An alternative to removing internal peptides for N-terminal peptide identification is to positively enrich for the peptides of interest. Salvesen and colleagues have developed a method for enrichment based on selective guanidinylation of lysine residues using O
-methylisourea (). O
-methylisourea inefficiently modifies protein α-amines, leaving them available for subsequent chemical biotinylation. After digestion with trypsin, N-terminal peptides can be isolated with immobilized streptavidin.[31
] In contrast to the two previous methods for N-terminal modification, this approach discards endogenously N-acetylated N-termini (~80% of all N-termini)[32
] and thus increases the sensitivity for detecting proteolytic cleavages. The authors used their technique to investigate mitochondrial transit peptides from yeast, mouse, and human cells. They found a total of 34 transit peptides from 27 proteins, only 10 of which had been previously annotated, demonstrating the utility of this method for N-terminal discovery. It should be noted that this method depends on highly efficient modification of lysine residues and
highly selective biotinylation of the remaining α-amines. Biotinylation of serine, threonine, or histidine side chains could lead to false identifications. In principle, database matching in the MS-analysis should distinguish real N-terminal peptides from spurious identifications, but incomplete peptide coverage, particularly close to the N-terminus, can introduce false positives.
We have employed subtiligase, an engineered variant of the bacterial protease subtilisin BPN’, to selectively label N-terminal peptides in a single step without lysine derivatization. Created by mutating the catalytic serine to a cysteine and modifying the geometry of this active site residue with a second point mutation (P225A), subtiligase has negligible amidase activity but remains active as an esterase.[33
] The thioester-enzyme intermediate formed with peptide ester substrates during the catalytic cycle is slowly hydrolyzed by water, but can be rapidly intercepted by N-terminal amines, allowing transfer of the N-terminal portion of the ester onto free amines. We have never observed transfer onto peptide or protein side chains, suggesting an enzymatic specificity thus far unachievable via small molecule approaches. Identification of N-terminal peptides was accomplished by treating cell lysates with subtiligase and a biotinylated-peptide ester ().[36
] The resulting peptide mixture was trypsinized, N-terminal peptides were captured on streptavidin beads, and the desired peptides were released by cleavage of a TEV protease site in the original biotinylated peptide. TEV protease cleavage leaves a dipeptide tag that can be used to confirm true positives. In typical runs, >95% of identified peptides contain this tag. Using this method on apoptotic Jurkat cells, we identified 333 caspase-like cleavage sites on 292 protein substrates.[36
] The broad scope of these substrates enabled the discovery of unexpected caspase enzymatic activities- most notably cleavage within helices and systematic cleavage of proteins within protein-protein complexes. Though highly specific, the efficiency of subtiligase N-terminal labeling is low, and thus this technique requires large amounts of material (typically 50–100 mg of a complex mixture per experiment) to identify numerous substrates. However, the highly enriched set of N-termini that result can be routinely analyzed in a single day via LC-MS/MS. Like all N-terminal identification procedures, the identification of one peptide per protein systematically excludes proteins with N-terminal peptides not readily identifiable via MS.