Clickable report tags for identification of modified peptides by mass spectrometry

The identification and quantification of modified peptides are critical for the functional characterization of post-translational protein modifications (PTMs) to elucidate their biological function. Nowadays, quantitative mass spectrometry coupled with various bioinformatic pipelines has been successfully used for the determination of a wide range of PTMs. However, direct characterization of low abundant protein PTMs in bottom-up proteomic workflow remains challenging. Here, we present the synthesis and evaluation of tandem mass spectrometry tags (TMT) which are introduced via click-chemistry into peptides bearing alkyne handles. The fragmentation properties of the two mass tags were validated and used for screening in a model system and analysis of AMPylated proteins. The presented tags provide a valuable tool for diagnostic peak generation to increase confidence in the identification of modified peptides and potentially for direct peptide-PTM quantification from various experimental conditions.     

Semisynthesis of Functional Glycosylphosphatidylinositol‐Anchored Proteins

Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell‐ and tissue‐specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one‐pot intein‐mediated ligation (OPL) to obtain GPI‐anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP1₁₉ were prepared. Glypiation did not alter the structure of eGFP and MSP1₁₉ proteins in solution, but it induced a strong pro‐inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections.     

Semisynthesis of Functional Glycosylphosphatidylinositol-Anchored Proteins

Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell- and tissue-specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one-pot intein-mediated ligation (OPL) to obtain GPI-anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP1₁₉ were prepared. Glypiation did not alter the structure of eGFP and MSP1₁₉ proteins in solution, but it induced a strong pro-inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections.     

A Set of Homo‐Oligomeric Standards Allows Accurate Protein Counting

Techniques based on fluorescence microscopy are increasingly used to count proteins in cells, but few stoichiometrically well‐defined standards are available to test their accuracy. A selection of bacterial homo‐oligomers were developed that contain 10–24 subunits and fully assemble when expressed in mammalian cells, and they can be used to easily validate/calibrate molecular counting methods. The utility of these standards was demonstrated by showing that nuclear pores contain 32 copies of the Nup107 complex.     

Coil-coated steel sheets measured by DMA using an immersion testing cell

The effect of humidity on the glass transition temperature of coatings is well known and analyzed. In this study, an analysis method is introduced for analyzing coil-coated metal sheets submerged in water and water/methyl ethyl ketone (MEK) by using an immersion cell coupled to a DMA. For this purpose, a model polyurethane acrylate coating was applied to zinc/magnesium-coated steel plates and measured before and after immersion in water and water/MEK by DMA and immersion cell. The results show a shift of glass transition temperature from 138 to 13 °C of the coating by storing the coated steel plates under water and immersion testing. A time-dependent diffusion of water from the cell into the coating until saturation can be observed. An increasing concentration of MEK within the immersion cell results in a greater shift of the glass transition temperature. Overall, the immersion cell seems to be a useful tool for in situ characterization of the behavior of coil coating varnishes in liquid environment (Foster et al. in Prog Organ Coat 51:244–249, 2004, van der Wel et al. in Prog Organ Coat 37:1–14, 1999).

     

Trace formulae and applications to class numbers

In this paper we compute the trace formula for Hecke operators acting on automorphic forms on the hyperbolic 3-space for the group PSL₂( $\mathcal{O}_K $ ) with $\mathcal{O}_K $ being the ring of integers of an imaginary quadratic number field K of class number H _K > 1. Furthermore, as a corollary we obtain an asymptotic result for class numbers of binary quadratic forms.     

Determination of anionic surfactants in lubrication fluids

A simple method is described for the isolation and determination of anionic surfactants in oils and emulsions used as lubricants in metal working processes. The oils were separated on silica gel solid phase extraction columsn by successive elution of solvents with increasing polarity as hexane, diisopropyl ether, acetic acid ethyl ester, acetone and methanol/NH₃ (8:1). The most polar methanol phase, which contains the surfactants, was titrated with Hyamine and the end point was determined potentiometrically using an ion sensitive electrode. The retention behaviour of sulfonates and sulfates on the sorbent has not been affected by other lubricants.     

SLAP: Small Labeling Pair for Single‐Molecule Super‐Resolution Imaging

Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small‐molecule recognition unit (Ni‐trisNTA) and the genetically encoded minimal protein His_6‐10‐tag. High‐density protein tracing by SLAP was demonstrated. This technique allows super‐resolution fluorescence imaging and fulfills the necessary sampling criteria for single‐molecule localization‐based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).