This feature was written using content from out recenty published Proteomics Playbook, which you can download for FREE here.
The sequence of a protein is critical as it largely dictates the protein’s three-dimensional conformation and, consequently, its biological function. By decoding the sequence of amino acids, researchers can gain insights into the protein’s role within the cell, its interactions with other molecules and its involvement in various physiological processes. Protein sequencing has been instrumental in determining the mechanisms of numerous biological processes, including enzyme catalysis and immune response.
Next-Generation Protein Sequencing
Next-generation protein sequencing (NGPS) refers to advanced techniques and technologies designed to determine the amino acid sequences of proteins with greater speed, accuracy and throughput than traditional methods. Traditional protein sequencing techniques, such as Edman degradation, sequentially removes and identifies the N-terminal amino acid from a peptide chain, which is a time-consuming and labour-intensive process. MS-based methods, while more modern, still typically involve complex sample preparation and extensive data analysis to deduce protein sequences indirectly through peptide mass fingerprints and tandem MS spectra.
In contrast, NGPS leverages innovative approaches, including but not limited to single-molecule sequencing and nanopore-based technologies, which can directly read amino acid sequences from intact proteins or peptides. These methods minimise sample preparation and provide real-time sequencing data with higher sensitivity and resolution. NGPS platforms can identify post-translational modifications (PTMs) and sequence heterogeneity more comprehensively, which are critical for understanding protein function, dynamics and interactions. By integrating advanced bioinformatics tools and machine learning algorithms, NGPS also enhances the ability to analyse large-scale proteomic data, thus enabling deeper insights into complex proteomes and facilitating applications in biomarker discovery, personalised medicine, and synthetic biology.
Fluorescent Labelling Approaches:
Recently, new sequencing approaches have adopted the Edman degradation technique and created NGPS methods that can sequence many peptides at once. One such technique, termed fluorosequencing, integrates Edman degradation, single-molecule microscopy and stable fluorophore chemistry and multiplexing strategies, allowing millions of fluorescently labelled peptides to be visualised and sequentially degraded on a glass flow cell. Each cycle of Edman degradation trims away the N-terminal amino acid and thereby alters the fluorescence intensity, producing unique signatures – a peptide fingerprint – that identifies individual peptides. This massively parallel method, like MS approaches, relies on the use of a reference proteome to piece together the partial sequences.
Nanopore-Based Approaches:
Nanopores are emerging as a powerful approach for NGPS by enabling the direct, real-time analysis of individual protein molecules. The core principle involves threading a protein or peptide through a nanoscale pore under an applied electric field. As the protein translocates through the nanopore, it disrupts the ionic current flowing through the pore, generating a unique electrical signal corresponding to the sequence of amino acids. These signal patterns are decoded to determine the primary structure of the protein. Notably, one of the major benefits of nanopore-based protein sequencing is its ability to provide long-read, label-free sequencing with minimal sample preparation.
Read more in the latest Proteomics Playbook, available soon for download here.
