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Aminooxy biotin

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Aminooxy-biotin is a bifunctional reagent that contains both an aminooxy group and a biotin group. The aminooxy group can be used for the chemoselective ligation of aldehydes and ketones, while the biotin group enables the detection and purification of the labeled molecules.

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Lab products found in correlation

Trypsin, by Promega (4 mentions) Pngase, by New England Biolabs (2 mentions) Ltq orbitrap xl, by Thermo Fisher Scientific (2 mentions) Nanoacquity uplc, by Waters Corporation (2 mentions) Aniline, by Merck Group (2 mentions) Sodium meta periodate, by Thermo Fisher Scientific (2 mentions) Iodoacetamide iaa, by Merck Group (2 mentions) Formic acid, by Thermo Fisher Scientific (1 mentions) G7 buffer, by New England Biolabs (1 mentions) High capacity streptavidin beads, by Thermo Fisher Scientific (1 mentions) Optima lc ms grade acetonitrile, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions) Pngase f, by New England Biolabs (1 mentions) Sodium periodate, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Sep pak, by Waters Corporation (1 mentions) Iodoacetamide, by Merck Group (1 mentions) Avasimibe, by Merck Group (1 mentions) Sodium metaperiodate, by Merck Group (1 mentions) Aniline hydrochloride, by Merck Group (1 mentions)

9 protocols using aminooxy biotin

1

Glycoproteome Enrichment and Analysis

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Fetal bovine serum was obtained from Atlanta Biologicals (Lawrenceville, GA). Tris-HCl, PBS, aniline, iodoacetamide, dithiothreitol (DTT), urea, protease inhibitor cocktail solution, sodium(meta)periodate, Triton X-100, SDS, NH4HCO3, and glycerol were purchased from Sigma-Aldrch (St. Louis, MO). NaCl and trifluoroacetic acid was purchased from EMD chemicals (Gibbstown, NJ). Trypsin was obtained from Promega (Madison, WI). PNGaseF and G7 buffer were purchased from New England Biolabs (Ipswich, MA). High capacity streptavidin beads were obtained from Thermo Scientific (Rockford, IL). Amino-oxy-biotin was purchased from Biotium (Hayward, CA). Optima LC/MS grade acetonitrile and formic acid were purchased from Fisher Scientific. Pierce C18 spin columns were purchased from Glygen Corp (Columbia, MD). Fluorescently labeled monoclonal antibodies to murine Gr1 (clone RB6-8C5), CD11b (clone M170), CD47 (clone miap301), and isotype-matched controls were from BioLegend (San Diego, CA). Deionized water used for all experiments was obtained from a Milli-Q A10 system.
Chauhan S., Danielson S., Clements V., Edwards N., Ostrand-Rosenberg S, & Fenselau C. (2016). Surface Glycoproteins of Exosomes Shed by Myeloid-Derived Suppressor Cells Contribute to Function. Journal of proteome research, 16(1), 238-246.
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2

Cell Surface Glycoproteome Enrichment

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PMP was performed as described previously14 (link). Briefly, surface sialic acid residues were oxidised, biotinylated with aminooxy-biotin (Biotium), and biotinylated cells incubated in a 1% Triton X-100 lysis buffer. Biotinylated glycoproteins were enriched with high affinity streptavidin agarose beads (Pierce) and washed extensively. Captured protein was reduced and alkylated then digested with trypsin on-bead overnight. Tryptic peptides were collected and fractionated. Glycopeptides were eluted using PNGase (New England Biolabs).
High pH reverse-phase high pressure liquid chromatography (HpRP-HPLC) was performed on tryptic peptides as described previously14 (link). LC-MSMS was performed using a NanoAcquity uPLC (Waters, MA, USA) coupled to an LTQ-OrbiTrap XL (Thermo, FL, UA). Raw MS files were processed using MaxQuant version 1.3.0.553 (link). Reversed decoy databases were used and the false discovery rate for both peptides and proteins were set at 0.01. Protein quantitation utilised razor and unique peptides and required a minimum of 2 ratio counts, with normalised protein ratios reported. Significance B values were calculated. We assessed the number of PM proteins identified as described previously14 (link).
Hoare M., Ito Y., Kang T.W., Weekes M.P., Matheson N.J., Patten D.A., Shetty S., Parry A.J., Menon S., Salama R., Antrobus R., Tomimatsu K., Howat W., Lehner P.J., Zender L, & Narita M. (2016). NOTCH1 mediates a switch between two distinct secretomes during senescence. Nature cell biology, 18(9), 979-992.
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3

