Nanodisc-based co-immunoprecipitation for mass spectrometric identification of membrane-interacting proteins

Abstract

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.

Fig. 1.

Strategy for nanodisc assisted detection and co-immunoprecipitation for isolation of interaction partners to membrane embedded molecules: Antibodies that recognize the epitope tag of nanodiscs are immobilized on sensorchip surfaces (A) or affinity beads (B, left). Potential binding activity can be monitored by surface plasmon resonance (SPR) by first capturing nanodiscs and then introducing a flow of a mixture, e.g. a cell lysate or culture medium, containing interaction partners of the nanodisc embedded molecule (A, right). Alternatively the binding components of the mixture can be isolated by incubating the nanodiscs with the mixture and isolating the nanodiscs and associated proteins with the antibody decorated affinity beads (B).

Fig. 2.

SPR analysis of binding activity in culture media of two E. coli strains. Media cleared after growth of enterotoxigenic E. coli H10407 (A) and the nonpathogenic E. coli MG1655 (B) were injected over ganglioside GM1 nanodiscs captured on antibody coated sensorchips. The presented sensorgrams are produced by reference subtraction of the signal of binding to 900 RU GM1-free (control) nanodiscs from the signal of binding to 940 RU GM1 nanodiscs.

Fig. 3.

SPR analysis of detergent based elution from nanodiscs captured by antibody. 6xHis-tagged Nanodiscs were injected over immobilized anti-tetra-His antibodies. Then the flow was changed to buffer without nanodiscs and subsequently an injection of detergent or buffer without detergent was injected (A). The remaining signal normalized to after detergent injection is reported in (B). The concentrations of detergent were as follows: Octyl glucopyranoside (OGP) 50 mm; cholate 25 mm; DHPC 5 mm; Triton X-100 (TX-100) 1 mm; P20 (tween 20) 0.1 mm.

Fig. 4.

Co-immunoprecipitation of proteins that bind to GM1 containing nanodiscs (A) and nanodiscs without GM1 (B). Fifteen microliters of the flow through, washes and eluents of 3xFLAG peptide incubation or DHPC incubation were subjected to SDS-PAGE as indicated. An aliquot of the FLAG M2 antibody that was used for immunoprecipitation is shown in (C) for comparison. The gel bands of the eluents are indicated with numbers that are also used in the text and Table I.

Fig. 5.

Identification of the protein that binds specifically to GM1 nanodiscs. The protein of band 1 in Fig. 4A was digested with trypsin after reduction and alkylation. A, shows the peptide mass map with peaks that are annotated to LTB indicated Signal denoted “T” correspond to commonly observed trypsin peptides. Signals denoted “K” correspond to commonly observed keratin peptides. Of the 40 searched peptides 6 were assigned to LTB, 3 to trypsin, and 6 to keratin. B, shows the annotated MALDI MS/MS spectrum of the peptide ion 1553.65.