Pseudotrypsin: A Little-Known Trypsin Proteoform

Abstract

Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin, which is produced by a limited autolysis of β-trypsin. An additional intra-chain split leads to pseudotrypsin (ψ-trypsin) with three chains interconnected by disulfide bonds, which can be isolated from the autolyzate by ion-exchange chromatography. Based on experimental data with artificial substrates, peptides, and protein standards, ψ-trypsin shows altered kinetic properties, thermodynamic stability and cleavage site preference (and partly also cleavage specificity) compared to the above-mentioned proteoforms. In our laboratory, we have analyzed the performance of bovine ψ-trypsin in the digestion of protein samples with a different complexity. It cleaves predominantly at the characteristic trypsin cleavage sites. However, in a comparison with common tryptic digestion, non-specific cleavages occur more frequently (mostly after the aromatic residues of Tyr and Phe) and more missed cleavages are generated. Because of the preferential cleavages after the basic residues and more developed side specificity, which is not expected to occur for the major trypsin forms (but may appear anyway because of their autolysis), ψ-trypsin produces valuable information, which is complementary in part to data based on a strictly specific trypsin digestion and thus can be unnoticed following common proteomics protocols.

Nonspecific cleavages arising from reconstitution of trypsin under mildly acidic conditions

“…The formation of pseudotrypsin (ψ-trypsin) [ 35 , 36 ], a known variant of trypsin generated from the autolytic bond opening between K176 and D177 following an interchain split between K131 and S132 that yields α-trypsin [ 26 , 37 ], is another cause of trypsin nonspecific cleavages. Coincident with our observations, pseudotrypsin also demonstrated a preference of cleavages after aromatic residues (Tyr, Phe, Trp) in addition to having characteristic trypsin properties [ 15 , 38 ]. However, autolytic activities of trypsin should be very limited at low pH environment such as 1 mM HCl or 50 mM acetic acid condition [ 28 ].…”

“…We obtained highly comparable mass distribution profiles between the two trypsins, but confident identification of +18 Da mass shifts were unattainable. To further corroborate that acidic conditions do not yield new forms of trypsin, a cation-exchange chromatography experiment was conducted to separate trypsin autolyzate [15,38]. We compared the UV profiles of Trypsin-1 reconstituted in 50 mM acetic acid versus that reconstituted in water, and obtained highly comparable profiles between the two conditions (detailed in S4 File), indicating that the observed increase of nontryptic activities upon acidic reconstitution of trypsins was not due to increased amount of pseudotrypsin.…”

“…Although the use of nonspecific cleavage products might contribute to improved protein sequence coverage and identification [ 11 , 12 ], they are also accompanied by unexpected peptides in the tryptic digestion profile. These nontryptic cleavages disperse the signals of the specifically cleaved peptides that are available for detection and increase the database searching workload for a complex sample matrix, potentially affecting accurate identification and quantitation [ 13 – 15 ].…”

“…Trypsin autolytic activities that yield pseudotrypsin were reported to be associated with nonspecific cleavages [ 15 , 25 – 27 ]; however, low pH conditions (such as 1 mM HCl, 50 mM acetic acid) should prevent such trypsin autolysis [ 28 ]. Herein we demonstrate that the reconstitution of several commercial trypsins in mildly acidic conditions (50 mM acetic acid or 1 mM HCl) can lead to dramatically increased nontryptic activities, giving rise to numerous semitryptic and nontryptic peptides.…”

What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

“…Maybe the epitome of this situation is represented by trypsin itself. After the classical activation of trypsinogen into trypsin [ 181 ], trypsin undergoes a series of self-cleavages [ 181 ], resulting into a protease with a low chymotrypsin activity [ 182 ] before final inactivation by autolysis. This simple example shows that besides regulating the half-lives of the proteins, cleavage can also alter their activity.…”