Due to scheduled maintenance work, this service may not be available on Monday January 22nd between 08.00 am and 9.00 am CEST
To improve security and privacy, we are moving our web pages and services from HTTP to HTTPS.
To give users of web services time to transition to HTTPS, we will support separate HTTP and HTTPS services until the end of 2017.
From January 2018 most HTTP traffic will be automatically redirected to HTTPS. [more...] View this page in https
Instructions for PeptideMass Peptide
This program is designed to calculate the theoretical masses of peptides
generated by the chemical or enzymatic cleavage of proteins, to assist in
the interpretation of peptide mass fingerprinting and peptide mapping
experiments. Protein sequences can be provided by the user or can be a code
name for a protein in the UniProt Knowledgebase (Swiss-Prot or TrEMBL)
. When proteins of
interest are specified from UniProtKB/Swiss-Prot, the program considers
all annotations for that protein in the UniProtKB/Swiss-Prot database, and uses these
in order to generate the correct peptide masses and warn users about
peptides that are not likely to be found when undertaking peptide mass
fingerprinting. Many protein post-translational modifications
the masses of peptides can thus be taken into consideration.
2. Using PeptideMass:
In the program there are a number of fields that must be filled in. Here we
will describe these fields, from the top to the bottom of the form.
In the following set of notes, instructions you should follow for filling in
the form on the computer will be shown with a . Background notes relevant
to that part of the form will be marked with a .
2.1. Protein Sequence to be Cleaved
Here you should specify the sequence of the protein you would like to
cleave. If this protein is known in UniProtKB, enter the
UniProtKB/Swiss-Prot ID code (e.g. ALBU_HUMAN
) or the protein accession number (e.g. P02769
If the protein
is not known in UniProtKB, you can enter the sequence of your
protein of interest, in single letter amino acid code, in either upper or
lower case. However, you can only specify one sequence at a time in this case.
Hint: Protein sequences from other sources (e.g. word processor programs or
other Web pages) can be copied and pasted directly into this field.
In such cases the program allows to specify post-translational modifications
along with the sequence, provided that the input
format is respected.
If there are spaces in your sequence,
these will be ignored. Note the residue J will be treated as either Ile or Leu,
which have the same average and monoisotopic masses.
The characters B, X, or Z (see Comment 5 of the
Compute pI/Mw documentation) are accepted, but no masses are computed for
peptides containing one or more of
these characters. These peptides are listed separately at the end of the output page.
2.2. Enzyme or Reagent to Use for Cleavage
Here you should select the enzyme or reagent with which you would like to
program to use in calculating the theoretical peptide masses. The rules
that are used to cleave the proteins are shown in Table 1
2.3. Modifications to Cysteines in Peptides
You can choose how you would like all cysteines in a protein to be
modified, before the theoretical masses of peptides are calculated.
Experimentally, proteins are usually subjected to reduction and then
alkylation with different reagents before they are used to generate
peptides. If you would like the masses of unmodified cysteines in your
peptides, check "nothing (in reduced form)" in the menu 'cysteines treated with'. If you would like all
cysteines to be theoretically reduced and alkylated, specify
the reagent to be used for alkylation.
You have a choice of iodoacetamide, iodoacetic acid and 4-vinyl pyridene.
If you wish to generate masses for peptides with acrylamide adducts, you can check
the box 'with acrylamide adducts'.
The program will then modify the theoretical masses of
Cys-containing peptides accordingly. Note that in proteins prepared by
polyacrylamide gel electrophoresis, it can be common for cysteines to have
reacted with free acrylamide monomers.
2.4. Modifications to Methionines in Peptides
You can request for all methionines in theoretical peptides to be
oxidised. If this option is selected, the program will modify the
theoretical masses of all Met-containing peptides accordingly. Note that
proteins prepared by gel electrophoresis often show this modification.
If several types of artifactual modifications are documented for one peptide
(e.g. alkylated cysteines, oxidized methionines, Homoserine lactones), a separate mass
of the peptide will be given for each of these modifications. For combinations
of modifications, the user is advised to refer to the
table of masses used by the ExPASy tools, or to use the FindMod tool.
2.5. Peptide Mass Range & Sorting
Here you can specify a low mass cutoff, such that any peptides below that
mass will not be shown in the results. You should then choose if you would
like the peptides to be sorted by their mass (from largest to smallest) or
by their chronological order in the protein.
