Characterization of histones and their post-translational modifications by mass spectrometry

Histone proteins and their accompanying post-translational modifications have received much attention for their ability to affect chromatin structure and, hence, regulate gene expression. Recently, mass spectrometry has become an important complementary tool for the analysis of histone variants and modification sites, for determining the degree of occupancy of these modifications and for quantifying differential expression of these modifications from various samples. Additionally, as advancements in mass spectrometry technologies continue, the ability to read entire 'histone codes' across large regions of histone polypeptides or intact protein is possible. As chromatin biology demands, mass spectrometry has adapted and continues as a key technology for the analysis of gene regulation networks involving histone modifications.     

Novel post-translational modifications of Smad2 identified by mass spectrometry

Smad2 is a crucial component of transforming growth factor-β (TGFβ) signaling, and is involved in the regulation of cell proliferation, death and differentiation. Phosphorylation, ubiquitylation and acetylation of Smad2 have been found to regulate its activity. We used mass spectrometry to search for novel post-translational modifications (PTMs) of Smad2. Peptide mass fingerprinting (PMF) indicated that Smad2 can be acetylated, methylated, citrullinated, phosphorylated and palmitoylated. Sequencing of selected peptides validated methylation at Gly122 and hydroxylation at Trp18 of Smad2. We also observed a novel, so far unidentified modification at Tyr128 and Tyr151. Our observations open for further exploration of biological importance of the detected PTMs. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry

Post-translational modifications generate tremendous diversity, complexity and heterogeneity of gene products, and their determination is one of the main challenges in proteomics research. Recent developments in mass spectrometry based approaches for systematic, qualitative and quantitative determination of modified proteins promise to bring new insights on the dynamics and spatio-temporal control of protein activities by post-translational modifications, and reveal their roles in biological processes and pathogenic conditions. Combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies and mass spectrometry are particularly attractive for systematic investigation of post-translationally modified proteins in proteomics.     

Schistosoma mansoni: In vitro schistosomicidal activity of piplartine

Schistosomiasis is one of the world’s greatly neglected tropical diseases, and its control is largely dependent on a single drug, praziquantel. Here, we report the in vitro effect of piplartine, an amide isolated from Piper tuberculatum (Piperaceae), on Schistosoma mansoni adult worms. A piplartine concentration of 15.8 μM reduced the motor activity of worms and caused their death within 24 h in a RPMI 1640 medium. Similarly, the highest sub-lethal concentration of piplartine (6.3 μM) caused a 75% reduction in egg production in spite of coupling. Additionally, piplartine induced morphological changes on the tegument, and a quantitative analysis carried out by confocal microscopy revealed an extensive tegumental destruction and damage in the tubercles. This damage was dose-dependent in the range of 15.8–630.2 μM. At doses higher than 157.6 μM, piplartine induced morphological changes in the oral and ventral sucker regions of the worms. It is the first time that the schistosomicidal activity has been reported for piplartine.     

Analysis of post-translational modifications in Alzheimer's disease by mass spectrometry

The roles of post-translational modifications (PTMs) in the onset and progression of disease have been extensively studied for decades. More specifically, various PTMs have been the focus of research in Alzheimer's disease (AD). The two most discussed hallmarks of the disease, senile plaques and tau tangles, are the result of PTMs of the amyloidβ protein precursor (AβPP) and the microtubule stabilizing protein: tau. While these modifications have been characterized indirectly by biochemical-based methods, the primary shortcoming to this research can be linked to a lack of a thorough molecular-based means of qualitative and quantitative analysis of many of these modifications within transgenic animal, and human samples. In this review, we discuss the important proteins and their associated PTMs linked to AD and the ways in which mass spectrometry has and will be utilized to analyze them. We also comment on novel ways in which molecular-based mass spectrometry methods should be employed going forward to resolve the interconnections of the PTMs involvement in various stages of AD pathology (preclinical, mild cognitive impairment, advanced-stage AD).     

Surface proteins on schistosomula and cercariae of Schistosoma mansoni

The surface proteins of schistosomula and cercariae of Schistosoma mansoni were examined by lactoperoxidase catalysed iodination followed by polyacrylamide gel electrophoresis. Two polypeptide chains were clearly labelled and they were present on both cercarial and schistosomular surfaces. A number of minor labelled components were also observed. The results suggest that membranes of the teguments of cercariae and schistosomula are similar.     

