Successful application of cross-linking combined with mass spectrometry for structural proteomics demands specifically designed cross-linking reagents to address challenges in the detection and assignment of crosslinks. A combination of affinity enrichment, isotopic coding, and cleavage of the cross-linker is beneficial for detection and identification of the peptide cross-links. Here we describe a novel cross-linker, cyanurbiotindipropionylsuccinimide (CBDPS), that allows affinity enrichment of cross-linker-containing peptides with avidin. Affinity enrichment eliminates interfering non-cross-linked peptides and allows the researcher to focus on the analysis of the cross-linked peptides. CBDPS is also isotopically coded and CID-cleavable. The cleaved fragments still contain a portion of the isotopic label and can therefore be distinguished from unlabeled fragments by their distinct isotopic signatures in the MS/MS spectra. This cleavage information has been incorporated into a program for the automatic analysis of the MS/MS spectra of the cross-links. This allows rapid determination of cross-link type in addition to facilitating identification of the individual peptides constituting the interpeptide cross-links. Thus, affinity enrichment combined with isotopic coding and CID cleavage allows in-depth mass spectrometric analysis of the peptide cross-links. We have characterized the performance of CBDPS on the 120-kDa protein heterodimer of HIV reverse transcriptase.Molecular & Cellular Proteomics 10: 10.1074/ mcp.M110.001420, 1-8, 2011.Cross-linking combined with mass spectrometric analysis is an attractive technique for obtaining structural information on proteins and protein complexes (1). Cross-linked proteins can be enzymatically digested, and the cross-linked peptides (cross-links) obtained can be analyzed by mass spectrometry to identify both the cross-linked peptides and the site of cross-linking. Unfortunately, ion signals from the cross-links are usually overwhelmed by ion signals from non-cross-linked or "free" peptides and often difficult to detect and to assign.The most straightforward way of simplifying the mixture is by using cross-linking reagents with affinity tags such as biotin that allow selective enrichment of all of the cross-linker-containing peptides in the digest. Despite the added inconvenience of the synthesis, several biotinylated cross-linking reagents have been reported recently (2-6).Even after the selective enrichment step, the detection of cross-links is still challenging. The most popular solution for facilitating specific detection of cross-links is isotopic coding of the cross-linking reagents (7). To enhance the signals from cross-links and to increase the likelihood of their identification, cross-links can be separated from the interfering free (non-cross-linked) peptides, thereby increasing their absolute and relative abundance and simplifying the subsequent mass spectrometric analysis. The final challenge results from the combinatorial nature of the possible interpeptide cros...
Background: Pf12 is the archetypal member of the 6-Cys protein family, members of which are important Plasmodium vaccine targets. Results: Purifying selection and apical localization of Pf12, crystal structure of tandem 6-Cys domains, and mass spectrometry of cross-linked Pf12-Pf41 heterodimer are shown. Conclusion: A functionally important role for Pf12 and potential for antiparallel heterodimer is provided. Significance: First full-length 6-Cys protein structure and first details of heterodimer organization are revealed.
Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2′-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me2 and H3K27me3 in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.
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