Capture and Qualitative Analysis of the Activated Fc Receptor Complex from Live Cells

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

This unit describes the isolation of activated Fc receptor complexes from RAW 264.7 macrophages using live?cell affinity receptor chromatography (LARC). The Fc receptor complex is activated and captured by IgG?coated microbeads on the surface of live macrophages. After the cells are disrupted, the receptor complexes are isolated by washing and sucrose gradient ultracentrifugation. Soluble proteins associated with the receptor complex are then eluted from the beads using a stepwise series of salt buffers and aqueous acetonitrile. The eluted proteins and the residual insoluble proteins on the beads can then be digested with trypsin and subjected to liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC?ESI?MS/MS). Controls include IgG?coated beads incubated with crude cell lysates or growth medium and beads coated with oxidized LDL or bovine serum albumin. Using this method, proteins present in IgG?FcR complexes can be distinguished from those in control scavenger receptor complexes (oxLDL or BSA). Thus, LARC is capable of detecting specific members of IgG receptor supramolecular complexes.

Keywords: receptor complex; live cells; cognate ligand; ligand coated microbeads; live?cell affinity receptor chromatography; LC?ESI?MS/MS; SQL

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Introduction
Basic Protocol 1: Live‐Cell Affinity Receptor Chromatography (LARC): Capturing Activated Fc Receptor Complexes from the Surface of Live Raw 264.7 Macrophages
Support Protocol 1: Preparation of Control Microbeads
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Live‐Cell Affinity Receptor Chromatography (LARC): Capturing Activated Fc Receptor Complexes from the Surface of Live Raw 264.7 Macrophages

Materials

RAW 264.7 macrophages (ATCC no. TIB‐70)
DMEM or alpha MEM (Cellgro)
5% fetal calf serum (FCS; PAA Laboratories)
10% (v/v) suspension of 2‐µm polystyrene beads (∼2.185 × 10¹⁰ microbeads/ml; Bangs Laboratories Inc., cat. no. PS05N; store at 4°C)
0.5 mg/ml human IgG (see recipe )
1× PBS ( appendix 2E ), 4°C
60% (w/v) sucrose in 1× PBS (store up to 72 hr at 4°C)
Isotonic experimental medium (see recipe ), 4°C
Homogenization buffer (see recipe ), 4°C
Series of NaCl solutions (50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, and 1000 mM) in 1× PBS (store up to 1 month at 4°C)
50% (v/v) acetonitrile in PBS made with HPLC‐grade water, 4°C
2% (w/v) SDS ( appendix 2E )
Bovine serum albumin (BSA) standards
5% and 50% (v/v) acetonitrile in 50 mM Tris‐HCl, pH 8.85, containing 200 mM urea
1 µg/µl trypsin (Roche) in 1% (v/v) acetic acid (store up to 2 weeks at 4°C)
80% (v/v) chloroform in 50 mM Tris, pH 8.5, made with HPLC‐grade water, 4°C
10% (v/v) formic acid
Dithiothreitol (DTT)
Buffer A: 5% (v/v) acetonitrile, 5% (v/v) formic acid (store up to 1 month in a tightly sealed bottle)
Buffer B: 65% (v/v) acetonitrile, 5% (v/v) formic acid (store up to 1 month in a tightly sealed bottle)

75‐cm² culture flasks
Cell culture wheel rotator (“Ferris wheel”)
Ultracentrifuge with TLA110 rotor (or equivalent) and 1.7‐ml ultracentrifuge tubes
Cell scraper
15‐ml Falcon tubes
French Press, precooled on ice
Light microscope and microscope slides
Microcentrifuge tubes
Syringe
Rotovap (Labconco)
C18 ZipTips (Millipore)
Electrospray mass spectrometer with:
- 20‐µl sample loop
- C18 LC‐MS column (300‐µm i.d., 15 cm; Vydek)
- Agilent 1100 HPLC pump
- Thermo LCQ ion trap
- SEQUEST software

Additional reagents and equipment for dot blotting (e.g., unit 3.4 ) and SDS‐PAGE (units 10.1 & 10.5 )

NOTE: Prepare buffers, especially buffers that contain a carbon source such as sucrose, fresh the day before use and independently for each replicate of the experiment.

