The Amino Acid Sequence: Decoding Protein's Blueprint (2025)

Amino acids are the building blocks of proteins and play a crucial role in metabolism. The sequence of amino acids in a protein is determined by the genetic code in the DNA of the cell, with each amino acid having unique properties such as size, charge, and hydrophobicity. This sequence guides the protein to fold into a specific three-dimensional structure, which is essential for its function. The primary structure of a protein is the linear sequence of amino acids linked by peptide bonds, while the secondary structure refers to local folding patterns such as alpha-helices and beta-sheets. The tertiary structure is the overall 3D shape resulting from further folding of the secondary structures, and the quaternary structure refers to the assembly of multiple protein subunits. To determine the amino acid sequence of a protein, selective hydrolysis is performed, followed by chromatography and sequencing techniques like Edman's method or mass spectrometry. De novo peptide sequencing uses tandem mass spectrometry and bioinformatics algorithms to determine the sequence without a reference database.

Amino acid sequencing methods

Amino acid sequencing is the process of identifying the arrangement of amino acids in proteins and peptides. There are two major direct methods of protein sequencing: mass spectrometry and Edman degradation using a protein sequencer.

Mass Spectrometry

Mass spectrometry methods are now the most widely used for protein sequencing and identification. Identification via mass spectrometry is increasingly preferred as it overcomes many of the established limitations of Edman degradation. However, there are various techniques within protein mass spectrometry that make amino acid sequencing via MS techniques harder to define in brief. MS-based amino acid sequencing can be done with or without reference to a database of known sequences. When a database or reference sequence is used, this is called protein identification, peptide sequence identification or peptide mapping.

In de novo peptide sequencing, the amino acid sequence of a peptide is determined via tandem mass spectrometry combined with bioinformatics algorithms, without a reference sequence or database. De novo protein sequencing compiles multiple overlapping de novo peptide sequences to derive a full-length protein sequence. The primary benefit of de novo sequencing over conventional MS-based sequence analysis is that it allows researchers to study proteins and peptides for which there is no reference sequence. Advanced techniques, including the use of multiple proteases, alternative fragmentation methods, liquid chromatography methods, high-resolution instruments, and machine-learning algorithms, allow rapid and highly accurate analysis of sequences and post-translational modifications.

Edman Degradation

Edman degradation is a classical method for determining the amino acid sequence of a protein. It is based on the selective cleavage of the N-terminal amino acid residue from a peptide chain without affecting the rest of the sequence. The cleaved amino acid is then identified, and the process can be repeated sequentially to determine the complete sequence of the protein. Edman degradation provides accurate N-terminal sequencing information and has been widely used and established as a reliable method for protein sequencing. Automated Edman sequencers are now in widespread use and are able to sequence peptides up to approximately 50 amino acids long. However, the accuracy of Edman degradation can be affected by certain amino acids, such as proline, which may require special treatment.

Other Methods

Before carrying out amino acid sequencing, it is often useful to determine the unordered composition of a protein by hydrolytically degrading it and then derivatizing the sample to make it more volatile and less reactive, thus increasing its suitability for analysis via ion-exchange chromatography. This can help in identifying errors and may elucidate ambiguous results. It may also offer insights into the right protease to use for protein digestion.

Other methods of peptide fragmentation in the mass spectrometer, such as ETD or ECD, may give complementary sequence information. Enzymatic cleavage is another method, which involves cleaving the protein at specific amino acid positions using proteolytic enzymes.

De novo protein sequencing

Proteins are made up of amino acids, and the sequence of these amino acids is determined by the types of amino acids involved and their arrangement. De novo sequencing is a method for determining the sequence of amino acids in a peptide without prior knowledge of the sequence or a reference database. This method is particularly useful when a reference sequence is unavailable.

De novo sequencing can be used to identify previously unknown peptide sequences and search for post-translational modifications or mutations. However, it may not always be able to derive a complete sequence and may have uncertainty in a portion of the derived sequence. It can also be challenging to determine the directionality of a sequence.

De novo sequencing uses mass spectrometry (MS) to analyze a target protein that has been digested into peptides. The MS spectra are then identified as amino acid sequences by de novo algorithms. Overlapping peptides are assembled into contigs, and repeating these steps should eventually assemble the full sequence of the target protein. However, there are several problems that can lead to insufficient or incorrect assembly of the protein sequence, such as instrumental errors, signal loss, and noise.

To overcome these challenges, various techniques have been developed, such as the use of multiple enzymes to generate overlapping peptides and the integration of top-down and bottom-up strategies. Advanced techniques, including the use of multiple proteases, alternative fragmentation methods, liquid chromatography methods, high-resolution instruments, and machine-learning algorithms, enable rapid and highly accurate analysis of sequences and post-translational modifications.

Overall, de novo protein sequencing is a powerful tool for studying proteins and peptides, especially those without reference sequences, and advancements in the field continue to improve its accuracy and robustness.

