= Trypsin cleavage specificity
Write a <peptide>'s <protein primary structure> from its <N-terminus> toward its <C-terminus>. The usual simplified <trypsin cleavage specificity> rule places a cut immediately after K (<lysine>) or R (<arginine>), unless the next <amino acid residue> is P (<proline>). The following <amino acid residue> is essential: a terminal K or R has no following <peptide bond> to cut. The inspected institutional documentation describes <proline> as usually blocking cleavage, and explicitly lists sequence contexts in which that blocking is negligible. It also warns that experimental results can differ from sequence-based predictions. Thus “never before <proline>” is too strong.<PeptideCutter - Cleavage specificities of selected enzymes and chemicals (ExPASy)>[PeptideCutter: Cleavage specificities of selected enzymes and chemicals, General model; Establishing a specificity model; Trypsin; Cleavage rules and exceptions]{ref}
For an original worked example, stipulate complete cleavage at all sites allowed by that simplified rule in the twelve-<amino acid residue> sequence
$$
\mathrm{AGKARPKAGRAK}\quad\longrightarrow\quad
\mathrm{AGK}\;|\;\mathrm{ARPK}\;|\;\mathrm{AGR}\;|\;\mathrm{AK}.
$$
A denotes <alanine>, G denotes <glycine>, and K, R and P have the meanings above. Cuts follow positions $3$, $7$ and $10$. Position $5$ is R, but its following P suppresses that cut in the stipulated rule; position $12$ is terminal K. Notice that the R in ARPK is retained inside the fragment, despite being a usually favoured <trypsin> target.
\Image[/trypsin-peptide-cleavage-map.png]
{title=<Trypsin cleavage specificity>: three selected cuts make four <peptide> fragments}
{description=Original sequence and prediction under the simplified K/R rule with suppression before <proline>. Blue plus signs identify predominantly positive <lysine> and <arginine> <amino acid side chains> near neutral <pH>, not the net <electric charge> of each fragment. The example is constructed, not an observed digest.}
Near neutral <pH>, the <lysine> and <arginine> <amino acid side chains> undergo predominant <protonation> and carry positive <electric charge>. This helps distinguish those <amino acid residues> from the uncharged letter symbols used to write the sequence; those symbols specify identities, not whole-fragment <electric charges>.<Organic Chemistry by John McMurry/26.1 Structures of Amino Acids>[Organic Chemistry by John McMurry; Table 26.1, lysine and arginine rows; concluding classification paragraph, side-chain protonation at pH 7.3]{ref}
Fragment accounting checks the prediction: lengths $3+4+3+2=12$ conserve the twelve <amino acid residues>. The original linear chain has eleven <peptide bonds>; the four fragments retain $2+3+2+1=8$, so three were hydrolysed. Three <water> molecules are consumed per original chain in this formal <stoichiometry>. More generally, cutting $m$ distinct bonds of one linear <peptide> makes $m+1$ fragments: each cut splits one connected piece into two. This argument assumes an unbranched chain without additional links joining the pieces.
A <missed trypsin cleavage> at position $7$ would instead give $\mathrm{AGK}\;|\;\mathrm{ARPKAGR}\;|\;\mathrm{AK}$, merging two neighbouring fragments without changing their order. Even a correct cleavage preference does not guarantee complete <proteolysis>; predicted fragment boundaries describe a model, while actual boundaries require evidence from the digest.
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