T., Mani R.S., Yocum A., Nyati M.K., Castilho R.M., Varambally S., Chinnaiyan A.M, & D’Silva N.J. (2014). TRIP13 promotes error-prone nonhomologous end joining and induces chemoresistance in head and neck cancer. Nature communications, 5, 4527.

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Optimized Proteomic Workflow with DDA and DIA

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For DDA and DIA, 2 μg of each sample were separated using a self-packed analytical PicoFrit column (75 μm × 50 cm length) (New Objective, Woburn, MA) packed with ReproSil-Pur 120A C18-AQ 1.9 μm (Dr. Maisch GmbH, Ammerbuch, Germany) with a 2-h segmented gradient using an EASY-nLC 1200 (Thermo Scientific). The datasets were acquired in a block randomized manner. The Spike-in-biol-var-OT and the MP-LFC-MS1var-OT were acquired on a Q Exactive HF mass spectrometer (Thermo Scientific) with methods modified from (21 (link)). The BiolDS-OT dataset was acquired on a Q Exactive HF-X mass spectrometer. The DIA method contained 43 DIA segments of 30,000 resolution with injection time set to auto and automatic gain control of 3*10⁶ and a survey scan of 120,000 resolution with 60ms max injection time and automatic gain control of 3*10⁶. The mass range was set to 350–1650 m/z. The default charge state was set to 3. Loop count 1 and normalized collision energy stepped at 25.5, 27, and 30. For the dataset with varying MS1 resolutions, the number of DIA segments was adapted to maintain a constant method cycle time. For the acquisition of the fractionated sample for the library, a DDA method was applied. The TOP15 method was modified from (24 (link)) (MS-Methods.xlsx).

Huang T., Bruderer R., Muntel J., Xuan Y., Vitek O, & Reiter L. (2019). Combining Precursor and Fragment Information for Improved Detection of Differential Abundance in Data Independent Acquisition. Molecular & Cellular Proteomics : MCP, 19(2), 421-430.

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Reversed Phase Peptide Separation

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Reversed phase separation of peptides was performed using a Surveyor liquid chromatography system (Thermo Scientific, Waltham, MA) as previously described ^{27 (link)}. Briefly, peptides were loaded onto desalting peptide trap (Michrom Bioresources, Auburn, CA) using an autosampler (Thermo Scientific). All MS analyses were performed using an LCQ Deca mass spectrometer (Thermo Scientific) equipped with a nanospray ionization source. Peptides were introduced into the mass spectrometer via a 75 μm ID/15 μm tip ID C18-packed PicoFrit®column (New Objective, Woburn, MA). The spray voltage was 2.0 kV and the heated capillary temperature was 200 °C. MS data was acquired using a top 3 data-dependent acquisition method with dynamic exclusion enabled. MS spectra was searched against a human database (downloaded on Nov. 29, 2007 from NCBI; 88,334 sequences) by using Sorcerer-SEQUEST (SageN Research, Milpitas, CA). The quality of peptide and protein assignments was assessed using PeptideProphet and ProteinProphet. Proteins with probabilities of ≥ 0.9 and ≥ 2 unique peptides were accepted as confidently identified peptides.

Yu L., Shen J., Mannoor K., Guarnera M, & Jiang F. (2014). Identification of ENO1 as a potential sputum biomarker for early stage lung cancer by shotgun proteomics. Clinical lung cancer, 15(5), 372-378.e1.

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Peptide Analysis by LC-MS/MS

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The LC/MS/MS analyses of samples were carried out using a Thermo Scientific Q-Exactive hybrid Quadrupole-Orbitrap Mass Spectrometer and Thermo Dionex UltiMate 3000 RSLCnano System (Poolchon Scientific, Frederick, MD, USA). Peptide mixtures from each sample were loaded onto a peptide trap cartridge at a flow rate of 5 μL/min. The trapped peptides were eluted onto a reversed-phase PicoFrit column (New Objective, Woburn, MA) using a linear gradient of acetonitrile (3–36%) in 0.1% formic acid. The elution duration was 120 min at a flow rate of 0.3 μl/min. Eluted peptides from the PicoFrit column were ionized and sprayed into the mass spectrometer, using a Nanospray Flex Ion Source ES071 (Thermo) under the following settings: spray voltage, 1.6 kV, capillary temperature, 250 °C.

Saddala M.S., Lennikov A., Grab D.J., Liu G.S., Tang S, & Huang H. (2018). Proteomics reveals ablation of PlGF increases antioxidant and neuroprotective proteins in the diabetic mouse retina. Scientific Reports, 8, 16728.

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Nanoflow LC-MS/MS Proteomics Workflow

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Using an Acquity M-class nanoLC system (Waters, Milford, MA, USA), 5 —L of the sample (1 —g) was loaded at 15 —L/min for 3 min onto a nanoEase Symmetry C18 trapping column (180 —m x 20 mm) before being washed onto a PicoFrit column (75 —m ID x 250 mm; New Objective, Woburn, MA, USA) packed with Magic C18AQ resin (Michrom Bioresources, Auburn, CA, USA). Peptides were eluted from the column and into the source of a Q Exactive™ Plus mass spectrometer (Thermo Scientific, Rockford, IL, USA) using the following program: 5–30% MS buffer B (98% acetonitrile + 0.2% formic acid) over 90 min, 30–80% MS buffer B over 3 min, 80% MS buffer B for 2 min, 80–5% for 3 min. The eluting peptides were ionized at 2000V. A Data Dependent MS/MS (dd-MS²) experiment was performed, with a survey scan of 350–1500 Da performed at 70,000 resolution for peptides of charge state 2+ or higher with an AGC target of 3e6 and maximum Injection Time of 50 ms. The Top 12 peptides were selectively fragmented in the HCD cell using an isolation window of 1.4 m/z, an AGC target of 1e5 and maximum injection time of 100 ms. Fragments were scanned in the Orbitrap analyzer (Thermo Scientific, Rockford, IL, USA) at 17,500 resolution and the product ion fragment masses measured over a mass range of 120–2000 Da. The mass of the precursor peptide was then excluded for 30 s.

Roediger B., Lee Q., Tikoo S., Cobbin J.C., Henderson J.M., Jormakka M., O’Rourke M.B., Padula M.P., Pinello N., Henry M., Wynne M., Santagostino S.F., Brayton C.F., Rasmussen L., Lisowski L., Tay S.S., Harris D.C., Bertram J.F., Dowling J.P., Bertolino P., Lai J.H., Wu W., Bachovchin W.W., Wong J.J., Gorrell M.D., Shaban B., Holmes E.C., Jolly C.J., Monette S, & Weninger W. (2018). An atypical parvovirus driving chronic tubulointerstitial nephropathy and kidney fibrosis. Cell, 175(2), 530-543.e24.

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