Recent advances in single-cell proteomics have solved many bottlenecks, such as throughput, sample recovery, and scalability via nano-scale sample handling. In this study, we aimed for a sensitive mass spectrometry analysis capable of handling single cells with a conventional mass spectrometry workflow without additional equipment. We achieved seamless cell lysis and TMT-labeling in a micro-HOLe Disc (microHOLD single-cell proteomics) by developing a mass-compatible single solution based on a zwitterionic detergent and a catalyst for single-cell lysis and tandem mass tag-labeling without a heat incubation step. This method was developed to avoid peptide loss by surface absorption and buffer or tube changes by collecting tandem mass tag-labeled peptide through microholes placed in the liquid chromatography injection vials in a single solution. We successfully applied the microHOLD single-cell proteomics method for the analysis of proteome reprogramming in hormone-sensitive prostate cells to develop castration-resistant prostate cancer cells. This novel single-cell proteomic method is not limited by cutting-edge nano-volume handling equipment and achieves high-throughput and ultrasensitive proteomic analysis of limited samples, such as single cells.