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Phos select iron affinity gel

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PHOS-Select iron affinity gel is a lab equipment product designed for the purification and enrichment of phosphorylated proteins and peptides. It utilizes iron-based affinity chromatography to selectively capture phosphorylated biomolecules from complex samples.

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17 protocols using phos select iron affinity gel

1

Phosphoproteomic Analysis of Drought Stress

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Frozen leaf tissue (100 mg) of the tetraploid plants with or without drought stress was used for phosphoproteomic analyses. Three parallel replicates were set up for the control group (TC) or drought stress group (TD). Phosphopeptides were enriched using immobilized metal-ion affinity chromatography (IMAC) resin (PHOS-Select™ Iron Affinity Gel, Sigma-Aldrich) (Bigeard et al., 2014 (link)). Proteomic analysis, including protein preparation, trypsin digestion, tandem mass tag (TMT) labeling, and liquid chromatography (LC)–mass spectrometry (MS)/mass spectrometry (MS) analysis was performed according to the detailed description of Jiang et al. (2021) (link). The entire phosphoproteomic analysis was done at PTM BIO Company (Hangzhou, China).
Jiang J., Yang N., Li L., Qin G., Ren K., Wang H., Deng J, & Ding D. (2022). Tetraploidy in Citrus wilsonii Enhances Drought Tolerance via Synergistic Regulation of Photosynthesis, Phosphorylation, and Hormonal Changes. Frontiers in Plant Science, 13, 875011.
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2

Phosphopeptide Enrichment for Mass Spectrometry

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SCX was performed on 10 mg of digested protein sample as described56 (link),57 (link). Phosphopeptides were enriched from SCX fractions using TiO2 beads as described56 (link),57 (link). For enrichment of phosphopeptides by immobilized metal affinity chromatography (IMAC), ten SCX fractions were dried and resuspended in 300 μl of 250 mM acetic acid, 30% acetonitrile (ACN) (v/v). Peptides were gently mixed with 80 μl of Phos-Select iron affinity gel (Sigma-Aldrich P9740) and incubated for 1.5 h using a tube rotator58 (link). The mixture was transferred into SigmaPrep spin columns and washed twice with 200 μl of 250 mM acetic acid, 30% ACN (v/v), then once with 200 μl distilled water. Bound phosphopeptides were eluted with 100 μl 400 mM NH4OH, 30% ACN by centrifugation (1 min at 8,200 × g). Flow-through and elution fractions were dried almost completely (5–6 μl) under vacuum and stored at −20 °C until LC-MS/MS analysis.
Huang C.Y., Gonzalez-Lopez C., Henry C., Mijakovic I, & Ryan K.R. (2020). hipBA toxin-antitoxin systems mediate persistence in Caulobacter crescentus. Scientific Reports, 10, 2865.
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3

Enrichment of Phosphorylated Peptides via PHOS-Select Affinity Chromatography

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PHOS-Select iron affinity gel (Sigma) was equilibrated with 5 × 1 ml IMAC load/wash buffer [0.25 M acetic acid, 30% (v/v) acetonitrile]. 500 µl IMAC load/wash buffer was added to completely dry samples and 50 µl of 50% PHOS-Select slurry was added followed by rotating at room temperature for 1 h. Following incubation with IMAC beads, the samples were transferred onto Mobicol ‘Classic’ spin columns (2B Scientific Ltd), centrifuged for 30 s at 1000×g, and the flow-through (unbound peptides) collected and frozen. The IMAC beads were washed twice with 200 µl of IMAC load/wash buffer, once with 200 µl of HPLC grade water and eluted twice with 100 µl of solution containing 22 µl ammonia solution (Fisher Chemical, 35% stock), 300 µl acetonitrile to a total volume of 1 ml with HPLC grade water. Eluates were concentrated to a small volume (15–20 µl) in a Speedvac centrifuge and submitted for mass spectrometry analysis.
Mitcheson D.F., Bottrill A.R., Carr K., Coxon C.R., Cano C., Golding B.T., Griffin R.J., Fry A.M., Doerig C., Bayliss R, & Tobin A.B. (2016). A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase. Malaria Journal, 15, 535.
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4

