Identifying Ca2+-binding sites in proteins by liquid chromatography-mass spectrometry using Ca2+-directed dissociations.

Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography-mass spectrometry in concert with calcium-directed collision-induced dissociations. Our method does not require any modifications to the liquid chromatography-mass spectrometry apparatus, uses standard digestion protocols, and can be applied to existing high-resolution MS data files. In contrast to NMR, our method is applicable to very small amounts of complex protein mixtures (femtomole level). Calcium-bound peptides can be identified using three criteria: (1) the calculated exact mass of the calcium containing peptide; (2) specific dissociations of the calcium-containing peptide from threonine and serine residues; and (3) the very similar retention times of the calcium-containing peptide and the free peptide.

Accurate molecular weight analysis of histones using FFE and RP-HPLC on monolithic capillary columns.

Due to their large diversity with respect to post-translational modifications (PTMs), the family of histones provides a major analytical challenge in current proteomics research. Their function has a large impact on the transcription of DNA, and as a result, on the expression of proteins. The variation in PTMs regulates transcription, and, as a result, many methods are being employed for the in-depth analysis of histones. In this paper, we present a separation strategy for histones based on free-flow electrophoresis (FFE) followed by an RP separation on capillary monolithic PS-DVB columns. The capillary columns are directly interfaced with an FT-ICR MS providing an online system for the detection and accurate molecular weight analysis of intact histones.

Isoform separation of a multi-acetylated protein using capillary polystyrene-divinylbenzene monolithic columns.

Capillary polystyrene-divinylbenzene (PS-DVB) monolithic columns were used to separate differentially acetylated intact IM9 protein isoforms. Compared to the unmodified form, the hydrophobic shift for intact acetylated isoforms was significant under standard reversed-phase conditions (32.5-45% acetonitrile in 10 min). The high chromatographic resolution of the PS-DVB monolithic columns resulted in peak widths at half height of 4-5s. This allowed us to nearly completely resolve a number of peaks greater than the number of possible acetylation sites. This observation suggested that not only the number, but also the location of the acetylations on the protein had a significant effect on the retention. Matrix-assisted laser desorption ionization time-of-flight MS and MS/MS were used to confirm the chromatographic separation of isoforms. It was found that the acetylations site, especially on the N-terminus, has an effect on the retention on the PS-DVB column.

Dimerization of peptides by calcium ions: investigation of a calcium-binding motif.

We investigated calcium-binding motifs of peptides and their recognition of active functionalities for coordination. This investigation generates the fundamentals to design carrier material for calcium-bound peptide-peptide interactions. Interactions of different peptides with active calcium domains were investigated. Evaluation of selectivity was performed by electrospray ionization mass spectrometry by infusing solutions containing two different peptides (P1 and P2) in the presence of calcium ions. In addition to signals for monomer species, intense dimer signals are observed for the heterodimer ions (P1 ⋯ Ca(2+) ⋯ P2) (⋯ represents the noncovalent binding of calcium with the peptide) in the positive ion mode and for ions ([P1-2H](2-) ⋯ Ca(2+) ⋯ [P2-2H](2-)) in the negative ion mode. Monitoring of the dissociation from these mass selected dimer ions via the kinetic method provides information on the calcium affinity order of different peptide sequences.