In a peptide analysis report, sequence information usually appears as a long string of letters written without spaces. Cryptic as it may look at first glance, this line is actually a simple notation: each letter represents an amino acid, and the order of the letters is the order of the residues in the chain. This article explains how to read that line and what information it provides.
How sequence information is obtained in the laboratory was covered in the article on mass spectrometry. The topic here is reading a sequence once it has been obtained.
Why a single letter?
There are two conventions for writing amino acids. Three-letter abbreviations evoke the name itself and are readable in short notations. One-letter codes, on the other hand, were developed so that long sequences can be written on a single line. Writing a protein of several hundred residues in three-letter codes triples the length of the lines, which is why databases, publications and analysis reports use the one-letter notation.
The choice of letters is not entirely arbitrary. Most amino acids take the first letter of their name. Where several names compete for the same letter, the initial was given to the most common one, and the others were assigned letters that evoke their pronunciation or structure. This is why some codes have to be memorized.
| Code | Amino acid | Code | Amino acid |
|---|---|---|---|
| A | Alanine | M | Methionine |
| R | Arginine | F | Phenylalanine |
| N | Asparagine | P | Proline |
| D | Aspartic acid | S | Serine |
| C | Cysteine | T | Threonine |
| E | Glutamic acid | W | Tryptophan |
| Q | Glutamine | Y | Tyrosine |
| G | Glycine | V | Valine |
| H | Histidine | K | Lysine |
| I | Isoleucine | L | Leucine |
A few letters not in the list are also used. X indicates that the residue at that position could not be determined. Letters such as B, Z and J are reserved for pairs of residues that cannot be told apart; for example, leucine and isoleucine cannot be distinguished under some measurement conditions, in which case a shared code may be used.
Reading direction and numbering
The two ends of a peptide chain are different. One end carries a free amino group and the other a free carboxyl group; these are called the amino terminus and the carboxyl terminus. Sequences are always written from the amino terminus to the carboxyl terminus. This rule provides the common ground needed to compare two sequences.
Position numbers are given in the same direction, counting the residue at the amino terminus as number one. Position information in a report is read according to this numbering. For example, the peptide identified in our reference batch is 36 residues long and carries six cysteines; the positions of these cysteines in the sequence show where disulfide bridges can form. The role of the bridges in the structure was described in the article on disulfide bridges.
There is a pitfall in numbering. A protein may be produced in the cell as a longer precursor and cleaved later; in that case, the first residue of the mature molecule corresponds to a different number in the precursor sequence. When comparing position information taken from the literature, it is worth checking which sequence the numbering refers to.
What can be read from a sequence?
A sequence carries the first clues about how the molecule behaves. Looking at the nature of the residues, the following inferences can be made:
- Length: The number of letters gives the number of residues. Since each residue contributes roughly 110 daltons on average, the approximate mass can also be estimated.
- Number of cysteines: The number of C letters sets the upper limit on the number of disulfide bridges that can form.
- Charge tendency: K, R and H carry basic side chains; D and E carry acidic ones. Their ratio gives an idea of the molecule's surface charge and affects its behaviour in chromatography.
- Absorbance: The presence of W and Y is the reason measurements can be made at 280 nanometres; details are given in the article on 280 nm.
- Sensitive residues: M and W are relatively prone to oxidation, and N and Q to structural change.
Proline residues in a sequence also deserve attention: because this residue restricts the flexibility of the chain, it often corresponds to a turn in the fold. Glycine, by contrast, as the residue with the smallest side chain, gives the chain local freedom. The placement of these two letters in the sequence can give a rough idea of where the structure bends.
None of these inferences gives the three-dimensional shape of the molecule. A sequence is the order of a chain; how that chain folds and how it binds to its target are studied with separate methods.
Comparing two sequences
When a new peptide is identified, the first step is to compare its sequence with known sequences. The comparison is carried out by aligning the two sequences letter by letter; where necessary, gaps are inserted to bring similar regions into register. The result is expressed with two measures: identity, the proportion of positions carrying the same letter; and similarity, a broader measure that also counts different but closely related residues.
In venom peptides, the most informative part of an alignment is the cysteine positions. In peptides belonging to the same family, the numbers of residues between the cysteines form a characteristic pattern, and this pattern can reveal a family relationship even when sequence similarity is low. Classification is therefore based not only on percentage similarity but also on whether the pattern is conserved. The naming scheme for the families is described in the article on α-KTx.
Reading sequence information in a report
The sequence line in a report is not an identity document on its own. The method by which the sequence was obtained, whether it was read in full or only in part, and whether any positions could not be determined are assessed together. A database match is also a statement of probability; the confidence measure for the match should be given in the report.
The length, number of cysteines and class of the peptide identified in the report for our reference batch, together with its target channel information, are published on the analysis page. This identification belongs to a single batch; the component content is measured anew for each batch.