Further studies on the carbohydrate metabolism of immature Schistosoma mansoni

Further studies on the carbohydrate metabolism of immature Schistosoma mansoni. International Journal for Parasitology 3: 783–787. Analysis of whole cercariae, cercarial bodies and tails and schistosomula shows the tail contains 26 per cent of the cercarial nitrogen, 46 per cent of the glycogen and 59 per cent of the cytochrome oxidase. 32 per cent of the total nitrogen and 22 per cent of the glycogen of the cercarial body is lost during penetration. Oxygen uptake of adult worms but not of cercariae increases between 30 and 37°C. A temperature of 41°C partly inhibits cercarial oxygen uptake but not that of adult worms. Incubation of schistosomula, 21-day-old and adult worms in nitrogen increases the rate of glycolysis.     

Schistosoma mansoni: Schistosomicidal effect of mefloquine and primaquine in vitro

We investigated the effects of the anti-malarials mefloquine and primaquine against the juvenile and adult life stages of Schistosoma mansoniin vitro. Cercariae were incubated with 0.5 μg/ml, 1 μg/ml and 2 μg/ml mefloquine or primaquine and with 1 μg/ml praziquantel for 12 h. Schistosomula, pre-adults and adults were incubated with 0.5 μg/ml, 1 μg/ml and 2 μg/ml mefloquine or primaquine and with 1 μg/ml praziquantel for 7 days. The viability status was classified as viable, damaged or dead and was checked every 3 h for cercariae and every 12 h for schistosomula, pre-adults and adults. Both, mefloquine and primaquine show time and dose-dependent schistosomicidal effects on the four life stages of S. mansoni. The promising in vitro effects on all stages of the blood fluke S. mansoni warrants further evaluation of both anti-malarials and their derivatives for their prophylactic and therapeutic values in early and late schistosomiasis in field trials.     

Biochemical properties of purified cathepsin B from Schistosoma mansoni

A previously described “major acidic proteinase” of adult Schistosoma mansoni, believed to play a key role in the parasite's metabolism, has been identified as a cathepsin B (Sm31). Purified Sm cathepsin B was not recognized by anti-Sm32 or anticathepsin L antibodies. The enzyme hydrolyzes the synthetic protease substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC as well as protein substrates. Its pH optimum is 3.0 with serum albumin, 4.0–5.0 with globin and 5.5–6.0 with the synthetic substrates. The enzyme was inactivated by cysteine proteinase inhibitors. Its activity against protein substrates would support the hypothesis that it plays a role in schistosome nutrition.     

Studies on preservation of schistosomula of Schistosoma mansoni and S. mattheei

Schistosomula were not damaged by exposure for 1 hr at room temperature to the cryoprotectant dimethylsulphoxide (DMSO) providing that concentrations greater than 10% were not used. Rapid dilution to remove the DMSO was less harmful to the organisms than was slow dilution. Schistosomula were not damaged by thermal shock (cooling in the absence of freezing) but were damaged by conditions produced by freezing. Although the freezing damage rendered schistosomula noninfective they retained flame cell activity and certain contractile properties in the oral sucker, gut, and musculature. The least damage was produced by slow cooling (at approximately 0.3 °C/min) and fast warming (approximately 300 °C/min). Schistosomula remained infective following freezing and slow cooling to −20 °C in DMSO (10%) and storage for 2 hr at this temperature but were damaged at temperatures below −26 °C and at −20 °C for longer time periods.     

Comprehensive identification of post-translational modifications of rat bone osteopontin by mass spectrometry

