Fig 1.
Peptide barcoding for new types of genotype–phenotype linkages.
A DNA fragment encoding a unique peptide barcode is fused with the C-terminal of each nanobody gene. Transformed Pichia pastoris produces nanobodies fused with peptide barcodes. These nanobodies are mixed with antigen-coated magnetic beads in one pot, and non-specific nanobodies are washed out. Peptide barcodes are cleaved by a specific protease (e.g., enterokinase) and eluted peptide barcodes are quantified by mass spectrometry.
Fig 2.
Generation of functional nanobodies fused with peptide barcodes.
(A) Design of nanobodies used in this study. A unique peptide barcode was fused at the C-terminal of each nanobody. These peptide barcodes were designed to have high detectability for LC–MS analysis. (B) SDS-PAGE analysis of the nanobodies produced by Pichia pastoris. The culture medium was applied to an SDS-PAGE gel, and proteins were stained by Coomassie Brilliant Blue. pPIC9K is a backbone vector used as a negative control. (C) The productivity of the nanobodies by P. pastoris. Each value represents the mean ± standard deviation of at least five biological replicates. Statistical analysis was performed by t-test. An asterisk indicates a significant difference (p < 0.05).
Fig 3.
Immunofluorescence staining of CD4.
Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, blue). CD4 was stained by each nanobody purified with anti-FLAG gels as a primary antibody and anti-DDDDK-tag mAb Alexa Fluor 488 (anti-FLAG-AF488) as a secondary antibody (green). CD4 was specifically stained by the anti-CD4-FLAG-Barcode 1. Wild-type HEK293 cells were used as negative control cells. Scale bars indicate 20 μm.
Table 1.
Kinetic parameters of the nanobodies measured by surface plasmon resonance.
Each value represents the mean ± standard deviation of three technical replicates.
Fig 4.
Quantification of binding activities of the barcoded nanobodies by LC–MS.
(A) Experimental scheme to quantify the binding activities of the nanobodies by LC–MS. The barcoded nanobodies are mixed with CD4-coated magnetic beads in one pot, and non-specific nanobodies are washed out. The peptide barcodes are cleaved on beads by a specific protease, enterokinase. Then, eluted peptide barcodes are quantified by mass spectrometry for the evaluation of binding activities. (B) The quantitativity of SRM of each peptide barcode. Synthetic peptide barcodes were serially diluted and quantified by LCMS-8060. Each peptide barcode was successfully analyzed with high sensitivity and quantitativity. Each value represents the mean ± standard deviation of three technical replicates. (C) Quantification of each peptide-barcoded nanobody. The peptide barcodes were cleaved from 250 fmol of each nanobody, and quantified by LCMS-8060. Each value represents the mean ± standard deviation of three independent experiments. (D) Quantification of the binding strength of the peptide-barcoded nanobodies. The amount of nanobodies bound on the CD4-coated magnetic beads was quantified by LCMS-8060. The peptide barcode fused with anti-CD4 nanobody was specifically detected. Each value represents the mean ± standard deviation of three independent experiments.
Table 2.
Parameters for SRM analysis of peptide barcodes.