IMAC Phosphopeptide Enrichment Protocol

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IMAC phosphopeptide enrichment was performed according to the published protocol (in-tube batch version) with the following minor modifications (Villen and Gygi, 2008 (link)). 20 μl of a 50% IMAC bead slurry composed of 1/3 commercial PHOS-select iron affinity gel (Sigma Aldrich, St Louis, MO), 1/3 in-house made Fe3+-NTA Superflow agarose and 1/3 in-house made Ga3+-NTA Superflow agarose was used for phosphopeptide enrichment (Ficarro et al., 2009 (link)). The IMAC slurry was washed three times with 10 bed volumes of 80% aq. ACN containing 0.1% TFA and phosphopeptide enrichment was performed in the same buffer.
Golkowski M., Lau H.T., Chan M., Kenerson H., Vidadala V.N., Shoemaker A., Maly D.J., Yeung R.S., Gujral T.S, & Ong S.E. (2020). Pharmacoproteomics identifies kinase pathways that drive the epithelial-mesenchymal transition and drug resistance in hepatocellular carcinoma. Cell systems, 11(2), 196-207.e7.
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5

Shotgun Proteomics of Enriched Phosphoproteins

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Samples were minced individually in liquid nitrogen. The enrichment was carried out using PHOS-Select iron affinity gel (Sigma, P9740) following the manufacturer's instructions. Shotgun proteomics analyses were performed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) operating in a data-dependent acquisition (DDA) mode. The resulting spectra from each fraction were searched separately against the UniProt database [25] . For protein identification, proteins with at least one unique peptide were identified at an FDR less than 1.0% at the peptide and protein level, respectively.
Yang M., Fan Y., Wu Z.Y., Gu J., Feng Z., Zhang Q., Han S., Zhang Z., Li X., Hsueh Y.C., Ni Y., Li X., Li J., Hu M., Li W., Gao H., Yang C., Zhang C., Zhang L., Zhu T., Cheng M., Ji F., Xu J., Cui H., Tan G., Zhang M.Q., Liang C., Liu Z., Song Y.Q., Niu G, & Wang K. (2021). DAGM: A novel modelling framework to assess the risk of HER2-negative breast cancer based on germline rare coding mutations. EBioMedicine, 69, 103446.
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6

Phosphopeptide Enrichment and Proteomics

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From each tissue homogenate, 2 mg of protein was used for protein digestion and for the subsequent enrichment of phosphopeptides. Samples were reduced with 5 mm dithiothreitol for 45 min at 37 °C and alkylated with 10 mm Iodoacetamide in the dark for 30 min at room temperature (RT) before digestion with 5 μg Lys‐C (Wako Chemicals, Richmond, VA, USA) for 2 h at RT. Samples were then diluted with 50 mm ammonium bicarbonate (NH4HCO3) to a final urea concentration of 1 m before digestion with trypsin (10 μg) over night for 37 °C. Of the resulting peptides, 70 μg was saved for full‐proteome analysis, while the remainder was used for phosphopeptide enrichment. Samples were enriched using either immobilized metal affinity chromatography (IMAC; n = 12 each of TN and NAT) or titanium dioxide (TiO2; n = 8 of TN and n = 10 of NAT). Samples for phospho‐proteomics were desalted on 50 mg Sep‐Pak Vac RP C18 cartridges (Waters, Milford, MA, USA). Phosphopeptides were enriched with PHOS‐Select Iron Affinity Gel (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions following the protocol described in Soderholm et al. [29 (link)] or using TiO2 beads as described in Casado et al. [30 (link)].
Amrutkar M., Verbeke C.S., Finstadsveen A.V., Dorg L., Labori K.J, & Gladhaug I.P. (2022). Neoadjuvant chemotherapy is associated with an altered metabolic profile and increased cancer stemness in patients with pancreatic ductal adenocarcinoma. Molecular Oncology, 17(1), 59-81.
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7

Phosphoproteome analysis of Arabidopsis seedlings

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Arabidopsis seedlings grown on MS agar plates in standard 16/8 h and 21/17°C day/night conditions were transferred to liquid MS media supplemented with 10 μM NAA (Sigma-Aldrich). Total protein extracts were precipitated with 0.1 M ammonium acetate in 100% methanol, reduced, alkylated and digested overnight with trypsin (Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate. Resulting peptides were vacuum-dried and re-suspended in 250 mM acetic acid with 30% acetonitrile for phosphopeptide enrichment with Phos-Select Iron Affinity Gel (Sigma-Aldrich) according to the protocol from Thingholm et al. (2008) (link). Eluted phosphopeptides were desalted and analyzed by nano LC-MS/MS on a TripleTOF 5600 (Sciex, Canada) coupled a NanoLC-2DPlus system with nanoFlex ChiP module (Eksigent, Sciex).
Dobrenel T., Mancera-Martínez E., Forzani C., Azzopardi M., Davanture M., Moreau M., Schepetilnikov M., Chicher J., Langella O., Zivy M., Robaglia C., Ryabova L.A., Hanson J, & Meyer C. (2016). The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6. Frontiers in Plant Science, 7, 1611.
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8

