Peptide Calculator

Peptide calculator reads a peptide or a protein fragment sequence and displays: average molecular weight with uncertainty, monoisotopic molecular weight, as well as mass divided by charge values for various stages of protonation and deprotonation (which is useful for mas-spec analysis of charged ions).

Using the peptide properties calculator analytical tool is very easy. Just place a sequence in the edit box, and press the “Calculate” button. This guide shows what is possible with peptide calculator and gives general tips on how to employ the analytical instrument to your advantage in the fields of biotechnology, molecular biology, and proteomics.

Amino Acids

The shortest way of encoding peptide and protein fragment sequences that peptide calculator understands is through the standard one-letter amino acid codes that are available for usual amino acids, like in the following example of a seven-amino acid peptide.

YGGFMRF

With one-letter amino acid code, dashes between adjoining amino acids are optional and can be used for clarity.

Y-G-G-F-M-R-F

When using three-letter amino acid code, dashes separating one amino-acid from the next are required. Following is the same peptide sequence in three-letter code.

Tyr-Gly-Gly-Phe-Met-Arg-Phe

Furthermore, peptide calculator allows mixing one-letter and three letter codes. This is useful when an unusual amino-acid, lacking one-letter code, needs to be inserted in otherwise one-letter encoded sequence. In the following example Norlvaline (Nva) is inserted after the fourth Phenylalanine residue.

YGGF-Nva-MRF

The same sequence in three-letter code is Tyr-Gly-Gly-Phe-Nva-Met-Arg-Phe.

While mixing one and three letter codes is practiced when space is at a premium (such as on small vial labels) in general it is recommended to stick to one way or the other. Fortunately peptide calculator will convert any sequence it understands to three-letter code, so that you do not have to do it manually.

For more information on amino acids, and a complete list of amino acids peptide calculator understands, please see the following article: Amino Acids.

Peptide Termini

By default peptide chains are terminated with carboxyl at C-terminal (normally on the right) and amine at N-terminal (normally on the left). These defaults are often referred to as free termini and can be skipped when writing peptide sequences. Other terminal types must be specified explicitly. Here are a few examples of different termini peptide calculator understands.

Ac-SYSMEH­FRWGKPVG — N-terminal capped with Acetyl

Phe-Met-Arg-Phe-NH2 — C-terminal  Amide (capped C-terminal)

Ac-ATQRLANF­LVHSSNNFGAIL­SSTNVGSNTY-NH2 — peptide both acetylated at N-terminal and amidated at C-terminal

5(6)-FAM-YPSKPDNPGE­DAPAEDMARY­YSALRHYINLI­TRQRY-NH2 — 5(6)-Carboxyfluorescein fluorescent dye on N-terminal and Amide on C-terminal

Biotin-KRREILSRRPSYR — N-terminal biotinylated peptide.

Isotopic Labeling

Stable isotope labeled amino acids, available commercially, allow researchers to stage experiments based on comparison of labeled and unlabeled peptides that while having the same chemical structure can be distinguished by mass.

You can specify isotopic labeling of an amino acid in peptide sequences in square braces to the left of the amino acid symbol. There should be no dashes or spaces between the square braces and the amino acid. For example, in the following sequence, the Alanine residue in the fifth position contains an atom of Nitrogen with nucleon number 15.

KCNT[15N1]ATCATQ

The natural abundance of ¹⁵N on our planet is estimated at less than 0.4%; as such ¹⁵N₁ labeled Alanine gives an approximate +1 mass shift in comparison to unlabeled Alanine.

When labeling different elements in an amino acid, separate them with comas inside the square braces, like in the following example peptide where all nine Carbons and one Nitrogen of the Phenylalanine residue are specific isotopes.

AKRHHGYKRK[13C9,15N1]FH-NH2

When applying isotopic labels, there are a couple of shortcuts. First, you do not have to write “1” after the element symbol if you are only labeling one atom. Second, if all atoms of a specified element in the amino acid are labeled (excluding attachments), then you can writhe “U-“ followed by the nucleon number and the element symbol. “U” stands for unified in unified isotopic labeling of the corresponding amino acid. Therefore (as Phenylalanine contains nine Carbons and one Nitrogen) the two sequences below are the exact equivalent to the labeled peptide sequence above.

AKRHHGYKRK[U-13C,U-15N]FH-NH2

AKRHHGYKRK[U-13C,15N]FH-NH2

Multiple amino acids in a peptide or protein fragment can also be labeled, like the following example demonstrates.

