About the MAP Format

3. Overview of the Format

A MAP format entry is structured similarly to a FASTA entry, with a header line followed by a sequence line. The key difference is the use of curly braces { } in the sequence to denote annotations or modifications of the standard residues. A MAP format entry consists of two parts: a header line and one or more sequence lines. This structure is similar to FASTA, with enhancements to include annotations:

3.1 Header Line

• It always starts with a > symbol.
• Right after the >, you'll find the protein's identifier (like a name or code).
• Then, you can optionally include metadata tags enclosed in curly braces {}. Each tag gives you a specific piece of information about the entire protein.
• Multiple tags are separated by spaces.

3.2 Sequence Lines

• These lines contain the one-letter codes for the amino acids in the protein.
• The magic of MAP happens here\! If an amino acid has a modification or annotation, it's immediately followed by a tag in curly braces {...} that describes it.
• These tags provide details about a specific amino acid without breaking the main sequence.

4. How MAP Extends the FASTA Format

MAP builds upon the simple structure of FASTA to add more layers of information.

4.1 Traditional FASTA Format

• It's a basic text format for representing sequences.
• Each entry has a header line (starting with >) with an identifier and maybe a description.
• Followed by sequence lines containing only the standard 20 amino acid letters (or nucleotide). No modifications are directly indicated in the sequence.

4.2 Extension of FASTA for MAP

• MAP keeps the basic FASTA structure, so it's still compatible with tools that read FASTA.
• Header Enhancement: MAP headers can include the same information as FASTA, but they can also contain structured metadata within curly braces {} to give more context about the whole protein.
• Sequence Enhancement: This is the key difference. MAP uses curly braces {} within the sequence line to directly annotate modifications or special features of individual amino acids.

5. Residue-Level Modifications & Annotations

Residue-level modifications refer to chemical changes or non-standard features of specific amino acids in the sequence. These tags, placed right after an amino acid in the sequence, describe specific changes or features of that amino acid. The general format for modification tags is {prefix:Code}.

• Cyclization (e.g., disulfide bonds or head-to-tail cyclization)
• Terminal modifications (modifications at the N-terminus or C-terminus of the protein)
• D-amino acids (non-standard chirality of amino acids)
• Common post-translational modifications (PTMs)
• Other non-natural chemical modifications (synthetic modifications, e.g., PEGylation)
• Non-natural amino acid residues (unnatural amino acids incorporated into the sequence)
• Conjugation of macromolecules (attachment of larger moieties like lipids or other biomolecules)
• Isotopic and fluorescent labels

5.1 Cyclization in Proteins/Peptides

Cyclization means forming a loop or ring structure within a peptide. The tag is {cyc:}.

5.2 Terminal Modifications

Proteins often undergo modifications at their N-terminus or C-terminus, which can alter stability, localization, or activity. For example, N-terminal acetylation and C-terminal amidation are common modifications in peptides and proteins that can protect against degradation or alter function. In MAP format, terminal modifications are indicated by {nt:Type} for N-terminal modifications and {ct:Type} for C-terminal modifications, placed at the respective end of the sequence.

• The {nt:...} tag is placed after the last character of the sequence if it denotes a modification of the N-terminus, because conceptually it modifies the start of the chain. (It might seem counterintuitive to place it at the end, but since the sequence is written N-to-C, we indicate it at the position of the N-terminus, which is before the first residue. In practice, we append the tag after the sequence string for formatting reasons, to avoid confusion at the very beginning of the line.)
• Similarly, a {ct:...} tag is placed at the end of the sequence to denote a C-terminal modification.

Common N-terminal modifications include amidation, formylation, acetylation, glycosylation, and methylation of the N-terminus. Common C-terminal modifications include amidation, glycosylation, and addition of other groups like a carboxylate modifications or peptide tags.

Examples of N and C-terminal modifications:

5.3 D-Amino Acids

This tag {d} Most amino acids in natural proteins are in the L configuration (chirality). indicates that an amino acid has a "mirror image" (D-form) structure instead of the usual L-form and are not commonly found in proteins produced by ribosomes, though they do occur in some peptides (like certain bacterial cell wall peptides and some neuropeptides). Incorporating D-amino acids into a peptide can dramatically increase resistance to proteases and change the peptide’s structure and activity

• In the MAP format, a D-amino acid is indicated by the tag {d} placed immediately after the amino acid letter. This tag signifies that the residue is the D-enantiomer instead of the normal L-form.

5.4 Post-Translational Modifications (PTMs)

• Post-translational modifications (PTMs) are covalent modifications to amino acids that occur after a protein has been synthesized (translated).
• PTMs are crucial for regulating protein function, stability, localization, and interactions. Common PTMs include phosphorylation, glycosylation, acetylation, methylation, ubiquitination, sumoylation, hydroxylation, and lipidation (among others).
• In MAP format, a post-translational modification on a specific residue is denoted with a {ptm:Code} tag immediately after that residue.
• The ptm prefix indicates a PTM, and the code specifies the type of modification. Here are some common PTM tags and their meanings:

5.5 Non-Natural Modifications (NNM)

Non-natural modifications (often abbreviated here as NNM) are chemical modifications that are not typical biological PTMs. These include synthetic modifications like attaching polymers, adding fluorescent probes (though we cover labels in 5.8 separately), or other chemical groups that are not gene-encoded modifications. These modifications are often used in drug development or protein engineering to enhance stability or to attach drugs/labels.