2.6. Modifications, Conflicts, Variants, and Alternative Splicing
Here you can specify which of the annotations in the UniProtKB/Swiss-Prot database
you would like to be taken into account, or warned of, during the
calculation of your peptide masses. If you select any of these, the program
will print them in the results output if they are known for your protein of
Note: This feature allows you to see which peptides in a protein are
likely to be unmodified, and thus easy to find in peptide mass
fingerprinting procedures, and which peptides may present problems. For a
full explanation of how these modifications are taken into account, see
sections 3.3. to 3.8. below.
2.7. Reset and Perform Buttons
Once you have filled in the form according to your needs, press the
Perform button. If you have made a mistake and would like all fields to be
reset to their default values, press the Reset button.
3. Special Features of PEPTIDE MASS:
The following section provides details as to how the program works, and how
it uses a maximum of information available from the UniProtKB/Swiss-Prot database to
calcluate the masses of peptides.
3.1. Mass Calculations
have been calculated to 4 or 5 decimal places for all amino acids and
Average isotopic and monoisotopic mass values
are available for all of these modifications.
The rules that are used to cut proteins in the program are as summarised in
. Note that to take into account partial cleavages,
it is possible to specify a maximum number (0, 1, 2, or 3) of missed cleavages to be considered.
Table 1: Cleavage rules for PEPTIDE MASS program.
Enzyme or Reagent
||C-terminal side of K or R
||if P is C-term to K or R
|Trypsin (C-term to K/R, even before P)||C-terminal side of K or R
|Trypsin (higher specificity)||C-terminal side of K or R
||if P is C-term to K or R; after K in CKY, DKD, CKH, CKD, KKR; after R in RRH, RRR, CRK, DRD, RRF, KRR
||C-terminal side of K
||N-terminal side of K
||C-terminal side of M
||C-terminal side of R
||if P is C-term to R
||N-terminal side of D
|Asp N + Lys C
||N-terminal side of D, C-terminal side of K
|Asp N + N-terminal Glu
||N-terminal side of D or E
|Asp N + Glu C (bicarbonate)
||N-terminal side of D, C-terminal side of E
|Glu C (bicarbonate)
||C-terminal side of E
||if P is C-term to E, or if E is C-term to E
|Glu C (phosphate)
||C-terminal side of D or E
||if P is C-term to D or E, or if E is C-term to D or E
|Glu C (phosphate) + Lys C
||C-terminal side of D, E and K
||if P is C-term to D or E, or if E is C-term to D or E
|Glu C (phosphate) + Trypsin
||C-terminal side of D, E, K, R
||if P is C-term to K, R, D, E, or if E is C-term to D or E
|Glu C (phosphate) + Chymotrypsin
||C-terminal side of F, L, M, W, Y, D, E
||if P is C-term to F, L, M, W, Y, D, E, or if E is C-term to D or E, not after Y if P is N-term to Y
|Microwave-assisted formic acid hydrolysis (C-term to D)
||C-terminal side of D
|Chymotrypsin (C-term to F/Y/W/M/L, not before P, not after Y if P is C-term to Y)
||C-terminal side of F, L, M, W, Y
||if P is C-term to F, L, M, W, Y, if P is N-term to Y
|Chymotrypsin (C-term to F/Y/W/, not before P, not after Y if P is C-term to Y)
||C-terminal side of F, Y, W
||if P is C-term to F, Y, W, if P is N-term to Y
|Trypsin/Chymotrypsin (C-term to K/R/F/Y/W, not before P, not after Y if P is C-term to Y)
||C-terminal side of K, R, F, Y, W
||if P is C-term to K, R, F, Y, W, if P is N-term to Y
|Pepsin (pH 1.3)
||C-terminal side of F, L
|Pepsin (pH > 2)
||C-terminal side of F, L, W, Y, A, E, Q
||C-terminal side of A, F, Y, W, L, I, V
||N-terminal side of A, F, I, L, M, V
||if D or E is N-term to A, F, I, L, M, V
Cyanogen Bromide (CNBr) cleaves at Met and
converts the Met into a modified amino acid called Homoserine lactone (HSL).
Unlike in the calculation of peptide masses in most other cases,
where 18 mass units are added after the addition of all amino acid
and modification masses to account for the N-terminal H on the NH3 group and the
C-terminal OH on the COOH group, peptides with a C-terminal Homoserine lactone
only have one extra hydrogen added at the N-terminus. This is because the OH at the
C-terminus is actually absent if there is a C-terminal Homoserine
3.3. Signal Sequences, Propeptides & Transit Peptides
and transit sequences
are all removed from
proteins before cleavage rules are applied. A message is shown at the top
of the list of results if any of these are present in a protein. All will be
removed before generating the masses of peptides from the mature protein.
