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H/D Exchange Calculator

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Tool description

Simple calculator providing information on number of fast and slow exchangeable hydrogens within peptide/protein. It also calculates accurate monoisotopic and average masses (the table with the values used for calculation can be found below). The calculator works with peptide/protein sequence(s) in one-letter amino acid code. The tool takes into account that peptide bond at the N-terminus of proline does not contain exchangeable backbone amide hydrogen. It does not simply calculate the number of exchangeable sites as “all sites-prolines” but takes care also for the position of proline (N-terminal proline residue exception).

Input fields

There are two ways how to upload your data. One is to copy-paste peptide/protein sequence into the field Sequence. If you wish to enter several sequences/peptides, enter one sequence per line or separate them by a space. If there is any character not representing the amino acid, it is reported above the result table and peptide is ignored. The field is case insensitive.

Second way is to paste entire protein sequence into the field Sequence and use the second field Peptide file for upload of a simple text file containing the limits of individual peptides. Peptide limits are represented either by “white space character" (space or tab) separated numbers denoting starting and ending position of the peptide or by the peptide sequence itself. Example Peptide file and Sequence can be downloaded here – PeptideFile.txt and Sequence.txt. Wrong limits (numbers lower than 1 or higher than sequence length or wrong formatting) are reported in result, if it is not the first line in the file. The first line of the Peptide file can be used for headers/any comments. Be sure that the pasted sequence is a continuous single line string and does not contain any spaces.

Charged mass — tick this box if you want to obtain table with multiply charged m/z values of the uploaded peptides (calculated only for the monoisotopic mass). Charge limit field sets the upper limit of the charge. E.g. by entering 4 the script will list m/z values of 1+, 2+, 3+ and 4+ peptides. If the Charged masses box is selected but no number entered only 1+ will be calculated. The upper limit is 10. The results are shown in two separate tables but are merged in the downloadable tab-delimited file (hyperlink bellow the tables - tab separated result file).

Uniqueness — tick this box if you want to check the uniqueness of masses of your peptides. Toleration (ppm) field sets the tolerance (in ppm) for two peptides to be considered as having the same mass. These results are not included in the tab separated result file.

Results

Len – length of the peptide

P, STYCDEHW, KQN, R – number of resides in each of those four groups. The amino acids are grouped based on the number of side chain exchangeable hydrogens

Backbone – number of exchangeable backbone amides

Fast exchangeable &ndash number of exchangeable side chains (fast exchanging)

Sum – total number of exchangeable hydrogens (peptide bond+side chain)

Mono mass – monoisotopic mass

Avg mass – average mass

Backbone deuterated mass – average mass of peptide fully deuterated on backbone amides

Fully deuterated mass – average mass of a peptide fully deuterated on all exchangeable sites

Tips and tricks

Results are displayed in the table suitable to print or copy to (for example) the word processing application. If you prefer to insert data into a spreadsheet, you should use tab separated result file which can be displayed/downloaded using the tab separated result file link present below the result table.

Monoisotopic MassAverage Mass
A71.037113671.0779
C103.0091845103.1429
D115.0269428115.0874
E129.0425928129.1140
F147.0684136147.1739
G57.021463657.0513
H137.0589116137.1393
I113.0840636113.1576
K128.0949626128.1723
L113.0840636113.1576
M131.0404843131.1961
N114.0429272114.1026
P97.052763697.1152
Q128.0585772128.1292
R156.1011106156.1857
S87.032028287.0773
T101.0476782101.1039
V99.068413699.1311
W186.0793126186.2099
Y163.0633282163.1733
H+1.0072760
Δ2H1.0062767
H2O18.0105618.01528