The fragment 60C70, identified in the extraction/excision experiments, is a part of the 3-strand, while the 53C61 fragment, localized using the HDX-MS approach, covers the amino acids sequence in loop L1. Cyst28 are studied and compared with the binding sites found for mAb Cyst10 that has almost no effect on hCC dimerization. In addition, hCC epitopes in complexes with native polyclonal antibodies extracted from human serum were studied. The results obtained with hydrogenCdeuterium exchange mass spectrometry (HDX MS) were compared with the previous Ryanodine findings made using the excision/extraction MS approach. The main results from the two complementary MS-based approaches are found to be in agreement with each other, with some differences being attributed to the specificity of each method. The findings of the current studies may be important for future design of hCC Ryanodine dimerization inhibitors. Electronic supplementary material The online version of this article (doi:10.1007/s00726-016-2316-y) contains supplementary material, which is available to authorized users. strain C41(DE3) and purified by ion-exchange chromatography as described previously (Szymaska et al. 2009). The protein purity was characterized by SDSCPAGE, Size Exclusion Chromatography, and Mass Spectrometry (see Supplementary Materials Physique?1). Isolation of natural antibodies against human cystatin C (NAbs) Isolation of NAbs was performed as described previously (Johnstone and Thorpe 1996). Briefly, 25?mg of IgG fraction from human serum was applied onto an hCC-Sepharose column equilibrated in PBS (pH 7.4) and incubated overnight at 4?C with gentle shaking. After washing with PBS, the affinityCbound antigenCantibody complex was dissociated with 10??500?l of 0.1?% aqueous TFA (pH 2.5). The isolated NAbs were analyzed by SDSCPAGE, and their concentration was determined by measuring the absorbance at 280?nm (NanoQuant, Infinite M200Pro, Tecan) using the extinction coefficient 400 was used for the MS analysis. Several LC MS/MS runs were carried out to identify the peptides in the hCC pepsin digest. The Mascot software (Matrix Science) was used to search MS/MS data in a database composed of the cystatin sequence using the following parameters: variable modificationsoxidation of methionine; enzyme settingnone; peptide and fragment mass tolerances of 5?ppm and 0.6?Da, respectively. Peptides with Mascot ion scores higher than 20 were further selected for HDX kinetic studies. In addition, each selected peptide was further validated by manual inspection of the MS/MS spectrum. The HDExaminer software (Sierra Analytics, Modesto, USA) was used to process all HDX-MS data. Results peptic peptides of human cystatin C: HDX experiment To assess the effect of the antibody binding to human cystatin C, HDX-MS analysis of the monomeric protein was performed. Unlabeled hCC Ryanodine was subjected to online pepsin digestion, desalting, chromatography, and tandem mass spectrometry analysis. To achieve high sequence coverage of peptides obtained after enzymatic Ryanodine digestion with pepsin, various digestion conditions (different denaturing reagents, variable enzyme: protein molar ratio) were tested. It was found that enzymatic digestion carried out in answer on ice was not effective enough. Therefore, digestion of the protein around the column was Ryanodine attempted. This experiment resulted in a sequence coverage of 93?% (43 peptic peptides presented in Fig.?2). From the digestion of the N-terminal fragment of human cystatin C, 9 fragments were obtained. The shortest of them had 9 amino acid (AA) residues, and the longest one28 AA residues. The majority of the peptides were about 15-AA long. The central part of the protein (29C64) was the most efficiently digested. Looking on the primary cystatin C structure (Fig.?2), one can notice that one of the digestion sites is located around residues 28/29, i.e., in the Rabbit Polyclonal to Cytochrome P450 19A1 central part of the -helix (Fig.?1). However,.