This study reports a copper(II)-functionalized *Mycobacterium smegmatis* porin A (MspA) nanopore with the N91H substitution, enabling the direct identification and quantification of all 20 proteinogenic amino acids. The nanopore's ability to coordinate copper ions and amino acids generates well-defined current signals, allowing for the detection of all 20 amino acids, including two post-translational modified amino acids and one unnatural amino acid. The system demonstrates high accuracy (99.1%) and signal recovery (30.9%) in validation tests. Additionally, it can quantitatively measure amino acids at the nanomolar range. The nanopore is also capable of real-time analysis of peptides, including those with clinically relevant modifications, neoantigens associated with cancer, and pathologically relevant peptides with single-amino acid differences. This technology offers a promising approach for single-molecule protein sequencing and the comprehensive understanding of proteome dynamics.This study reports a copper(II)-functionalized *Mycobacterium smegmatis* porin A (MspA) nanopore with the N91H substitution, enabling the direct identification and quantification of all 20 proteinogenic amino acids. The nanopore's ability to coordinate copper ions and amino acids generates well-defined current signals, allowing for the detection of all 20 amino acids, including two post-translational modified amino acids and one unnatural amino acid. The system demonstrates high accuracy (99.1%) and signal recovery (30.9%) in validation tests. Additionally, it can quantitatively measure amino acids at the nanomolar range. The nanopore is also capable of real-time analysis of peptides, including those with clinically relevant modifications, neoantigens associated with cancer, and pathologically relevant peptides with single-amino acid differences. This technology offers a promising approach for single-molecule protein sequencing and the comprehensive understanding of proteome dynamics.