Endoproteinase Lys-C digestion

Table 5.9 Peptides detected during the LC-electrospray-MS (LC-ESMS) analysis of the endoproteinase Lys-C digest from native cytochrome c". Reprinted from Biochim. Biophys. Acta, 1412, Klarskov, K., Leys, D., Backers, K., Costa, H. S., Santos, H., Gnisez, Y. and Van Beenmen, J. J., Cytochrome c" from the obligate methylotroph Methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph Rhodobacter sphaeroides", 47-55, Copyright (1999), with permission from Elsevier Science...

A. Limited endoproteinase Lys-C digestion Figure 4A shows SDS-PAGE of digests generated by limited proteolysis (nonreducing conditions, for 15 min [lane 1] or 2 h [lane 2]) with Lys-C protease. The SCF polypeptide has... 

Figure 4. SDS-PAGE of peptide products of SDS-nondissociable dimer derived from chemical and proteolytic cleavages. A, endoproteinase Lys-C digestion. Lanes 1 and 2 (nonreducing), products at 40 and 10 pg lanes 3 and 4, as lanes 1 and 2, but reducing. B, CNBr cleavage of Met-oxidized dimer. Lane 1 (nonreducing), oxidized dimer lane 2 (nonreducing), cleavage product lanes 3 and 4, as lanes 1 and 2, but reducing.

Fig. 1. Reversed phase HPLC chromatogram of an endoproteinase Lys C digest of PrP 27-30, showing absorbance at 214 and 280 nm versus time (min). The gradient line shows acetonitrile content. The peptide compositions shown for each peak were derived from the experiments described in Section III. Peptides were identified containing all residues between 74 and 231, with the exception of an anticipated tetrapeptide, residues 107-110. (Reproduced with permission from Stahl, N. et al. in Prusiner, S.B., Collinge, J., Powell, J., and Anderton, B. [1992], Prion Diseases of Humans and Animals, pp. 361-379. Copyright Elsevier Science).

FIGURE 10.4 Matrix-assisted laser desorption mass spectra of (a) peptide fragments produced by endoproteinase Lys-C digestion of melittin, (b) Endo Lys-C fragments isolated by immunopre-cipitation with mab 83144, (c) chymotrypsin fra ents of melittin, and (d) chymotrypsin fragments of melittin immunoprecipitated with antimelittin antibody mab 83144. Peaks labeled with an asterisk are dynorphan A,.,3 added as internal mass calibrant, and peaks labeled II, 12, and I are impurities. Reprinted with permission from reference 10. 

Such a process led to the full assignment of the sequence of the cytochrome c" protein shown in Figure 5.17, in which the polypeptide fragments generated by enzymatic digestion with endoproteinase Lys-C and Glu-C are also indicated. 

As shown in Table I, 76% of the 39 laboratories that participated in this study routinely carry out in situ PVDF and/or in-gel digests, and trypsin (78%) or endoproteinase Lys-C (43%) are the two most frequently used enzymes. The most commonly cited protocols that were routinely used included those by... 

Enzymatic digestion was performed at a 1/20 (w/w) ratio of each enzyme (Endo-Lys C and chymotrypsin) to substrate. Endoproteinase Lys-C (Endo Lys-C, Wako) digestion was performed according to Riviere et al. (1991). The protein... 

Limited proteolysis bv endoproteinase Lvs-C Sample was reconstituted in 20 mM Tris-HCl buffer, pH 7.5 (1 mg/ml) and digested with endoproteinase Lys-C (enzyme-to-substrate ratio= 1 100) at 25 C. At 15 min and 2 h, sample aliquots (1(K) il each) were taken and digestion stopped by adding 5 j,l of 20% TFA. Samples of 5-20 p,g were dried completely and subjected to SDS-PAGE as described below. 

The primary structure was assessed by peptide mapping and N- and C-terminal sequencing. N-terminal sequence analysis showed that a single sequence was detected, MKAIFVLNAA, which corresponds exactly to the first 10 amino acids at the N terminus of P40 as predicted from the DNA sequence. Reverse phase HPLC analysis of a digestion of P40 with a lysine-specific endopeptidase, i.e. endoproteinase Lys-C, was used for identification and primary structure confirmation (Fig. 9). Endoproteinase Lys-C hydolyzes peptide bonds at the carboxylic side of lysine residues. The seventeen peaks resolved were characterized by mass spectrometry, allowing the confirmation of 99 % of the primary sequence. 

When P40 was digested with endoproteinase Lys-C in the presence of the detergent used to refold and purify the protein, several peptides identified by LC-MS analysis contained 3 to 4 theoretical fragments. These peptides were localized in the membrane domain of P40 and resulted from an incomplete digestion of this part of the protein. This was mostly due to the non accessibility of some lysine residues in the P-barrel formed in the presence of the detergent. On the basis of the two-dimensional model of the P-barrel domain of E. coli OmpA (Vogel and Jahnig, 1986 Pautsch and Schulz,... 

Fig. 9 Localization of the site of PEGylation by C4-HPLC peptide mapping following digestion with endoproteinase Lys-C to release peptide 1-19. Left unmodified IFN-/3-la. Right PEGylated IV N-p-la. Arrowheads (pointing to peak APS) mark the elution position of the peptide that has disappeared in the PEGylated protein, right panel. Reproduced from [55]...

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