Proteomic sequencing is a method used to study and characterize proteins in a biological sample.
It involves identifying the exact order of amino acids in a protein to reveal its structure and function in biological processes.
Proteins are essential biological molecules involved in structural roles, metabolic processes, transport, signaling, and regulatory functions.
Proteomic sequencing helps in understanding complex cellular processes and mechanisms of diseases.
Genetic information flows from DNA to RNA, which is then used to produce proteins.
While amino acid sequences can be inferred from gene sequences, this approach cannot provide insights into post-translational modifications, protein folding, or interactions.
These additional factors are crucial for determining protein function.
High-throughput and direct proteomic methods are necessary to obtain detailed and accurate information about proteins.
Proteomic sequencing serves as a direct approach to studying protein structure, function, interactions, and modifications within biological systems.
What is Proteomics?
All the proteins expressed in an organism at a given time are collectively called its proteome.
The study of the proteome is known as proteomics.
Proteomics focuses on understanding the interactions and specific functions of proteins within an organism.
It helps reveal how proteins contribute to biological processes and the development of diseases.
There are three main approaches used in proteomics to study proteins in a sample:
Top-down Proteomics involves separating and directly analyzing intact proteins.
This approach is valuable for studying post-translational modifications (PTMs) and protein isoforms, as it allows direct examination of whole proteins.
However, it faces difficulties in analyzing complex protein mixtures.
Bottom-up Proteomics requires digesting proteins into smaller peptides before analysis.
These peptides are then separated and identified using techniques like chromatography and mass spectrometry.
This method is suitable for identifying and quantifying a large number of proteins, as peptides are easier to analyze.
However, it does not provide information about the structure or modifications of intact proteins.
Middle-down Proteomics is a newer approach that falls between top-down and bottom-up methods.
In this method, proteins are partially digested to produce larger peptide fragments.
It allows more detailed analysis of protein structure and post-translational modifications compared to bottom-up proteomics.
Principle of Proteomic Sequencing
Proteomic sequencing involves identifying and characterizing proteins to determine their structure, function, and interactions.
The core principle of proteomic sequencing is to separate, identify, and quantify proteins present in a biological sample.
The process begins with the isolation of the target protein from the sample.
The isolated protein is broken down into smaller fragments known as peptides.
These peptides are analyzed using techniques such as mass spectrometry and Edman degradation.
These analytical methods help determine the amino acid sequence of the peptides.
The complete protein sequence is then reconstructed by comparing the obtained sequences with known protein databases.
Steps of Proteomic Sequencing
1. Sample Preparation
Proteins of interest are extracted from biological samples.
Extraction involves the use of organic solvents, detergents, and other lysis methods to release proteins from cells.
Organic solvents and detergents are removed through lyophilization to enhance protein detection.
2. Protein Separation
Proteins are separated using gel-based or chromatography-based techniques.
Gel-based methods include 1-Dimensional Electrophoresis (1-DE), which separates proteins by molecular weight, and 2-Dimensional Electrophoresis (2-DE), which separates by molecular mass and isoelectric point for better resolution.
Difference Gel Electrophoresis (DIGE) is a 2-DE variation that uses fluorescent dyes to compare multiple protein samples on the same gel.
Chromatography-based methods help separate proteins from complex mixtures and include Ion Exchange Chromatography (IEC), Size Exclusion Chromatography (SEC), Affinity Chromatography, and Liquid Chromatography (LC).
3. Sequencing
Following separation and purification, sequencing is performed on both ends of the polypeptide.
Individual peptide fragments are sequenced using techniques such as Edman degradation and mass spectrometry.
Advanced methods like fluorosequencing, tunneling current, and nanopore-based protein sequencing are also being used.
4. Data Analysis
Raw sequencing data is preprocessed and normalized to eliminate experimental biases.
Peptide sequences are aligned with known protein databases using computational algorithms.
This allows for protein identification, quantification, and analysis of protein interactions and post-translational modifications.
Methods of Proteomic Sequencing
a. Mass Spectrometry (MS)
MS is a commonly used method in proteomic sequencing that measures the mass-to-charge ratio of peptide ions and determines the chemical structures of proteins.
