Computing H/D-Exchange rates of single residues from data of proteolytic fragments

<p>Abstract</p> <p>Background</p> <p>Protein conformation and protein/protein interaction can be elucidated by solution-phase Hydrogen/Deuterium exchange (sHDX) coupled to high-resolution mass analysis of the digested protein or protein complex. In sHDX experiments mutant proteins are compared to wild-type proteins or a ligand is added to the protein and compared to the wild-type protein (or mutant). The number of deuteriums incorporated into the polypeptides generated from the protease digest of the protein is related to the solvent accessibility of amide protons within the original protein construct.</p> <p>Results</p> <p>In this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based on combinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX data of overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residue resolution.</p> <p>Conclusions</p> <p>With our new method it is possible to automatically determine deuterium exchange with higher spatial resolution than the level of digested fragments.</p

3-Trifluoromethyl-3-aryldiazirine photolabels with enhanced ambient light stability

Ambient light stable 3-trifluoromethyl-3-aryldiazirine photolabels were designed, synthesized and evaluated. In addition to the enhancement to ambient light stability, the modified photolabels were equally efficient in labeling biological targets. The new photolabels also increase the aqueous solubility of the photoaffinity labels by several folds.</p

Site-specific human histone H3 methylation stability: fast K4me3 turnover

We employ stable‐isotope labeling and quantitative mass spectrometry to track histone methylation stability. We show that H3 trimethyl K9 and K27 are slow to be established on new histones and slow to disappear from old histones, with half‐lives of multiple cell divisions. By contrast, the transcription‐associated marks K4me3 and K36me3 turn over far more rapidly, with half‐lives of 6.8 h and 57 h, respectively. Inhibition of demethylases increases K9 and K36 methylation, with K9 showing the largest and most robust increase. We interpret different turnover rates in light of genome‐wide localization data and transcription‐dependent nucleosome rearrangements proximal to the transcription start site

Analysis of Intact Protein Isoforms by Mass Spectrometry

The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we discuss the basic technology for “top-down” intact protein analysis. Furthermore, examples of studies involved with the qualitative and quantitative analysis of full-length polypeptides are provided

New Reagents for Enhanced Liquid Chromatographic Separation and Charging of Intact Protein Ions for Electrospray Ionization Mass Spectrometry

Electrospray ionization produces multiply charged ions, thereby lowering the mass-to-charge ratio for peptides and small proteins to a range readily accessed by quadrupole ion trap, orbitrap, and ion cyclotron resonance (ICR) mass analyzers (m/z = 400−2000). For Fourier transform mass analyzers (orbitrap and ICR), higher charge also improves signal-to-noise ratio, mass resolution, and mass accuracy. Addition of m-nitrobenzyl alcohol (m-NBA) or sulfolane has previously been shown to increase the charge states of proteins. Moreover, polar aprotic dimethylformamide (DMF) improves chromatographic separation of proteolytic peptides for mass analysis of solution-phase protein hydrogen/deuterium exchange for improved (78−96%) sequence coverage. Here, we show that addition of each of the various modifiers (DMF, thiodiglycol, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone) can significantly increase the charge states of proteins up to 78 kDa. Moreover, incorporation of the same modifiers into reversed-phase liquid chromatography solvents improves sensitivity, charging, and chromatographic resolution for intact proteins