MLSDE­DFKAVFG­MTRSA[15N]FA­NLPLWKQQN­[3H]LKKEKGLF

Once again, labels of an amino acid are specified in square braces before the amino acid. Using dashes can bring more clarity to the way the sequence looks:

MLSDE­DFKAVFG­MTRSA-[15N]F-ANLPL­WKQQN-[3H]L-KKEKGLF

The same sequence in three-letter amino-acid code would look like the following.

Met-Leu-Ser-Asp-Glu-Asp-Phe-Lys-Ala-Val-Phe-Gly-Met-Thr-Arg-Ser-Ala-[15N1]Phe-Ala-Asn-Leu-Pro-Leu-Trp-Lys-Gln-Gln-Asn-[3H1]Leu-Lys-Lys-Glu-Lys-Gly-Leu-Phe

Peptide calculator allows isotopic labeling of any combination of elements in any number of amino acids of a peptide or protein fragment sequence.

Sidechain Attachments

One of the most important if not the most important property that makes one amino-acid different from another is the amino acid sidechain. Sidechains is where many peptide and protein sequence modifications happen, including post-translational modifications.

Whenever there is something attached to the sidechain of an amino acid in peptide sequence, you can specify the attached moiety in round braces immediately after (to the right of) the amino acid. Let us review a few examples of peptides with sidechain attachments that peptide calculator can read.

Asp-Arg-Val-Tyr(PO3)-Ile-His-Pro-Phe

Thr-Tyr-Ile-Cys(PhCH2)-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu

5(6)-FAM-GGAK(Ac)RHRKVL-NH2

Ac-EE-K(Biotin)-GQST-S(PO3)-SHS-K(Ac)-Nle-STEG-K(5-TAMRA)-EE-NH2

In the first example above, the fourth residue is phosphorylated Tyrosine. In the second example, Benzyl is attached to the Cysteine sidechain thiol functional group. The third, fluorescent labeled peptide has its Lysine residue acetylated. And the last, twenty-mer capped peptide features four sidechain modifications: Biotin on the third residue Lysine; the eighth residue is Phosphoserine, the twelfth residue is acetylated Lysine, and Tetramethyl­rhodamine is attached to the sidechain of the eighteenth residue Lysine.

Branched Peptides

Not only simple moieties can attach to amino acid sidechains in peptides or proteins but also whole new chains of amino acids; the result is branched peptides, also known as isopeptides.

To feed peptide calculator with a branched peptide in linear notation, simply specify the branch sequence in round braces after the amino acid at which branching occurs. Here is an example.

YARK(TGNLD)DMRPNA

The sidechain of the fourth Lysine residue of the main chain of the YARKDMRPNA peptide has the DLNGT sub-sequence attached (inverted due to the attachment on the C-terminal side). Figure 1 shows two-dimensional representation of this branched peptide.

Get more detailed information from the article about branched peptides.

Cyclic Peptides

Cyclic peptides are peptides with sequences that form cyclic ring structures; head-to-tail cyclization and disulfide bridge forming are two common arrangements.

To specify a head-to-tail cyclic peptide, enclose the entire sequence in round braces, and write the word “cyclo” before it, like follows.

cyclo(Val-Orn-Leu-D-Phe-Pro)

Disulfide bridges are specified in square braces in front of the peptide sequence (leave a space between the closing square brace and the beginning of the sequence to help distinguishing instructions that apply to the whole peptide from possible labels that apply to the first amino acid residue only). Following is an example of a peptide with three disulfide bridges.

[Cys8-Cys28,Cys14-Cys33,Cys18-Cys35] GVEIN­VKCSGSPQ­CLKPCKDA­GMRFGKCMN­RKCHCTPK

If only the first and last Cysteine residues in a sequence form a bridge, there is a shorthand notation of simply adding the word “Cyclic” in front of the peptide. As such the following peptide reference…

Cyclic AGCKNF­FWKTYTSC

…is the exact equivalent of…

[Cys3-Cys14] AGCKNF­FWKTYTSC

Cyclic peptides article contains material on these and other cyclization methods peptide calculator can work with.

Conclusion

This guide showed a few examples of what is possible with peptide properties calculator on peptidenexus.com. The goal of this analytical tool is to be as unobtrusive as possible and lean on conventions already developed by peptide and protein scientists and researchers.

Please do not hesitate to contact us, with your feedback or ideas. Thank you.