Analytical biochemistry

  • Article
    | Open Access

    Humans are exposed to many xenobiotic chemicals, but identification of low abundance xenobiotic exposures is limited by a lack of authentic standards for xenobiotic metabolites. Here the authors develop methods for enzymatic generation of diverse xenobiotic metabolites for use with high-resolution mass spectrometry for biology-based chemical identification.

    • Ken H. Liu
    • , Choon M. Lee
    •  & Dean P. Jones
  • Article
    | Open Access

    Existing methods for multimeric protein complex quantification in single cells suffer from limited selectivity and sensitivity. Here the authors report Single-cell protein Interaction Fractionation Through Electrophoresis and immunoassay Readout (SIFTER) and use this to probe the effects of cellular stress.

    • Julea Vlassakis
    • , Louise L. Hansen
    •  & Amy E. Herr
  • Article
    | Open Access

    Many current immunoassays require multiple washing, incubation and optimization steps. Here the authors present Ratiometric Plug-and-Play Immunodiagnostics (RAPPID), a generic assay platform that uses ratiometric bioluminescent detection to allow sandwich immunoassays to be performed directly in solution.

    • Yan Ni
    • , Bas J. H. M. Rosier
    •  & Maarten Merkx
  • Article
    | Open Access

    Predicting chromatographic retention times (RTs) has proven beneficial in proteomics but has not yet been achieved for crosslinked peptides. Here, the authors develop an RT prediction tool for crosslinked peptides and leverage predicted RTs to increase identifications in crosslinking mass spectrometry studies.

    • Sven H. Giese
    • , Ludwig R. Sinn
    •  & Juri Rappsilber
  • Article
    | Open Access

    RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in eukaryotes. Here, the authors present three cryo-EM structure of S. pombe Pol I in different functional states among them a dimer structure and discuss conserved and organism-specific features of Pol I.

    • Florian B. Heiss
    • , Julia L. Daiß
    •  & Christoph Engel
  • Article
    | Open Access

    Mass spectrometry-based proteomics typically relies on highly sensitive nano-flow liquid chromatography (LC) but this can reduce robustness and reproducibility. Here, the authors show that micro-flow LC enables robust and reproducible high-throughput proteomics experiments at a very moderate loss of sensitivity.

    • Yangyang Bian
    • , Runsheng Zheng
    •  & Bernhard Kuster
  • Article
    | Open Access

    Imaging mass spectrometry is a powerful emerging tool for mapping the spatial distribution of biomolecules across tissue surfaces. Here the authors showcase an automated technology for deep proteome imaging that utilizes ultrasensitive microfluidics and a mass spectrometry workflow to analyze tissue voxels, generating quantitative cell-type-specific images.

    • Paul D. Piehowski
    • , Ying Zhu
    •  & Kristin E. Burnum-Johnson
  • Article
    | Open Access

    Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Here authors find and structurally characterize the selective interaction between CLCa and the actin motor protein myosin VI which act together to generate the force that leads to invagination and fission at the apical surface.

    • Matteo Biancospino
    • , Gwen R. Buel
    •  & Simona Polo
  • Article
    | Open Access

    The use of antibodies to capture and profile exosomes limits the number of target proteins that can be detected. Here the authors develop a proximity-dependent barcoding assay that allows profiling of 38 surface proteins on individual exosomes from heterogeneous samples such as serum and seminal fluid.

    • Di Wu
    • , Junhong Yan
    •  & Masood Kamali-Moghaddam
  • Article
    | Open Access

    Electrophoretic mobility shift assays are widely used in gel electrophoresis to study binding interactions between different molecular species, but these assays access only a subset of reaction possibilities. Here, the authors develop a band-collision gel electrophoresis (BCGE) approach that demonstrates a much wider variety of reaction types.

    • Dimitri A. Bikos
    •  & Thomas G. Mason
  • Article
    | Open Access

    Digital proximity ligation assay (dPLA) can measure proteins and mRNAs in single cells, but is not compatible with cell imaging and cannot quantify rare proteins due to a high dilution factor. Here the authors present an automated microfluidic device that combines live-cell imaging, chemical stimulation, and dPLA in a smaller reaction volume.

