Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation

Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using 14N/15N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers

Kvinnokonventionen- självklar men frånvarande. : En kvalitativ studie om implementering av Kvinnokonventionen

The purpose of this study is to examine the implementation of the Convention on the elimination of all forms of discrimination against women, CEDAW, in Uppsala local council and county council. The aim is to examine how the respondents, in their daily work, understand and implement the convention as well as how they consider the prerequisites for a successful implementation. The study was conducted using qualitative interviews with three civil servants and four politicians. The theoretical approach was implementation theory whose concepts willingness, comprehension and capability were used as tools for the analysis. The results portray CEDAW as a well-known treaty amongst the respondents but at the same time absent in local politics and everyday work. The convention is perceived as an abstract and ideological document which reveals the respondents’ insufficient knowledge about CEDAW as a human rights treaty and the state´s obligation to eliminate discrimination against women. The results also indicate that unclear instructions and responsibility distribution as well as lack of resources, such as time and knowledge, obstruct successful implementation of CEDAW. In conclusion, gender equality is neither a prioritised issue in Uppsala local council and county council and nor seen as a human right.

Simulation of helium flow through ion guide with COMSOL multiphysics

The program COMSOL Multiphysics was used to simulate a flow of helium gas transporting ionized fission products out of an ion guide. Two important parameters to study from the simulation was the evacuation time and velocity of the ions. The mean evacuation time was shown to be 0.1173s, and the velocity of a single particle peaked at 2500m/s, 1000-1500m/s being more common

Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase.

The molecular mechanism whereby the small heat-shock protein (sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase (CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,3'-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross-linker captured Hsp21 in dodecameric form, even at increased temperature (47 degrees C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21

Karta öfver Provinsen Gestrikland hörande till Gefleborgs Län, upprättad ar 1857 af L.E. Ahrman

Échelle(s) : 1 Mil [=Om. 106 ; 1:100 000

Site-specific transamidation and deamidation of the small heat-shock protein Hsp20 by tissue transglutaminase

Contains fulltext : 32990.pdf (publisher's version ) (Closed access

Thiol-exchange in DTSSP crosslinked peptides is proportional to cysteine content and precisely controlled in crosslink detection by two-step LC-MALDI MSMS

Contains fulltext : 92496.pdf (publisher's version ) (Closed access)12 p