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.
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 ⋯ Ca2+ ⋯ P2) (⋯ represents the noncovalent binding of calcium with the peptide) in the positive ion mode and for ions ([P1-2H]2− ⋯ Ca2+ ⋯ [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.
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 ( Calcium-dependent protein interactions mostly organized in protein networks are responsible for the regulation of cell cycle progression, cell growth, differentiation, secretion, and cytoskeletal organization (1-3). As many of these proteins are linked to various pathological conditions, they are clinically important. The speed at which calcium can have an interplay between various cellular components is impressive and comes notably detectable in neurological processes and in muscle contraction. Calcium binding sites in proteins can be determined by NMR spectroscopy (4, 5). For example, by such NMR measurements, the Ca 2ϩ -binding sites of the tellurite-resistance protein TerD from Klebsiella pneumoniae were found to be formed in part by a highly conserved motif of 13 residues specified by the sequence GDN(R/L)TG(E/A)GDGDDE (4).Although NMR is the gold standard to study calcium binding in proteins, this approach has several drawbacks. For instance, protein size is limited (Ͻ 30 kDa) and proteins should be pure and isotopically labeled. In addition, although the information content is high, NMR is relatively insensitive compared with other techniques such as MS and fluorescence spectroscopy, and relatively large quantities of material (typically 0.5 ml at 0.5-1.0 mM in biological samples) are needed, although efforts are devoted to improve sensitivity in NMR, such as stripline NMR (6).In bottom-up proteomics, proteolytic peptides, generated by enzymatic digestion of complex protein mixtures, are sequenced by MS-based methods (MS/MS (7, 8)) using collision-induced dissociations. Because of the even higher complexity of these peptide mixtures, liquid chromatography (LC) 1 is used to separate the peptides prior to sequencing. In such an LC-MS/MS procedure, many peptides can be identified belonging to the same protein. It has been stated (9) that by this procedure more peptides are analyzed than strictly necessary for identification purposes, but it can equally well be argued that such large coverages enable more reliable protein identifications; moreover, these larger coverages allow the detection of post-translational modifications, including specific calcium complexation as described here.Considering the need of identifying calcium-bound proteins in complex biological samples at...
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