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Figures

Figure 19.22.1 Flowchart illustrating capture and analysis of activated Fc receptor complexes from live cells. At each step, representative samples are boiled in SDS‐PAGE buffer for protein assays and SDS‐PAGE to ensure sample quality. Replicate samples prepared in parallel (without boiling in SDS) are trypsin‐digested for 8 hr (in Tris, pH 8.85, 200 mM urea, 5% acetonitrile), then reduced and redigested, before the peptides are collected by ZipTip for LC‐ESI‐MS/MS. ACN, acetonitrile; CHL, chloroform.

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Figure 19.22.2 Protein content of individual sample fractions following LARC. Receptor complexes bound to ligand‐coated beads were purified by sucrose gradient ultracentrifugation and washed in 1× PBS (W). Beads were collected by centrifugation and resuspended in PBS (PBS), then proteins were eluted with a salt step gradient (50 to 1000 mM NaCl in 1× PBS) followed by 50% acetonitrile in PBS (ACN). Protein was quantified using the Dumbroff assay. Liquid samples were mixed 1:10 in 10× SDS‐PAGE sample buffer and solid samples were dissolved in 2× SDS‐PAGE sample buffer, then samples were boiled and spotted onto filter paper. Experimental samples (middle row) were analyzed along with controls for nonspecific binding (bottom) and a BSA standard curve (top). Additional labels: IgG, IgG‐opsonized beads presented to live cells; control, same IgG‐opsonized beads incubated with crude homogenates; beads, beads boiled in SDS; HB, homogenization buffer; H, HEPES buffer (blank); R, growth medium.

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Figure 19.22.3 Silver‐stained gels of elution fractions of a LARC experiment performed using RAW macrophages incubated with IgG‐coated beads. Panels A‐C: Proteins were extracted from the ligand‐coated beads through a salt step gradient followed by elution with an organic solvent (acetonitrile, ACN). Beads were then boiled in SDS to elute any remaining proteins. Controls were run alongside the experiment to account for nonspecific binding. Panel D: After cells were disrupted using a French press, beads were pelleted, and the supernatant (homogenization buffer) was collected for gel analysis. The washes before protein extraction using the salt step gradient were also analyzed. Protein profiles of the isotonic experimental media and growth media are also shown. Controls were run alongside the experiment to account for nonspecific binding. The sample labels are shown. See Figure for further details.

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Figure 19.22.4 LC‐MS/MS TIC traces of 150 mM NaCl extractions of a LARC experiment performed with RAW macrophages incubated with LARC‐coated microbeads alongside controls. (A ) IgG beads incubated with live cells; (B ) IgG beads incubated with crude extracts; (C ) IgG beads incubated with growth medium.

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Figure 19.22.5 Representative MS/MS spectra of proteins identified with IgG beads bound to live cells versus control beads. (A ) Cyclin‐dependent kinase 9 (18699998), LADFGLARAFSLAKNSQPNR, MH+2177.45, 3+. (B ) Catenin alpha‐like 1 (31542343), LGLLSSDADCEIEK, MH+ 1493.66, 3+. (C ) Phosphatidylinositol‐3‐phospatase‐associated protein (27370470), TVSVNEGYRVSDRLPAYFVVPTPLPEDDVR, MH+ 3392.76, 3+. (D ) Transketolase (11066098), LGQSDPAPLQHQVDIYQK, MH+ 2038.25, 3+. (E ) Ribose 5‐phosphate isomerase A (6677767), WHKGIPIEVIPMAYVPVSR, MH+ 2193.64, 3+. Ribose 5‐phosphate was found only in the crude homogenate control, whereas the other proteins were observed predominantly in the LARC sample.

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Literature Cited

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