DIRECT PEPTIDESEQUENCING AFTER TLC ON SILICA GEL WITHOUT
Regine Kraft, Albrecht Otto, Gerhard Etzold
Akademie der Wissenschaften der DDR,
Zentralinstitut für Molekularbiologie
The purification and separation of peptides is frequently
performed on silica gel because this material is widely free
of amino acids or proteins. Therefore, the determination of
the composition or the sequence of amino acids are practi-
cally not impaired. Inspired by the method published by
Fishbein et al. (1) we describe
Bromein, a cysteine proteinase inhibitor from pineapple stem, is a unique double-chain inhibitor. The 27.5-kDa precursor protein is processed by the removal of three interchain, two interdomain, and two terminal-flanking peptides, thus resulting in the release of mature isoinhibitors of approximately 6 kDa. To characterize the processing of the interchain peptide Thr15-Ser-Ser-Ser-Asp, we expressed a single-chain precursor with this peptide and monitored proteolytic cleavage by the target proteinase bromelain. By peptide sequencing and mass spectrometric analysis, the initial cleavage was found to occur in vitro between the light-chain and interchain peptides; subsequent trimming formed the terminal-ragged peptides Thr15–Lys60, Ser17–Lys60, Ser18–Lys60, and Asp19–Lys60. However, bromelain did not show any cleavage activity between the interchain and heavy-chain peptides. We also discovered that cleavage between the light-chain and interchain peptides is essential for the single-chain inhibitor to exhibit full inhibitory activity. Notably, the incompletely processed intermediates showed higher inhibitory activity than either the native bromein or the single-chain precursor. Bromein is also known to weakly inhibit the serine proteinases chymotrypsin and trypsin; however, a recombinant single-chain inhibitor with the interchain peptide was no longer able to inhibit these serine proteinases.
Two heptapeptides have been prepared by Fmoc methodology using Wang resin as solid support. For attachment of the first amino acid, several coupling systems were evaluated, and DIC/DMAP system could give yields of >99% and low levels of racemization. The selection of scavenger combination to deprotect side chains revealed that H2O/p-cresol was good at scavenging trityl and 1,2-ethanedithiol was highly efficient for scavenging t-butyl. Through shortening the preactivation time to 5 min, the racemization which occurred during formation of amide bonds coupled by HBTU was minimized. The crude peptides were characterized by RP-HPLC and MS, and sequenced by MS/MS to acquire reliable amino acid sequence information.
degradation of collagen IV using nanospray ionization
Fourier transform ion cyclotron resonance mass spec-
trometry (FTICR MS) after separation by nanoscale liquid
chromatography (nanoLC). The combination of FTICR
MS analysis and de novo peptidesequencing resulted in
the identification of 38 specific collagen IV fragments of
which several peptides included the integrin-binding
motif RGD/E known from numerous mammalian immune-
related proteins. Custom-synthesized peptides corre-
sponding either to the presently identified collagen
peptide GIRGEHyp or to a well
The regulated secretory pathway of neurons is the major source of extracellular Aβ that accumulates in Alzheimer's disease (AD). Extracellular Aβ secreted from that pathway is generated by β-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major β-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with Aβ in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to Aβ in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular Aβ released by the regulated secretory pathway, but CA074Me had no effect on Aβ released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal β-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate β-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as β-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce Aβ in AD.
matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer, Rapid Commun. Mass Spectrom., 2008, 22, 1823–1833 http://dx.doi.org/10.1002/rcm.3555  Nilsson S., Wetterhall M., Bergquist J., Nyholm L., Markides K.E., A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/mass spectrometry, Rapid Commun. Mass Spectrom., 2001, 15, 1997–2000 http://dx.doi.org/10.1002/rcm.466  Keough T., Youngquist R.S., Lacey M.P., A method for high-sensitivity peptidesequencing using postsource
.G., Thomson, B., Wilm, M., and Mann, M. ( 1997 ). Rapid ‘ de novo ’ peptidesequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 11 , 1015 –1024. Tetlow , N., Robinson, A., Mantle, T., and Board, P. ( 2004 ). Polymorphism of human mu class glutathione transferases. Pharmacogenetics 14 , 359 –368. Tezel , G., Nagasaka, T., Shimono, Y., and Takahashi, M. ( 2002 ). Differential expression of RET finger protein in testicular germ cell tumors. Pathol. Int. 52 , 623 –627. Thomas
. Stüber, S. Zaiss, R. Ehring, P. Zabel 303
Aminopeptidase M in the Sequence Analysis of Peptides
K.-D. Jany, J. Czech, G. Pfleiderer 327
Fast Atom Bombardment Mass Spectrometry. A New Technique
for PeptideSequencing. A Review
W. Schäfer 337
Perspectives in the Circular Dichroic Analysis of Protein
A. Wollmer, W. Straßburger, U. Glatter 361
Spin-Labelled Amino Acids, Peptides and Proteins -
Synthesis and Application
H.R. Wenzel, H. Tschesche, E. von Goldammer 385
The Ultrastructure of Macromolecular Complexes Studied
98 (2001) 2894–2898. http://dx.doi.org/10.1073/pnas.041616498  Schwan, W.R. and Kopecko, D.J. Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene. Infect. Immun.
65 (1997) 4754–4760.  Dubiel, W., Ferrell, K. and Rechsteiner, M. Peptidesequencing identifies MSS1, a modulator of HIV Tat-mediated transactivation, as subunit 7 of the 26 S protease. FEBS Lett.
323 (1993) 276–278. http://dx.doi.org/10