In MAP, we use the {nnm:Code} tag for non-natural modifications. Some examples:

5.6 Non-Natural Residues (NNR)

Non-natural residues (NNRs) are amino acids not among the standard 20 genetically encoded amino acids. These could be amino acid analogs or synthetic amino acids incorporated via chemical peptide synthesis or specialized translational machinery (such as tRNA suppressor techniques in synthetic biology). NNRs allow for fine-tuned control of protein properties, such as adding functional groups not normally present, enhancing stability, or creating binding sites.

In MAP format, non-natural amino acids are indicated with the {nnr:Code} tag at the position where the non-natural amino acid occurs. Typically, the one-letter code of the standard amino acid is not used for these; instead, the sequence might show a placeholder or the preceding residue, but since we still need a letter in the sequence for FASTA compatibility, one approach is to use a similar letter or X for unknown. However, a clearer approach in MAP is to use a specific single-letter code if available (for example, many unnatural amino acids do not have standard one-letter codes, so one might choose a letter like X or Z to represent them and then clarify with the tag). For this manual, we will assume the sequence uses an appropriate placeholder letter (like X) and the {nnr:Name} tag clarifies what that residue is. Alternatively, some unnatural amino acids have been assigned one-letter codes in certain contexts (like U for selenocysteine, O for pyrrolysine, etc., which are actually natural but rare amino acids). For truly non-natural, often X is used as placeholder.

Common examples of NNR tags (the code after nnr: is usually a short abbreviation of the residue name):

5.7 Conjugation of Macromolecules

Proteins can be conjugated to other molecules such as polymers, lipids, or other biomolecules (like attaching a protein to a DNA, or to a drug molecule). These conjugations often involve a covalent bond between a specific amino acid and another molecular entity. In the MAP format, we denote such attachments with the {conj:Code} tag, where conj stands for conjugation.

5.8 Isotopic & Fluorescent Labeling

Isotopic labeling and fluorescent labeling are special cases of modifications often used in experimental contexts:

• Isotopic labeling: incorporating stable isotopes like ^13C, ^15N, ^2H (deuterium), or ^18O into specific positions in the protein for NMR studies or mass spectrometry. These do not change the chemical structure in a way that alters the amino acid letter (it’s still the same amino acid, just heavier), but it is useful to annotate them.
• Fluorescent labeling:attaching a fluorescent dye (such as fluorescein, rhodamine, Cy5, FITC, etc.) to a residue (commonly lysine or cysteine) for detection or imaging purposes.

In MAP format, these can be represented with an {iso:Code} tag (think of iso as indicating either isotope or possibly "label"). We choose iso as a prefix to cover these labeling cases for simplicity.

Complete example:

>Peptide010
AK{iso:Fluorescein}DEFA{iso:13C}HIK

This indicates the lysine (K) is labeled with a fluorescein dye, and the alanine (A) later in the sequence is ^13C-labeled. In practice, one might label multiple residues or uniformly label a protein. The MAP format allows indicating specific labeled sites if needed.

Having covered various chemical modifications and special residues (Sections 5.1 through 5.8), we now move on to annotation of functional sites—namely, residues that interact with other molecules—and to indicating sequence variants. These are not chemical modifications per se, but rather annotations of residue function or changes in the primary sequence.

6. Residue Annotations (Interaction Sites)

Beyond chemical modifications, it is often useful to annotate residues that have certain functional roles or interactions. For instance, an enzyme active site, a DNA-binding residue, or a metal-binding site are not "modified" in a chemical sense, but we want to mark them as important. The MAP format includes an {IR:...} tag for Interacting Residues or Important Residues, which can denote interactions with various biomolecules or ligands.

The prefix IR stands for "Interaction Residue" (or one might think "Interacts with..."). The code following IR: specifies the type of interaction or the molecule with which the residue interacts.

Complete example usage in a sequence with multiple interactions::

>Peptide011
ACD{IR:ATP}FK{IR:PI}HI{IR:miR-21}AT{IR:DNA}

Breaking down this example:

• D{IR:ATP} – aspartate interacting with ATP.
• K{IR:PI} – lysine interacting with "PI". Here, PI could stand for phosphatidylinositol (a lipid) or inorganic phosphate. Given context, it likely means a phosphatidylinositol (a lipid signaling molecule), showing that the format could allow such abbreviations.
• I{IR:miR-21} – isoleucine interacting with miR-21, which implies this residue is involved in binding a microRNA (miR-21). This is an example of using a specific molecule name (miR-21) in the tag.
• T{IR:DNA} – threonine interacting with DNA.

This illustrates that the IR tag can be customized for a wide range of interaction partners. The MAP format user should ensure that any custom codes used after IR: are understandable (perhaps by providing a legend or comments elsewhere, since the format itself doesn’t enforce a controlled vocabulary for the ligand names). In summary, the {IR:...} tags allow annotation of residues by their interaction partners, enriching the sequence with functional information beyond just the amino acid modifications.