An automated screening method is presented that uses MALDI in-source decay (MALDI-ISD) of disulfide bonds for identification of disulfide-linked peptides in MALDI mass spectra. Peptides released by ISD of a disulfide bond can be detected at an m/z ratio that corresponds to the singly protonated peptide with a reduced cysteine residue. Therefore, screening of peak lists for signal patterns that fulfill the equation, m/z (peak A) + m/z (peak B) - m/z (H2 + H+) = m/z (peak C), facilitated identification of putative ISD fragments of disulfide-linked peptides (peaks A and B) and their precursors (peak C). Signals (peak C) from putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the existence of a disulfide bond. Using this method, we identified all 4 disulfide bonds in RNAseA and 8 two-disulfide clusters comprising 16 out of the 17 disulfide bonds in BSA. The presented screening method accelerated the identification of disulfide bonds in RNAseA and BSA, because the number of MS/MS spectra to be acquired was reduced by 1 order of magnitude. Less than 5% of the signals selected for LIFT-TOF/TOF-MS did not correspond to disulfide-linked peptides. Furthermore, the number of possible assignments for disulfide-linked peptides was reduced by 2-3 orders of magnitude, because knowledge of the mechanism of disulfide bond fragmentation by ISD permitted use of stricter rules for the interpretation of mass spectra. Therefore, interpretation of MS/ MS spectra of disulfide-linked peptides was considerably simplified in comparison to conventional approaches.

The use of highly purified MAb dimers and a panel of characterizations methods enabled to obtain a clear picture about molecular architecture and function of dimers.

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The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysinet-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal a-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure-selective modification of charged residues as an efficient approach in the structure-function evaluation of ion channels, and X-ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.