    • Jing Lin
    • , Christian Jordi
    •  & Savaş Tay
  • Article
    | Open Access

    Acyl carrier proteins (ACPs), a universal and highly conserved carrier of acyl intermediates during fatty acid and polyketide synthesis, are difficult to visualise. Here, the authors developed a facile, Raman spectroscopy-based method to detect ACP-substrate interactions.

    • Samuel C. Epstein
    • , Adam R. Huff
    •  & Louise K. Charkoudian
  • Article
    | Open Access

    Simultaneous quantification of DNA, RNA and protein at the single cell level has not yet been possible. Here the authors introduce a molecular labelling and detection strategy to quantify synthesis of these biomolecules and couple it to transient cell states through parallel quantification of state-dependent biomolecules.

    • Samuel C. Kimmey
    • , Luciene Borges
    •  & Sean C. Bendall
  • Article
    | Open Access

    The GTPase dynamin catalyzes membrane fission but activity of the dynamin-related ATPase EHD1 is unknown. Here, using in vitro reconstitution assays and molecular dynamics simulations, the authors report that EHD1 hydrolyzes ATP to remodel, causing fission of membrane tubes and that this is necessary for endocytic recycling.

    • Raunaq Deo
    • , Manish S. Kushwah
    •  & Thomas J. Pucadyil
  • Article
    | Open Access

    Ubiquitination and ubiquitin-like modifications of proteins regulate multiple cellular processes but identifying substrates of specific E2 and E3 enzymes remains challenging. Here, the authors conjugate E2 enzymes with enrichable ubiquitin derivatives to identify substrates of specific E2/E3 pairs by mass spectrometry.

    • Gábor Bakos
    • , Lu Yu
    •  & Jörg Mansfeld
  • Article
    | Open Access

    The specific glycosylation patterns of biological drugs often impact the efficacy and safety of the therapeutic product. Here the authors describe a native mass spectrometry approach that allows the resolution of highly complex glycosylation patterns on large proteins, which they apply to the therapeutic Fc-fusion protein Etanercept.

    • Therese Wohlschlager
    • , Kai Scheffler
    •  & Christian G. Huber
  • Article
    | Open Access

    The heat shock protein 90 (Hsp90) chaperone undergoes large conformational changes during its functional cycle. Here the authors combine in vivo, biochemical, biophysical and computational approaches and provide insights into the allosteric regulation of Hsp90 by identifying and characterizing a switch point in the Hsp90 middle domain.

    • Daniel Andreas Rutz
    • , Qi Luo
    •  & Johannes Buchner
  • Article
    | Open Access

    Spatial localization of genetic information is important for tissue heterogeneity but difficult to capture with current analytical techniques. Here the authors present “Pixelated RT-LAMP”, an approach that uses parallel on-chip reactions to provide the distribution of target sequences directly from tissue.

    • A. Ganguli
    • , A. Ornob
    •  & R. Bashir
  • Article
    | Open Access

    Levels of the enzyme myeloperoxidase in the blood are considered a biomarker for the severity of cardiovascular disease. Here the authors report a rapid and inexpensive method for measuring myeloperoxidase activity in human plasma by luminescence, after adsorption of the enzyme to a polymer surface.

    • Reece J. Goiffon
    • , Sara C. Martinez
    •  & David Piwnica-Worms
  • Article
    | Open Access

    Diagnostic microfluidic devices often require complicated optical systems and computers to quantify results. Here, Qin and colleagues link enzymatic biomarker detection with the displacement of ink, resulting in a device that displays quantitative results as bar graphs directly on the device.

    • Yujun Song
    • , Yuanqing Zhang
    •  & Lidong Qin
  • Article
    | Open Access

    The influenza virus life cycle relies on sialidases, which are classified as group-1 or group-2, depending on the flexibility of the '150-loop'. In this study, chemical compounds are developed, which lock open the '150-loop', selectively inhibiting the activity of group-1 sialidases.

    • Santosh Rudrawar
    • , Jeffrey C. Dyason
    •  & Mark von Itzstein