Hence, introduction of bias may be avoided by using the barcoding assay. The lower sensitivity of detecting HaloTag fusion proteins compared to that of HaloTag alone suggests that the size and/or structure of protein could affect the sensitivity of the barcoding assay. used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage. == INTRODUCTION == Protein profiling is a major strategy used in post-transcriptome assays to assign a function to uncharacterized protein-coding genes. It is important not only for gene characterization in PTZ-343 basic biological studies but also for medical diagnosis, e.g. for antibody-based assays of an immune system disorder, such as autoimmune diseases. Approaches based on physical protein interactions include enzyme-linked immunosorbent assay (ELISA), protein microarrays, affinity purification-mass spectrometry, and yeast two-hybrid system. These approaches are used to characterize cellular signaling PTZ-343 networks and facilitate candidate biomarker discovery (13). Conventional protein profiling technologies involve the use of such dedicated platforms as a mass spectrometer or SFRP2 microarray platform (412). Next-generation sequencing (NGS) for investigating genome dynamics has rapidly emerged in the last decade. It is widely available and indispensable technology worldwide. Protein profiling involving NGS has been used to identify target protein molecules, e.g. for proteinprotein interaction (PPI) analysis and antibody-transcriptome profiling (1316). NGS technologies not only increase the number of target molecules that can be assayed at any time but also facilitate detection of target molecules present in low copies because of the nucleic acid amplification involved, regardless of the observed amplification bias (17). However, to address the NGS-associated amplification bias, multiplexed molecular barcoding methods that minimize the bias have been proposed (1719). Protein conjugation to DNA molecules is increasingly used for antibody labeling (4,13,15), proximity ligation (20,21), and cell imaging (22,23). Generally, the target protein is conjugated to another molecule (DNA or a fluorophore) modified by an activated ester, such asN-hydroxysuccinimide, via the amino group of the target protein. The formation of an ester-amine by covalent bonding in a pH-dependent manner can be used to modify proteinsin vivo(24,25). On the other hand, conjugation reaction via click chemistry is rapidly emerging for many organic reactions in the biological field because of several advantages, such as pH-insensitivity and reactivity in water with no apparent toxicity (26,27). Here, we report the development of a proteinoligonucleotide conjugation method involving a high-affinity capture tag, HaloTag, to link proteins to DNA oligonucleotides, and its application in protein profiling, including antigenantibody interactions. == MATERIALS AND METHODS == == Preparation of a barcoded HaloTag protein complex == The initial preparation of the HaloTag-barcoded-protein was performed using a first-generation set of custom proteins, HaloTag protein G (1 g/l, Kazusa DNA Research Institute, Japan), NanoLuc-HaloTag (8 g/l, NL-HaloTag; Promega, USA), HaloTag-FOS proto-oncogene proteins (40 ng/l, HaloTag-FOS; Cell Free Science, Japan), and HaloTag-Glutathione S-transferase (3 g/l, HaloTag-GST; PTZ-343 Promega). DNA encoding the protein identifier to identify the protein type (Figure1A: red, 8 bp; andSupplementary Table S1), semi-random bases for molecule counting (Figure1A: blue, 30 bp; andSupplementary Table S1), and the amplification base for polymerase chain reaction (PCR) reaction (Figure1A: black, 31 bp, 2; andSupplementary Table S1) were prepared with amine modification byN-hydroxysuccinimide at 5-end of the barcode DNA (Figure1A, Eurofins, Japan). The HaloTag ligandoligonucleotide complex was formed using the amido bond-based method (28). Briefly, 300 l 100 M DNA was mixed with 90 l 0.9 M sodium bicarbonate solution and 20 l 50 mM HaloTag O4 ester ligand.