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...

Fig. 18a-c. Base peak chromatograms for the LC/MS analyses of a cytochrome c Lys-C digest (0.7 pmol injected) on a a poly(styrene-co-divinylbenzene) monolith-filled needle b Vydac C18-packed needle c Poros R2-packed needle. (Reprinted with permission from [128]. Copyright 1998 American Chemical Society)... 

Figure 10.6 Peptide mapping of PIXY321. (A) Cleavage of the primary sequence with Lys-C endoprotease results in 14 theoretical fragments. (B) As detailed in the text, LC-MS analysis of the Lys-C digest shows that a few peptides, notably LI, L7, and L14, elute at several retention times due to heterogeneity of both their glycan structures and amino acid sequence. The insets illustrate the complexity of the mass spectra of glycopeptide LI eluting under peaks 17 (inset C) and 18 (inset D).

Figure 22 Electropherograms for the Lys-C digest of transferring on (A) etched and (B) unetched capillaries. Conditions 50 mM phosphate buffer, pH 3.0 field 300 V/cm temperature, 28°C /, 56 cm. (Reprinted from Ref. 99, with permission.)...

Figure 5. Stepped collision energy scanning LC-ESMS of 500 pmol 6-casein Lys-C digest spiked with the Pam3Cys showing the TIC and the summed marker-ion traces for m/z 114,239,256 and 257.

Figure 1. Peptide maps of Stem Cell Factor Lys-C digests. A and B in-solution digests containing Tween-20 and Triton respectively C. In-gel method and D PVDF method.

Figure 2. MALDI-MS analysis of unfractionated SCF Lys-C digests from in-gel (upper) and PVDF (lower) methods. Note prompt fragmentation of (1+7) in lower (12).

Figure 3. Peptide maps of EPO Lys-C digests from A. PVDF and B. in-gel methods. Table FV. Predicted EPO peptides derived from Lys-C digestion...

Figure 4 Peptide map of an unknown 3SkD PVDF-bound protein after Lys-C digestion. Sequencing yields were in the 20 pmol range for the numbered peptides, with sequences of 7,24, 17, and 11 amino acids obtained.

Figure 5 Peptide map of actin, a 40kD protein after in-gel Lys-C digestion. Peptides 1,3, and 5 were sequenced on a standard sequencer. Pepdd 2 and 4 were sequenced on a modified instrument.

In order to compare peptide recovery from proteolysis in solution, in gel and on PVDF membrane, we digested the same amount of BSA (2.5 ig each, 35 pmol). Figure 1 shows chromatograms of peptides from Lys-C digestion of reduced and denatured BSA without alkylation (A), or with the addition of either iodoacetamide (B), or 4-vinylpyridine (C). As was demonstrated before (Stone et al. 1989), it is clear that BSA was not efficiently digested, as indicated by the low intensity of the peptide peaks when it was not alkylated. On the other hand, alkylated BSA, either by iodoacetamide (B) or 4-vinylpyridine (C), were digested much better, as indicated by the higher intensity of the peaks and the number of peaks. 

The Lys-C digestion of BSA in zinc chloride stained gel (D) and Ponceau S stained gel (E) are also shown in Figure 1. These peptide maps demonstrated that the gels stained with zinc chloride and... 

Figure 1. Comparison of Lys-C digestion of 35 pmol BSA (A), carboxamidomethylated BSA (B), pyridylethylated BSA (C) in solution, and carboxamidomethylated BSA in gel with zinc chloride staining (D), and Ponceau S staining (E).

Figure 1. Comparison of peptide separations from Cytochrome C Lys-C digested...

Figure 4. LC-MS analysis of Cytochrome C Lys-C digestion mixtures in the absence (a) and presence (b) of SDS. The base peak profile of the maw spectral analyses is displayed. The insets I and II show the corresponding m/z spectra acquired over peak I and n.

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).

Experiments with synthetic peptides established that the hydrophilic tetrapeptide Thr-Asn-Met-Lys was not retained on the C18 HPLC column. The HPLC column flow-through from the Lys-C digest was derivatized to add a cholate group to the Atterminus. The increased hydrophobicify allowed the derivatized peptide to be repurified by HPLC and analyzed by... 

Lys C digestion of the Lys-Pro bonds at Lys27, LyslOl, and Lysl04 was incomplete and the A/ terminal portion from full length PrP < was man-... 

Figure 5. Mapping of protein polymorphisms by mass spectrometry. A Major Urinary Protein from Mus spretus was purified and Lys-C digestion fragments were mass measured by MALDl-ToF mass spectrometry. Four of the peptides had the same masses as the equivalent protein from Mus domesticus. However, for two of the remainder, of novel mass, tandem mass spectrometry led to the identification of an amino acid change that was consistent with the new peptide mass. (J is used to define leucine or isoleucine, which cannot be distinguished by this type of mass spectrometry).

Lys-C-digested wheat in Fig.3 is a consequence of the assay method, and reflects failure... 

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