In this method, peptides are first ionized using an ion source.
The two main ionization methods used in mass spectrometry are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI).
Once ionized, peptides are separated and analyzed using different mass analyzers like Time-of-Flight (TOF) and Ion Trap.
For more detailed sequencing, tandem mass spectrometry (MS/MS) is used, which involves two stages of mass spectrometry.
MS can also be combined with methods like liquid chromatography (LC-MS) to analyze complex mixtures of proteins.
b. Edman Degradation
It is a method that sequentially removes and identifies amino acids from the N-terminal end of a peptide.
It is considered the first protein sequencing method.
The process begins with a Phenylisothiocyanate (PITC) reaction which reacts with the protein of interest at the N-terminus and forms a stable Phenylthiocarbamyl (PTC) derivative.
Then, the N-terminal amino acid is cleaved using TFA (trifluoroacetic acid) and identified using chromatographic methods or mass spectrometry.
This is repeated to sequence the entire protein.
It allows sequencing of peptides by keeping the protein intact and provides accurate sequence data, but it is less efficient for larger proteins.
c. Nanopore sequencing
Nanopore sequencing was originally developed for DNA and RNA analysis, but advances in nanopore design have made it possible to use this technology in proteomics.
This method involves passing the proteins through a nanopore and measuring the electrical current as the protein moves through the nanopore.
Each amino acid or modification causes a unique disruption in the current, which allows the identification of specific residues.
Motor proteins and electro-osmotic flow can be used to control the speed of peptides, which helps overcome challenges associated with the rapid and uneven movement of proteins during sequencing.
Nanopore sequencing for proteomics is still in the experimental stage but it has potential applications in real-time and high-throughput protein analysis.
d. Fluorosequencing
This method uses fluorescence to detect and sequence peptides or proteins.
In this method, the protein is tagged with a fluorescent marker and sequenced.
The fluorescent signals from each amino acid are detected and analyzed.
It can provide detailed sequence information for small peptides, but is limited by the need for fluorescent labeling and is not widely used for large-scale sequencing.
e. Tunneling current-based sequencing
This method involves using a tunneling current to measure changes as peptides pass through a nanopore and provides sequence information.
The peptide sequence is determined by the electrical signals generated as individual amino acids move through the nanopore.
It allows high-throughput sequencing, but this technology is still under development.
Advantages of Proteomic Sequencing
Proteomic sequencing helps to identify and quantify proteins in complex biological samples.
It provides direct information on the proteins present in a sample, unlike genome sequencing which only predicts protein sequences.
It can detect low-abundance proteins, including potential biomarkers for diseases.
It aids in understanding signaling pathways and the mechanisms of diseases.
Proteomics can distinguish protein isoforms and alternative splice variants.
It can identify chemical modifications that regulate protein function, which cannot be predicted by DNA or RNA sequencing.
Limitations of Proteomic Sequencing
It is difficult to sequence larger or highly modified proteins, limiting the analysis of complex protein structures.
Sample preparation is often difficult and time-consuming; proteins must be carefully extracted, purified, and digested before sequencing.
Large-scale proteomic studies can be expensive, especially when using advanced techniques like mass spectrometry.
The Edman degradation method is less effective for sequencing long or chemically modified proteins.
It is challenging to fully capture and analyze the wide variety of proteomes due to their complexity and dynamic nature.
Proteomic sequencing produces large amounts of data, which requires specialized bioinformatics tools and expertise for analysis.
Applications of Proteomic Sequencing
Proteomic sequencing has applications in identifying novel proteins or isoforms.
It is used to identify protein biomarkers that can assist in disease diagnosis.
It plays a role in personalized medicine by helping to tailor disease treatment to individual patients based on their genetic profiles.
Proteomic sequencing can analyze post-translational modifications such as phosphorylation and glycosylation.
It is useful in drug discovery and development by helping to identify disease-related proteins.
It contributes to the development of biopharmaceuticals, recombinant proteins, and industrial enzymes.
References
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Methods and techniques for protein sequencing – Creative Proteomics. Retrieved from https://www.creative-proteomics.com/resource/methods-and-techniques-for-protein-sequencing.htm
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