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Home > Peptide Sequencing Techniques > Enzyme and Chemical Fragmentations


Enzyme and Chemical Fragmentation

Enzyme Fragmentations: TRPS, TRMA, TRDK, TREI, PEPS, CHYT, THML

Chemical Fragmentations: ASPH, BRCN, NAAM, NBS3, NBS4

Fragmentations are carried out with either enzymes or chemicals which cause peptide bonds to break next to particular aminoacids.

Peptides are fragmented primarily to reduce their size, since information from exopeptidase digestion is more easily interpreted in fragments shorter than 6 to 8 aminoacids. Occasionally, structural information may be infered when the number of fragments produced is fewer than expected.

The products of a fragmentation are listed reproducibly, that is always in the same order (of decreasing length of chain). When a fragment produced is already present in memory, it is identified by its current fragment number; if a fragment has previously been prepared, but is not now present, it is given a new identification number.

Yields on all fragmentation experiments (successful or not) are 80% (based on moles).

The minimum quantity of fragments reported is 0.1 mg. If in doubt whether a fragmentation will occur or not, try out the experiment on about 1 µ-mole, then repeat the fragmentation with most of your peptide. It is useful to reserve 0.1 mg for comparison or naming.

To reduce a peptide to fragments 2 to 5 aminoaminocids in length may require several fragmentations in succession; after each fragmentation, the composition and N-terminus of each fragment must be determined.

The quantity of a peptide to be fragmented should be enough for the required analysis and N-terminus determination and can be estimated at about 1 µ-mole per successive fragmentation + 2 µ-mole for the final determination (this includes some allowance for losses).

When selecting a fragmentation, the reagent should be capable of causing hydrolysis at either only 1 site or at 2 sites on the C-side of an known aminoacid, since: 

N------------CO--N------------CO--N------------C 
^(1)                ^(2)^(3)                 ^(4)^(5)                 ^(6)

gives:

N(1)---C(2): both N(1) and C(2) termini are known
N(3)---C(4): C(4) terminus known, N(3) terminus not known
N(5)---C(6): neither N(5), nor C(6) terminus (usually) known

    If more than 3 fragments were to be produced, there would be at least 2 fragments with a known C and an unknown N terminus whose relative mutual position would be indeterminate. Before attempting any fragmentation, all possible outcomes of the experiment and alternative strategies should be considered.

    Fragmentation by an enzyme will give products containing a minimum of 2 aminoacids each.

    This means that no fragmentation by an enzyme will occur if the bond to be broken is the bond to the N- or the C-terminus aminoacid of the peptide.

    With this proviso, PepSeq enzymes (differing slightly from real ones) always either:

    • Catalyse in 80% yield the breaking of bonds at the C- or N-terminus of all succeptible aminoacids irrespective of their neighbours; or
    • In other cases catalyse the breaking of the bonds of particular amino-acids in a pseudo-random but reproducible manner which depends on the structure of the substrate.

    Chemical reagents may produce single aminoacids as fragments.

    PepSeq chemical reagents do not degrade the chemical properties of the fragments produced so that these can be subjected to further identification procedures.


    Enzyme Fragmentation

    TRPS: Treatment with trypsin. 

    Catalyses hydrolysis at the C-terminal of arginine and lysine (Arg and Lys), these being basic aminoacids.

    TRMA: Acylation with maleic anhydride, followed by trypsin.

    Catalyses hydrolysis at the C-terminal of arginine (Arg) only.

    Treatment with maleic anhydride converts the basic amino group on lysine (that makes it succeptible to hydrolysis by the enzyme) to the neutral amide. The more basic guanidino group of arginine is not affected.

    TRDK: Treatment with cyclohexandione, followed by trypsin.

    Catalyses hydrolysis at the C-terminal of lysine (Lys) only.

    Cyclohexandione forms a 5-membered ring with the guanidino group on arginine (that makes it susceptible to hydrolysis by the enzyme) and neutralizes it. The less basic amino group of lysine is not affected.

    TREI: Treatment with ethyleneimine, followed by trypsin.

    Catalyses hydrolysis at the C-terminals of arginine, cysteine and lysine (Arg, Cys and Lys).

    Treatment with ethyleneimine attaches a 2-aminoethyl group to the sulphur atom of cysteine. The resulting aminoacid is basic and so is susceptible to hydrolysis by trypsin.

    PEPS: Treatment with pepsin.

    Catalyses hydrolysis at the C-terminals of leucine, tryptophan and tyrosine (Leu, Trp and Tyr); also occasionally (about once in three times) at histidine and methionine (His and Met).

    CHYT: Treatment with chymotrypsin.

    Catalyses hydrolysis at the C-terminals of phenylalanine, tryptophan and tyrosine (Phe, Trp and Tyr); also occasionally (about once in three times) at aspartic acid, glutamic acid, histidine, leucine, methionine and valine (Asp, Glu, His, Leu, Met and Val).

    THML: Treatment with thermolysin.

    Catalyses hydrolysis at the N-terminal of alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine and valine (Ala, Ile, Leu, Met, Phe, Tyr and Val).


    Chemical Fragmentation

    ASPH: Mild hydrolysis with 0.25M acetic acid, 60 hrs reflux.

    Both the C- and N-terminal bonds of aspartic acid are hydrolysed (Asp).

    BRCN: Treatment with cyanogen bromide.

    Cleaves the C-terminal peptide link of methionine (Met).

    NAAM: Hydrolysis with Na metal in liquid ammonia.

    Hydrolyses at the N-terminal of proline (Pro).

    NBS3: Treatment with N-bromosuccinimide at pH 3.5 .

    Cleaves the C-terminal peptide link of tryptophan (Trp).

    NBS4: Treatment with N-bromosuccinimide at pH 4.5 .

    Cleaves the C-terminal bonds of histidine, tryptophan and tyrosine (His, Trp and Tyr).


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