Aspartic Protease Nepenthesin‑1 as a Tool for Digestion in Hydrogen/Deuterium Exchange Mass Spectrometry

Hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) utilizes enzymatic digestion of proteins to localize the information about altered exchange patterns in protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. Recently, it was shown that there is an interesting group of proteolytic enzymes from carnivorous pitcher plants of the genus <i>Nepenthes</i>. In this report, we describe successful immobilization and the use of one of these enzymes, nepenthesin-1, in HXMS workflow. In contrast to pepsin, it has different cleavage specificities, and despite its high inherent susceptibility to reducing and denaturing agents, it is very stable upon immobilization and withstands even high concentration of guanidine hydrochloride and reducing agents. We show that denaturing agents can alter digestion by reducing protease activity and/or substrate solubility, and additionally, they influence the trapping of proteolytic peptides onto the reversed phase resin

Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen–antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology

Identification of the exact site of oxidation in the peptide ⁴⁵²DAFSYGAVQQSIDSR⁴⁶⁶ by LC-ESI-MS/MS.

<p>The nearly complete series of C-terminal y-ions in the MS/MS spectrum of the double-charged ion of 830.4 confirms the peptide sequence. The +16 Da mass shift found for the ion y₁₃ further indicates that Phe454 is oxidised during substrate turnover and by H₂O₂ treatment.</p

Oxidation of methionines.

<p>Methionine-containing peptide fragments detected that show a mass increase of 16 or 32 Da after proteolytic digestion of <i>Tm</i>POx (inactivated during turnover of 100 mM D-glucose, treated with endogenous H₂O₂ or unaffected). Proteolytic digestion was performed with trypsin or Asp-N protease as indicated. The last column indicates whether the particular Met is considerably oxidised during substrate turnover and by H₂O₂ treatment compared to the unaffected sample (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.g002" target="_blank">Fig 2</a>).</p

Active-site geometry of pyranose oxidase from <i>T</i>. <i>multicolor</i>.

<p>In the closed form of the active-site loop of pyranose oxidase from <i>T</i>. <i>multicolor</i> (<i>Tm</i>POx, PDB code 1TT0; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>]), which is thought to be relevant for the oxidative half-reaction of POx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>], Phe454 is positioned in the direct vicinity of the isoalloxazine ring and the C4a/N5 locus, at which oxygen is reduced. The figure was generated using PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>).</p

Accessibility of methionine residues.

<p>Surface of the <i>T</i>. <i>multicolor</i> POx monomer in the vicinity of (<b>A</b>) Met417, showing the surface-exposed sulphur atom which is oxidised by H₂O₂, and (<b>B</b>) Met380 with its sulphur-containing side chain pointing towards the interior of the polypeptide matrix, in which it is buried and hence is not accessible from the surface.</p

Mass spectrometric identification of methionine residues oxidized by H₂O₂ during <i>Tm</i>POx inactivation.

<p>MALDI MS spectra were measured for unaffected POx (<b>A</b>), for POx inactivated during D-glucose oxidation (<b>B</b>) or for POx inactivated by endogenous H₂O₂ (<b>C</b>). The selected MALDI spectra in the left panel illustrate that Met497 of the tryptic peptide ITDAYNMPQPTFDFR with a theoretical MH⁺ of 1815.8 was extensively oxidised in <i>Tm</i>POx inactivated either during substrate turnover (<b>B</b>, left panel) or by H₂O₂ treatment (<b>C</b>, left panel). In contrast, some methionine residues were found not to be oxidised during <i>Tm</i>POx inactivation as shown for Met74 of the peptide VAMFDIGEIDSGLK having a MH⁺ of 1494.8 (right panel). The small signals at m/z 1510.8 are related to the oxidized form of the peptide generated due to the presence of air oxygen.</p

Apparent kinetic constants of GalOx produced in <i>E. coli</i> for several electron donor substrates.

<p>Apparent kinetic constants of GalOx produced in <i>E. coli</i> for several electron donor substrates.</p

Purification of recombinant GalOx expressed in <i>E. coli.</i>

<p>Purification of recombinant GalOx expressed in <i>E. coli.</i></p

Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i>

<p>Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i></p