BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation

Abstract

Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.

Keywords: APEX; BioID; biotinylation; peptide; protein−protein interactions; proximity-dependent biotinylation; subcellular proteome.

Figure 1

Overview of BioSITe. (A) Schematic of biotinylation labeling techniques commonly used to identify protein–protein interactions in vivo. A bait protein (green) fused to an engineered biotinylation enzyme (APEX2 or BioID, purple) biotinylates proteins in the proximity of the enzymes. Biotinylation, red circles with “B”; interacting proteins, brown/black; labeling radius, blue. (B) In conventional methods for candidate protein identification, biotinylated proteins are generally captured by streptavidin (green) conjugated to beads under denaturing conditions. Proteins bound to the beads are subsequently digested, generating nonbiotinylated peptides (brown and gray), which readily elute from the beads and can be identified by LC–MS/MS, and biotinylated peptides (cyan) that remain tightly bound to the beads. In BioSITe, proteins are digested prior to enrichment and biotinylated peptides are captured using by anti-biotin antibodies coupled to beads. (C) MS/MS spectra of SHC-transforming protein 1 (SHC1) biotinylated peptide detected by LC–MS/MS. Fragment ions adjacent to the biotin modification that confirm the site of biotinylation are indicated in red. (D) Lack of overlap of biotinylated peptides identified from BirA*-BCR-ABL detected by the conventional method or BioSITe. (E) Overlap of biotinylated proteins identified by BioSITe (green) and by the conventional method of on-bead digestion (purple).

Figure 2

Mapping of biotinylation sites. (A) Biotinylated proteins identified by BioSITe were grouped by the degree of biotinylation. (B) 3D models of representative proteins identified in the study. GRB2 and CRK are homology models based on their human homologues (PDB ID 1GRI and 2EYZ, respectively), while the structure of STAT5A was taken from PDB ID 1Y1U. The other domain structures from each protein were modeled using their human homologues from PDB. Lysine residues that are biotinylated upon interaction with BirA*-BCR-ABL are highlighted as red sticks and labeled with their position. The biotinylated lysines are colored in red and the functional domains are indicated (CC, coiled coil; ROC, Ras of complex proteins; PH, pleckstrin homology; SH3, Src-homology 3; SH2, Src-homology 2; PID, phosphotyrosine interaction domain; DBD, DNA-binding domain).

Figure 3

Overview of quantitative BioSITe. (A) Experimental workflow for differential interactome analysis of BirA*-p210 and BirA*-p190. Ba/F3 cells expressing BirA*-p210 and BirA*-p190 were incubated overnight with media containing light (green) or heavy biotin containing four deuterium atoms (red), respectively. Equal amounts of cell lysates from each condition were mixed and digested into peptides. Biotinylated peptides were enriched using BioSITe and analyzed by LC–MS/MS. (B) Plot of relative abundance of the biotinylated sites between BirA*-p210 and BirA*-p190. Identified biotinylated sites and the corresponding proteins are plotted according to their log₂ intensity ratios (BirA*-p190/BirA*-p210).

Figure 4

Application of BioSITe to the APEX system and biotin-based click chemistry (A) APEX2 constructs targeted to either the mitochondrial intermembrane space (IMS-APEX2) or cytoplasm (NES-APEX2) were expressed in HEK293T cells. APEX2 leads to labeling of proximal proteins in the intermembrane space or the cytoplasm with biotin-phenol (BP, red) (OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane). (B) MS/MS spectrum of a biotin-phenol-labeled peptide corresponding to COX assembly mitochondrial protein 2 homologue (CMC2). Peaks labeled in red indicate fragment ions series with or without mass shift by biotin modification. All peaks containing biotin are labeled with “Bio”. (C) Overlap of proteins identified by BioSITe (yellow) with that from a previous study identifying IMS proteins using a conventional in-gel digestion method (Hung et al., blue). The proteins unique to the BioSITe IMS-APEX2 experiment were compared with published mitochondrial protein database MitoCarta 2.0 (red). (D) Overlap of biotin-phenol-labeled peptides enriched by BioSITe (yellow) with that from Hung et al. (blue). (E) Predicted topology of mitochondrial transmembrane proteins of translocase of inner mitochondrial membrane 50 (TIMM50) (top), NADH: ubiquinone oxidoreductase subunit A8 (NDUFB8) (middle), and coiled-coil domain containing 51 (CCDC51) (bottom). Matrix-facing (orange), transmembrane (green), and intermembrane space domains are indicated. Cartoon depictions of these proteins embedded in the inner membrane (green) are shown in gray with biotinylation sites detected by BioSITe are colored in red and black, detected by Lee et al. using the Matrix-APEX2 construct. (F) Schematic overview of enrichment method for O-GlcNAcylated peptides. After LysC digestion, an azide-modified monosaccharide (GalNAz) is added to the O-GlcNAc motif using galactosyltransferase1 GalT1. Biotinylation of O-GlacNAc motif subsequently mediated by click chemistry reaction between the azide group and of GalNAz and the alkyne group of biotin DIBO alkene. Biotin is used to enrich peptides with O-GlcNAc motif using BioSITe. (G) MS/MS spectra of a O-GlcNAc modified peptide corresponding to nuclear pore complex protein NUP153 (NUP153). Ion peaks at m/z 216.08, 345.15, and 447.16, labeled signature ions, correspond to fragments from click chemistry reagents.