β‐Glucuronidase enzymes have been attached to a porous silicon surface through a direct silicon–carbon bond based linking system (see Figure). The attached enzymes display high activity and the photoluminescent (PL) properties and surface stability of the porous silicon are retained. Quenching of the PL is observed upon enzymatic breakdown of the substrate, leading to the possibility of a new class of chemical and biological sensors.
[M + Cu]+ peptide ions formed by matrix-assisted laser desorption/ionization from direct desorption off a copper sample stage have sufficient internal energy to undergo metastable ion dissociation in a time-of-flight mass spectrometer. On the basis of fragmentation chemistry of peptides containing an N-terminal arginine, we propose the primary Cu+ ion binding site is the N-terminal arginine with Cu+ binding to the guanidine group of arginine and the N-terminal amine. The principal decay products of [M + Cu]+ peptide ions containing an N-terminal arginine are [a(n) + Cu - H]+ and [b(n) + Cu - H]+ fragments. We show evidence to suggest that [a(n) + Cu - H]+ fragment ions are formed by elimination of CO from [b(n) + Cu - H]+ ions and by direct backbone cleavage. We conclude that Cu+ ionizes the peptide by attaching to the N-terminal arginine residue; however, fragmentation occurs remote from the Cu+ ion attachment site involving metal ion promoted deprotonation to generate a new site of protonation. That is, the fragmentation reactions of [M + Cu]+ ions can be described in terms of a "mobile proton" model. Furthermore, proline residues that are adjacent to the N-terminal arginine do not inhibit formation of [b(n) + Cu - H]+ ion, whereas proline residues that are distant to the charge carrying arginine inhibit formation of [b(n) + Cu - H]+ ions. An unusual fragment ion, [c(n) + Cu + H]+, is also observed for peptides containing lysine, glutamine, or asparagine in close proximity to the Cu+ carrying N-terminal arginine. Mechanisms for formation of this fragment ion are also proposed.
An experimental approach is described for determining protein-small molecule non-covalent ligand binding sites and protein conformational changes induced by ligand binding. The methodology utilizes time resolved limited proteolysis and the high throughput analysis capability of MALDI TOF MS to determine the binding site in a tetanus toxin C-fragment (51 kDa)-doxorubicin (543 Da) non-covalent complex. Comparing relative ion abundances of peptides released from the time resolved limited proteolysis of tetanus toxin C-fragment (TetC) and the TetC-doxorubicin complex every 10 min from 10 to 120 min of digestion revealed that the binding of doxorubicin induced a significant change in surface topology of TetC. Four of the twenty-nine peptides observed by MALDI MS, including amino acids 351-360, 299 -304, 305-311 and 312-316, had a lower abundance in the TetC-doxorubicin complex relative to TetC from 10 to 100 min of digestion. A decrease in ion abundance suggests doxorubicin obstructs the access of the protease to one or both termini of these peptides, identifying doxorubicin binding site(s). Conversely, five peptide ions, including amino acids 335-350, 364 -375, 364 -376, 281-298, and 316 -328, all had a greater abundance in the digest of the complex, indicating an increase in accessibility to these sites. These five peptides flank regions of decreased ion abundance, suggesting that doxorubicin not only binds to the surface, but also induces a conformational change in TetC. (J Am Soc Mass Spectrom 2003, 14, 460 -470)
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