Sialic Acid Metabolism Profiling

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hCMEC/D3 cells were trypsinized, washed in PBS, and treated with NanA, NanC, or buffer for 1 h in a 37 °C water bath. After washing in PBS containing 0.1 % (w/v) BSA, cells were incubated in 1 mM sodium periodate (Sigma) in PBS on ice for 30 min at 10 × 106 cells mL−1. Oxidation was quenched by addition of an equal volume of 1 mM glycerol before washing with PBS. Cells were then incubated in PBS, pH 6.7, containing 5 % (w/v) BSA, 250 μM aminooxy-biotin (Biotium), and 10 mM aniline (Sigma) to perform the oxime ligation. Incubation occurred for 90 minutes at 4 °C with constant rotation. For immunoblot analysis, cells were washed in PBS containing 0.1 % BSA, lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 % (v/v) NP-40, 0.5 % (w/v) sodium deoxycholate, 0.1 % (w/v) sodium dodecyl sulfate (SDS), and protease inhibitor cocktail (Roche)), run on a 10 % SDS-PAGE gel, and transferred overnight at 4 °C, 87 mA to a PVDF membrane.
McCombs J.E, & Kohler J.J. (2016). Pneumococcal neuraminidase substrates identified through comparative proteomics enabled by chemoselective labeling. Bioconjugate chemistry, 27(4), 1013-1022.
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4

Cell Surface Glycoproteome Enrichment

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PMP was performed as described previously14 (link). Briefly, surface sialic acid residues were oxidised, biotinylated with aminooxy-biotin (Biotium), and biotinylated cells incubated in a 1% Triton X-100 lysis buffer. Biotinylated glycoproteins were enriched with high affinity streptavidin agarose beads (Pierce) and washed extensively. Captured protein was reduced and alkylated then digested with trypsin on-bead overnight. Tryptic peptides were collected and fractionated. Glycopeptides were eluted using PNGase (New England Biolabs).
High pH reverse-phase high pressure liquid chromatography (HpRP-HPLC) was performed on tryptic peptides as described previously14 (link). LC-MSMS was performed using a NanoAcquity uPLC (Waters, MA, USA) coupled to an LTQ-OrbiTrap XL (Thermo, FL, UA). Raw MS files were processed using MaxQuant version 1.3.0.553 (link). Reversed decoy databases were used and the false discovery rate for both peptides and proteins were set at 0.01. Protein quantitation utilised razor and unique peptides and required a minimum of 2 ratio counts, with normalised protein ratios reported. Significance B values were calculated. We assessed the number of PM proteins identified as described previously14 (link).
Hoare M., Ito Y., Kang T.W., Weekes M.P., Matheson N.J., Patten D.A., Shetty S., Parry A.J., Menon S., Salama R., Antrobus R., Tomimatsu K., Howat W., Lehner P.J., Zender L, & Narita M. (2016). NOTCH1 mediates a switch between two distinct secretomes during senescence. Nature cell biology, 18(9), 979-992.
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5

Plasma Membrane Profiling of Enriched Erythrocytes

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We have described plasma membrane profiling previously10 (link),26 (link), and details of the method used is recapitulated here with modifications for this study. For ~1.5 × 109 of each enriched erythrocyte sample, surface sialic acid residues were oxidized with sodium meta-periodate (Thermo) then biotinylated with aminooxy-biotin (Biotium). After quenching, cells were incubated in 1% (v/v) Triton X-100 lysis buffer (10 mM Tris HCl, 1.6% Triton, 150 mM NaCl). Biotinylated glycoproteins were enriched with high affinity streptavidin agarose beads (Pierce) and washed extensively. Captured protein was denatured with dithiothreitol (DTT), alkylated with iodoacetamide (Sigma) and digested on-bead with trypsin (Promega) in 200 mM HEPES pH 8.5 for 3 h. Tryptic peptides were collected and labelled using TMT reagents. The reaction was quenched with hydroxylamine, and TMT-labelled samples combined in a 1:1:1:1:1:1:1:1:1:1 ratio. Labelled peptides were subjected to C18 solid-phase extraction (Sep-Pak, Waters) and vacuum-centrifuged to near-dryness.
Ravenhill B.J., Kanjee U., Ahouidi A., Nobre L., Williamson J., Goldberg J.M., Antrobus R., Dieye T., Duraisingh M.T, & Weekes M.P. (2019). Quantitative comparative analysis of human erythrocyte surface proteins between individuals from two genetically distinct populations. Communications Biology, 2, 350.
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6

Enrichment and Analysis of Cell Surface Proteins

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Cell surface proteins were enriched and analyzed by mass spectrometry as previously described.14 (link) Briefly, live cells were surface protein labeled with biotin through oxidation of terminal sialic acids on glycans with aminooxy-biotin (Biotium, USA). Cells were then lysed and streptavidin pulldown was performed using streptavidin agarose beads (Pierce, USA). On-bead digestion with trypsin (Promega) was performed and peptides were analyzed using an Impact II mass spectrometer for data acquisition and MaxQuant (ver 1.5.8.3) for data analysis.
Wang S.S., Davenport A.J., Iliopoulos M., Hughes-Parry H.E., Watson K.A., Arcucci V., Mulazzani M., Eisenstat D.D., Hansford J.R., Cross R.S, & Jenkins M.R. (2023). HER2 chimeric antigen receptor T cell immunotherapy is an effective treatment for diffuse intrinsic pontine glioma. Neuro-Oncology Advances, 5(1), vdad024.
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7