3.4. Chains and Polypeptides that Produce Multiple Mature Proteins
If there are known chains
that are created from any database entry, these
are considered as different polypeptides (e.g. A2HS_HUMAN
). Thus in the
list of results, there will be a different list of peptides for each of
these chains. The same applies to any proteins which are known to form
multiple mature peptides
or proteins from a single initial polypeptide (e.g.
3.5. Protein Post-Translational Modifications
All documented post-translational modifications of a protein in the
UniProtKB/Swiss-Prot database (including the annotations MOD_RES
), are considered by the program. It will indicate the peptide
which will carry the modification, detailing the type of modification and
the number of the residue that carries it. The type of the modification is usually
represented by the first four letters
of the code used in UniProtKB/Swiss-Prot
that post-translational modification.
For modifications that are simple and discrete (acetylation, amidation, biotin, C-mannosylation,
deamidation, dimethylation, farnesylation, formylation, geranyl-geranyl,
gamma-carboxyglutamic acid, O-GlcNac, hydroxylation, methylation,
myristoylation, palmitoylation, phosphorylation, pyrrolidone carboxylic acid,
sulfatation and trimethylation),
the predicted modified mass of
the peptide will be given. Currently, if there is more than one type of
modification in any peptide, a separate mass of the peptide will be given
for each type of modification. Thus there can be more than one modified mass
for a single peptide. For complex post-translational modifications, including
N- and O- glycosylation and phosphatidyl inositol glycan anchors, it is
difficult or impossible to predict the modified mass of a peptide. No
predicted theoretical mass is supplied in such cases.
3.6. Conflicts in the Database
If there are known conflicts in the database, which may represent database
errors, these are shown under the "conflict" column, corresponding to the
peptide that may be affected. In effect this is a caution that such
peptides may not necessarily be found in preparations of a sample. It is
currently not possible (apart from manually modifying the sequence and
re-cutting with the program) for the program to give you the masses of any
alternative peptides that would be created by the conflict. However, to
facilitate further investigation of such cases, the conflict itself is shown
in the "conflict" column (e.g. 168: F -> K). If there is more than one
residue changed in any peptide, the number represents the number of the
first changed residue. If there is a residue conflict that is a missing
amino acid, it will be represented as, for example: 168-169 MISS.
Refer to the user manual to see how conflicts are annotated
3.7. Variants in the Database
If there are known variants in the database, representing isoforms of the
same protein, these will be shown corresponding to the peptide that may be
affected. To facilitate further investigation of such cases, the variant
itself is shown in the "variant" column (e.g. 46: L -> S). If there is more
than one residue changed in any peptide, the number represents the number of
the first changed residue (e.g. 48: GLVVR -> PSSCARV).
Refer to the user manual to see how variants are annotated
3.8. Alternative Splicing in the Database
If there are known protein isoforms which correspond to
differentially spliced versions of a single mRNA species (e.g Q02040
these will be shown in the "varsplice" column corresponding to the peptide
that may be affected. The program will show the numbers of the residues
that may be affected in the peptide, by either the addition of extra
residues, or by the deletion of some residues from that area. The letter
code of the amino acids within any particular affected peptide are not shown.
If you wish to perform a theoretical digest of one of the annotated
splice isoforms, you can click on the IsoId in the NiceProt view of the underlying
UniProtKB entry, e.g. the short isoform Q02040-2
. This leads to a page displaying the sequence of this isoform
and containing links for the direct submission of that sequence to a number of tools,
Refer to the UniProtKB entry view manual to see how alternative splicing is annotated
3.9. Hypertext Link in Results Page
If a protein from the UniProtKB is the protein under study, a
hypertext link will be provided to the protein at the top of the results
page. This link allows the user to effortlessly retrieve the full
UniProtKB listing for that protein. Note that if the user wishes to, for
example, change the sequence from a UniProtKB/Swiss-Prot entry to take into account a
variant, it is easy for the sequence to be copied from the UniProtKB/Swiss-Prot entry,
pasted into the sequence field (see 2.1.
), modified as necessary, and then
cleaved as required.
In case of known chains or multiple mature peptides
(see 3.4.), hypertext links are provided to a page that
highlights the subsequence in question.