IMAC Phosphopeptide Enrichment Protocol

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IMAC phosphopeptide enrichment was performed according to the published protocol (in-tube batch version) with the following minor modifications (Villén and Gygi, 2008 ). 20 μl of a 50% IMAC bead slurry composed of 1/3 commercial PHOS-select iron affinity gel (Sigma Aldrich, St Louis, MO), 1/3 in-house made Fe3+-NTA Superflow agarose and 1/3 in-house made Ga3+-NTA Superflow agarose was used for phosphopeptide enrichment (Ficarro et al., 2009 (link)). The IMAC slurry was washed three times with 10 bed volumes of 80% aq. ACN containing 0.1% TFA and phosphopeptide enrichment was performed in the same buffer.
Bello T., Chan M., Golkowski M., Xue A.G., Khasnavis N., Ceribelli M., Ong S.E., Thomas C.J, & Gujral T.S. (2021). KiRNet: Kinase-centered network propagation of pharmacological screen results. Cell Reports Methods, 1(2), 100007.
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9

Phosphopeptide Enrichment and Identification

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Phosphopeptides in each fraction were enriched using IMAC resin (Sigma; PHOS-Select™ Iron Affinity Gel). Briefly, the tryptic peptides from each fraction were redissolved in IMAC binding buffer. IMAC resin (20 μL slurry/mg of digested protein) was washed twice with binding buffer. Phosphopeptides were incubated with the IMAC resin for 30 minutes at room temperature, with gentle agitation every 5 minutes. After incubation, the IMAC resin was washed twice with buffer 1 (0.1% acetic acid, 100 mM NaCl, pH 3.0), followed by 2 washes with buffer 2 (30% acetonitrile, 0.1% acetic acid, pH 3.0), and 1 wash with H2O. Phosphopeptides were eluted with 20% aqueous acetonitrile containing 200 mM ammonium hydroxide and concentrated by vacuum centrifugation. Concentrated peptides were diluted with 15 μL of 2% aqueous acetonitrile with 0.1% formic acid. LC-MS/MS and statistical analyses for MS data are described in Supplementary Materials and Methods.
Kim J.Y., Welsh E.A., Fang B., Bai Y., Kinose F., Eschrich S.A., Koomen J.M, & Haura E.B. (2016). Phosphoproteomics reveals MAPK inhibitors enhance MET- and EGFR-driven AKT signaling in KRAS-mutant lung cancer. Molecular cancer research : MCR, 14(10), 1019-1029.
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10

Proteomic Analysis of Mouse MA10 Leydig Tumor Cells

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Mouse MA10 Leydig tumor cells were a kind gift of Dr. Mario Ascoli (University of Iowa). Rolipram was purchased from Tocris Bioscience (Bristol, UK). PF-04957325 was a kind gift of Pfizer Inc. (Groton, CT). Isotope labeled amino acids for SILAC were obtained from Cambridge Isotope Labs (Andover, MA) or Sigma-Isotec (St Louis, MO). Custom RPMI medium (−Lys/−Arg) was obtained from Caisson Labs (North Logan, UT). Dialyzed FBS and PHOS-Select Iron affinity gel were from Sigma (St. Louis, MO). C18 reverse phase material for nano-LC columns (3 µm Reprosil-C18.aq) was obtained from Dr. Maisch (Ammerbuch, DE). Protease and phosphatase inhibitor cocktails as well as the Pierce 660 nm protein assay were purchased from Thermo Scientific (Rockford, IL). Sequencing grade endoproteinase LysC and Trypsin were obtained from Promega (Madison, WI). Oasis C18 cartridges for peptide extraction were from Waters (Milford, MA).
Golkowski M., Shimizu-Albergine M., Suh H.W., Beavo J.A, & Ong S.E. (2015). Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics. Cellular signalling, 28(7), 764-778.
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