What are the Methods of Protein Sequencing?
The methods of protein sequencing are different techniques for revealing the amino acid sequence of proteins. Protein sequencing provides a direct means to determine the amino acid sequence of proteins, forming a cornerstone for studying protein structure and function. Sequencing facilitates the prediction of three-dimensional protein structures, offering insights into their biological functions and mechanisms of interaction.
Currently, the primary methods of protein sequencing include mass spectrometry-based de novo sequencing, Edman degradation sequencing, and nanopore sequencing.
Workflow of Each Method
1. Mass Spectrometry-Based De Novo Sequencing
This method utilizes mass spectrometry to analyze peptide fragments and deduce their amino acid sequences based on MS/MS spectra. De novo sequencing algorithms are applied to directly interpret peptide sequences, which are subsequently assembled into the complete protein sequence. The workflow includes:
(1) Sample Preparation: Minimize protein degradation during this step to preserve sample integrity.
(2) Protein Digestion: Select appropriate enzymes (e.g., trypsin) to cleave proteins into peptides.
(3) Peptide Separation: Employ chromatographic techniques to separate peptides before analysis.
(4) Mass Spectrometry: Ensure the instrument's stability and high performance for reliable data acquisition.
(5) Data Analysis: Use advanced algorithms to accurately sequence peptides and assemble the full protein sequence.
Mass Spectrometry-Based De Novo Sequencing is versatile for analyzing proteins of various sizes and can also accurately detect post-translational modifications (PTMs). The methods of protein sequencing are constantly evolving, among which mass spectrometry de novo sequencing has become one of the most widely used technologies.
2. Edman Degradation Sequencing
Edman degradation sequencing employs a series of chemical reactions to remove and identify amino acid residues at the N-terminal end of the polypeptide chain (free α-amino residues). Meanwhile, the next residue in the sequence is exposed for subsequent reactions. By repeating this process, the amino acid sequence is determined from the N-terminus to the C-terminus. For longer protein sequences, the sample can be first cleaved into smaller peptide fragments, which are then individually sequenced and assembled to obtain the complete sequence. The limitations of Edman degradation method are quite obvious:
(1) Large proteins cannot be sequenced using Edman degradation.
(2) Proteins with chemically modified N-termini are unsuitable for this method.
3. Nanopore Sequencing
This emerging technique digests peptides into individual amino acids using aminopeptidases. The amino acids are then identified based on electrical signals as they pass through nanopores. Machine learning algorithms reconstruct the peptide sequence from these signals. The method is still in development and is currently limited to single peptide fragment analysis.
Edman degradation sequencing and nanopore sequencing are two supplementary means for the specific scenario sequence analysis in the methods of protein sequencing.
Case Studies
Monoclonal Gammopathy of Undetermined Significance (MGUS): MGUS is characterized by the presence of a monoclonal antibody (M-protein) in serum. Researchers applied liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze IgG1-type M-protein in human serum. By using multiple proteases for digestion and employing de novo sequencing methods, they reconstructed the heavy and light chain sequences of M-protein. Glycosylation levels in the complementarity-determining regions (CDRs) were also characterized, providing insights into the antibody's functional properties.
Mass spectrometry-based de novo protein sequencing is widely used due to its ability to determine amino acid sequences without prior knowledge or reference databases. This technology is particularly valuable for identifying novel proteins, including therapeutic antibodies, which are indispensable tools in medical diagnostics and therapeutics. In a word, choosing appropriate methods of protein sequencing is crucial for different research needs, as it directly affects the accuracy of experiments and the reliability of results.
MtoZ Biolabs leverages expertise and literature published in Nature Biotechnology and specialized proteomics journals such as Journal of Proteome Research (JPR), has developed software for antibody sequence analysis. The software integrates cutting-edge algorithms to ensure accurate protein sequencing/antibody sequencing. Utilizing Orbitrap Eclipse and Orbitrap Fusion Lumos mass spectrometers, this approach delivers the highest resolution and sensitivity, ensuring reliable results. The typical turnaround time for protein sequencing/antibody sequencing projects is 3–4 weeks. Free project evaluation!
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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