Protein identification

The sequence of amino acids in a protein is dictated by the types of amino acids involved and the order in which they are arranged. There are 20 amino acids in the human body, and they combine in numerous ways to form more than 40,000 known proteins.

There are several methods for identifying proteins, including:

• Edman Degradation: This technique sequences amino acids in a peptide by labelling the amino-terminal residue and cleaving it from the peptide without disrupting the peptide bonds between other residues.
• Mass Spectrometry: This technique measures the mass-to-charge ratio of charged particles to determine the masses of particles and the elemental composition of a sample of molecules. It is often used in conjunction with peptide mass fingerprinting, where the masses of proteolytic peptides are used as input to search a database of predicted masses from the digestion of known proteins.
• Nanopore Peptide Profiling: Nanopores are single-molecule sensors used in nucleic acid analysis. An engineered Fragaceatoxin C nanopore can identify individual proteins by measuring peptide spectra produced from hydrolysed proteins. This method is low-cost and portable.
• Chromatography: This is a common technique used for purification, identification, and quantification of protein mixtures. High-performance liquid chromatography and thin-layer chromatography are two frequently used chromatic methods for protein separation.
• Two-dimensional Gel Electrophoresis: This gel-based method is used to analyse complex samples to characterise the full range of proteins present.
• Western Blotting: This technique identifies proteins extracted from cells, separates them by size, and uses primary and secondary antibodies to target proteins, providing identification based on biological associations.
• Immunoassays: These tests identify proteins by their interactions with specific antibodies, including ELISA, Western blotting, and immunoprecipitation.
• Size Exclusion Chromatography: This method uses beads of specific dimensions packed in a column to separate proteins according to size.
• De novo peptide sequencing: This approach determines the amino acid sequence of a peptide via tandem mass spectrometry combined with bioinformatics algorithms, without a reference sequence or database.

These methods can be used to identify proteins and determine their amino acid sequences, which is valuable for understanding protein function and developing biological therapies.

Peptide sequence identification

The sequence of amino acids in a protein is determined by the gene encoding it. During transcription, the DNA gene sequence is copied into messenger ribonucleic acid (mRNA), which contains the information for producing a specific peptide or protein. The amino acid sequence of a peptide can be determined through de novo peptide sequencing, which involves tandem mass spectrometry combined with bioinformatics algorithms, without a reference sequence or database.

There are numerous tools available for biological sequence comparisons and searches, including PEPMatch, which can identify short peptide sequence matches in large sets of proteins. This is particularly useful for immunologists who need to find matches for linear peptide T-cell epitopes. The utility of such tools is critical in applications such as identifying conservation across viral epitopes, identifying putative epitope targets for allergens, and finding matches for cancer-associated neoepitopes to examine the role of tolerance in tumour recognition.

In addition to de novo peptide sequencing, peptide sequence identification can also be achieved through database or reference sequence matching. This is known as protein identification, peptide sequence identification, or peptide mapping. The choice of sequence database can impact the taxonomic and functional results in studies, such as those on gut microbiota.

Peptides can also be classified based on their production, such as peptones, which are derived from the breakdown of animal milk or meat, or milk peptides, which are formed during milk protein digestion in the gastrointestinal tract or through bacterial fermentation. Another class is ribosomal peptides, which are produced by cellular ribosomes in a translation process of RNA into mRNA, forming an amino acid sequence. Non-ribosomal peptides, on the other hand, are formed by enzymes that are not ribosomes and undergo post-translational modifications after the amino acid sequences are connected.

Protein structure

The sequence of amino acids in a protein is determined by the genetic code in the DNA of the cell. Each amino acid has unique properties, such as size, charge, and hydrophobicity, which influence how they interact with other amino acids in the protein chain. These interactions cause the protein to fold into a specific shape, which is crucial for its function.

The structure of a protein can be described at four levels. The primary structure is the linear sequence of amino acids, linked by peptide bonds. This sequence is directly encoded by the DNA. The secondary structure refers to local folding patterns within the protein, such as alpha-helices and beta-sheets, which are stabilised by hydrogen bonds between the backbone atoms of the amino acids. The tertiary structure is the overall three-dimensional shape of the protein, resulting from further folding of the secondary structures. This is stabilised by various types of bonds and interactions between the side chains of the amino acids, including hydrogen bonds, ionic bonds, disulphide bridges, and hydrophobic interactions. Finally, the quaternary structure refers to the assembly of multiple protein subunits into a larger complex.

There are various methods to determine the amino acid sequence of a protein. One method is to first carry out selective hydrolysis, and then separate the resulting oligopeptides by chromatography. The individual peptides can then be sequenced by Edman’s method or mass spectrometry. Another method is de novo peptide sequencing, where the amino acid sequence is determined via tandem mass spectrometry combined with bioinformatics algorithms, without a reference sequence or database. This method is useful for studying proteins and peptides for which there is no reference sequence.

Frequently asked questions