Osteopontin (OPN) is a highly modified protein that is found in many tissues and has been associated with a variety of physiological and pathological processes. Bone OPN is a potent inhibitor of hydroxyapatite crystal formation and stimulates bone resorption by osteoclasts; these activities, as well as others, are dependent upon phosphorylation of the protein. We have used mass spectrometry (MS) to perform a comprehensive analysis of the post-translational modification of OPN purified from rat bone. Matrix-assisted laser desorption time-of-flight (MALDI-TOF) MS showed masses of 37.6 and 36.8 kDa before and after enzymatic dephosphorylation, respectively, corresponding to a content of approximately 10.4 phosphate groups. Using proteolytic digestion and tandem MS, we localized 29 sites of phosphorylation: S10, S11, S46, S47, T50, S60, S62, S65, S146, T154, S160, S164, S167, S193, S196, S203, S220, S223, S232, S241, S245, S257, S262, S267, S278, S290, S295, S296, and S297. In addition, Y150 was shown to be sulfated and T107, T110, T116, and T121 are O-glycosylated. No glycan was detected at the potential N-glycosylation site. Other modifications, including deamidation, oxidation, and carbamylation, are also present. A 36-amino acid sequence from residues 67-102 could not be analyzed in detail, even after sialidase treatment, presumably because of the presence of a large number of acidic residues. In comparison to the previously characterized cow milk isoform, rat bone OPN is sulfated and has an additional site of glycosylation, many different sites of phosphorylation, and a lower overall phosphate content.     

Glycotope analysis in miracidia and primary sporocysts of Schistosoma mansoni: Differential expression during the miracidium-to-sporocyst transformation

Fucosylated carbohydrate epitopes (glycotopes) expressed by larval and adult schistosomes are thought to modulate the host immune response and possibly mediate parasite evasion in intermediate and definitive hosts. While previous studies showed glycotope expression is developmentally and stage-specifically regulated, relatively little is known regarding their occurrence in miracidia and primary sporocysts. In this study, previously defined monoclonal antibodies were used in confocal laser scanning microscopy, standard epifluorescence microscopy and Western blot analyses to investigate the developmental expression of the following glycotopes in miracidia and primary sporocysts of Schistosoma mansoni: GalNAcβ1-4GlcNAc (LDN), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF), Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and the truncated trimannosyl N-glycan Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan). All but Lewis X were variously expressed by miracidia and sporocysts of S. mansoni. Most notably, α3-fucosylated LDN (F-LDN, F-LDN-F, LDN-F) was prominently expressed on the larval surface and amongst glycoproteins released during larval transformation and early sporocyst development, possibly implying a role for these glycotopes in snail–schistosome interactions. Interestingly, Fucα2Fucα3-subsituted LDN (LDN-DF, DF-LDN-DF) and LDN-F were heterogeneously surface-expressed on individuals of a given larval population, particularly amongst miracidia. In contrast, LDN and TriMan primarily localised in internal somatic tissues and exhibited only minor surface expression. Immunoblots indicate that glycotopes occur on overlapping but distinct protein sets in both larval stages, further demonstrating the underlying complexity of schistosome glycosylation. Additionally, sharing of specific larval glycotopes with Biomphalaria glabrata suggests an evolutionary convergence of carbohydrate expression between schistosomes and their snail host.     

Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry

Methods for the simultaneous identification of interacting proteins and post-translational modifications of the focal adhesion adapter protein, paxillin, are presented. The strategy includes (1) lower-level, transient transfection of FLAG-GFP-Paxillin into HEK293 cells, (2) incubation of cells with phosphatase inhibitors prior to lysis, (3) purification of paxillin by anti-FLAG immunoprecipitation, (4) analysis of peptides generated from on-beads digestion using LTQ-FT or LTQ-ETD mass spectrometry, and (5) enrichment of phosphopeptide methyl esters with IMAC. Using the above strategies, we identify 29 phosphorylation sites (19 novel and 10 previously reported) and a novel glycosylation site on Ser 74. Furthermore, with this method, we simultaneously detect 10 co-purifying proteins which are present in focal adhesion complexes. Extension of these methods to other substrates should facilitate generation of global phosphorylation maps and protein-protein interactions for any protein of interest.     

Electron capture dissociation mass spectrometry in characterization of post-translational modifications

Electron capture dissociation (ECD) represents a significant advance in tandem mass spectrometry for the identification and characterization of post-translational modifications (PTMs) of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally induced dissociation and infrared multi-photon dissociation, ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation. This unique attribute offers ECD as an attractive alternative for detection and localization of PTMs. The success and rapid adoption of ECD recently led to the culmination of The 1st International Uppsala Symposium on Electron Capture Dissociation of Biomolecules and Related Phenomena (October 19-22, 2003, Stockholm, Sweden). Herein, we present a general overview of the ECD technique as well as selected applications in characterization of post-translationally modified polypeptides.