Plasma Membrane Profiling of Adherent Cells

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Plasma membrane profiling was performed as described previously, with minor modifications for adherent cells (Weekes et al., 2010; Weekes et al., 2012 ). Briefly, one 150cm2 flask of HCMV-infected HFFFs per condition was washed twice with ice-cold PBS. Sialic acid residues were oxidized with sodium meta-periodate (Thermo) then biotinylated with aminooxy-biotin (Biotium). The reaction was quenched, and the biotinylated cells scraped into 1% Triton X-100 lysis buffer. Biotinylated glycoproteins were enriched with high-affinity streptavidin agarose beads (Pierce) and washed extensively. Captured protein was denatured with DTT, alkylated with iodoacetamide (IAA, Sigma) and digested on-bead with trypsin (Promega) in 100 mM HEPES pH 8.5 for 3h. Tryptic peptides were collected. Ting et al. (2011) (link) suggest that LysC digestion is preferable for quantitative proteomic analysis using TMT, because it guarantees labeling of both N-termini and C-terminal lysine side chains on virtually every peptide (Ting et al., 2011 (link)). In practice TMT labeling is compatible with analysis of even tryptic peptides that may only bear TMT labels on their N terminus.
Weekes M.P., Tomasec P., Huttlin E.L., Fielding C.A., Nusinow D., Stanton R.J., Wang E.C., Aicheler R., Murrell I., Wilkinson G.W., Lehner P.J, & Gygi S.P. (2014). Quantitative Temporal Viromics: An Approach to Investigate Host-Pathogen Interaction. Cell, 157(6), 1460-1472.
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8

Cholesterol Homeostasis Modulation Assay

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The following compounds were used: 25-hydroxycholesterol (25HC, Sigma-Aldrich, St. Louis, MO), DAPI (Sigma-Aldrich), aminooxybiotin (Biotium, Fremont, CA), aniline hydrochloride (Sigma-Aldrich), avasimibe (Sigma-Aldrich), cholesterol (Sigma-Aldrich), doxycycline (Sigma-Aldrich), filipin III (Santa Cruz Biotechnology, Dallas, TX), gel filtration markers kit (MWGF1000, Sigma-Aldrich), GGTI 298 trifluoroacetate salt hydrate (Sigma-Aldrich), Hygromycin B Gold (InvivoGen, San Diego, CA), IAA (iodoacetamide, Sigma-Aldrich), lipoprotein-depleted fetal bovine serum (LPDS, Kalen Biomedical, Germantown, MD), LMNG (lauryl maltose neopentyl glycol, Anatrace, Maumee, OH), methyl-β-cyclodextrin (MBCD, Sigma-Aldrich), MG132 (Merck Chemicals Ltd, Darmstadt, Germany), N-ethylmaleamide (NEM, Thermo Fisher Scientific), propidium iodide (Sigma-Aldrich), puromycin (Invivogen), SILAC Lys-8-Arg-10 kit (282986444, Silantes GmbH, Munich, Germany), sodium-meta-periodate (Sigma-Aldrich) and ZA (zaragozic acid A, Santa Cruz Biotechnology).
Volkmar N., Thezenas M.L., Louie S.M., Juszkiewicz S., Nomura D.K., Hegde R.S., Kessler B.M, & Christianson J.C. (2019). The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis. Journal of Cell Science, 132(2), jcs223453.
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9

Sialylation analysis of hCMEC/D3 cells

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hCMEC/D3 cells were trypsinized, washed in PBS, and treated with NanA, NanC, or buffer for 1 h in a 37 °C water bath. Cells were then washed in PBS containing 0.1 % (w/v) BSA. Oxidation and oxime ligation was performed by incubating cells in PBS, pH 6.7, containing 5 % (w/v) BSA, 50 U mL−1 galactose oxidase from Dactylium dendoides (Sigma), 250 μM aminooxy-biotin (Biotium), and 10 mM aniline (Sigma) for 30 min in a 37 °C water bath at 10 × 106 cells mL−1. For immunoblot analysis, cells were washed in PBS containing 0.1 % BSA, lysed in RIPA buffer, run on a 10 % SDS-PAGE gel, and transferred overnight at 4 °C, 87 mA to a PVDF membrane.
McCombs J.E, & Kohler J.J. (2016). Pneumococcal neuraminidase substrates identified through comparative proteomics enabled by chemoselective labeling. Bioconjugate chemistry, 27(4), 1013-1022.
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