Complete NMR Assignment of Succinimide and Its Detection and Quantification in Peptides and Intact Proteins

Detecting and quantifying post-translational modifications (PTMs) in full-length proteins is a challenge, especially in the case of spontaneously occurring, nonenzymatic PTMs. Such a PTM is the formation of succinimide (Snn) in a protein that occurs spontaneously in prone primary sequences and leads typically to an equilibrium between Snn and its hydrolysis products isoaspartate (isoAsp) and aspartate. In order to detect these modifications in proteins by NMR spectroscopy, chemical shift assignments of reference compounds are required. We used peptide synthesis and 2D NMR spectroscopy to assign all ¹H and ¹³C chemical shifts of Snn and isoAsp and found characteristic chemical shift correlations. To provide chemical shift reference data suitable for comparison with data of denatured proteins, we repeated the assignment in 7 M urea (pH 2.3) and in DMSO. Most characteristic of Snn are the two downfield shifted carbonyl chemical shifts, the chemical shift correlations of Cβ-Hβ of Snn and Cα-Hα of the succeeding residue which are clearly distinct from random coil chemical shift correlations. The characteristic 2D NMR fingerprints of Snn were used to detect and quantify this PTM in the model protein lysozyme, the biotherapeutic filgrastim, and the Fc part of immunoglobulin G1. Mass spectrometry (MS) was applied as an additional independent method. The orthogonality of the NMR and MS techniques allows cross-validation, which is especially important to search for subtle PTMs in proteins. Studying PTMs by NMR spectroscopy is a promising method to analyze proteins and peptides from natural sources, recombinant expression, or chemical synthesis

Immunoreactivity of selected phage clones corresponding to different fragments of the <i>N. meningitidis</i> NadA antigen.

<p>Immunoreactivity of selected phage clones corresponding to different fragments of the <i>N. meningitidis</i> NadA antigen.</p

Enrichment of phage clones predicted to display authentic NadA fragments on their surface after selection with a serum pool from volunteers immunized with the Bexero vaccine.

<p>Frequency values reported in the vertical axis in panels A–C refer to the occurrence, per single amino acid position, of sequences predicted to express authentic NadA fragments, relative to those predicted to express irrelevant or no polypeptides. The inset in figure A reports the same data with a higher y-axis magnification. The horizontal axis reports the amino acid positions of the translated NadA sequence. A, unselected library; B and C, library outputs after one and two rounds of selection, respectively. D, Cumulative enrichment factors for each amino acid position derived from NadA fragments obtained after one (blue line) and two (red line) rounds of selection; colored bars in the horizontal axis refer to NadA domains; the area between the dashed vertical lines correspond to the cell binding region of NadA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114159#pone.0114159-Tavano1" target="_blank">[18]</a>. E and F, enrichment factors of NadA fragments after one and two rounds of selection, respectively. Only the fragments laying in the upper quartile of enrichment factors values are shown.</p

Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.

<p>Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.</p

Properties of the antigen-specific phage library before and after selection with a pool of serum samples from volunteers immunized with the Bexero anti-MenB vaccine.

<p>A–C, abundance of “natural frame” <i>nadA</i> fragments in the library before (A) and after the first and second rounds of selection (B and C, respectively). Each point represents the number of unique fragments (vertical axis) displaying the number of copies indicated in the horizontal axis; D–F, <i>nadA</i> fragment length distribution before (D) and after the first and second rounds of selection (E and F, respectively).</p

Schematic outline of the epitope mapping approach.

<p>The gene encoding the antigen is fragmented by DNAse digestion and the gene fragments are inserted into lambda phage vectors. The phage library is mixed with immune serum and phage particles binding to immunoglobulins are separated using Protein-G coated magnetic beads. The inserts of the phage population obtained after selection are massively sequenced and compared with those of the original unselected library using an <i>ad hoc</i> developed software which identifies the region(s) of the antigen targeted by serum antibodies.</p

Marginal means (adjusted prevalence) for significant gender*city interactions in lifetime drinkers and people with AUD in the past 12 months.

<p>Note: No women in Madrid met the criteria for past 12-month AUD.</p

Past 12-month and lifetime alcohol use patterns (use of alcohol, and consumption indicator: Index^*) in the overall sample and the subsample of lifetime drinkers (people who reported drinking any type of alcoholic beverage at least 12 times during their lifetime).

<p>Past 12-month and lifetime alcohol use patterns (use of alcohol, and consumption indicator: Index^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196574#t002fn002" target="_blank">*</a>}) in the overall sample and the subsample of lifetime drinkers (people who reported drinking any type of alcoholic beverage at least 12 times during their lifetime).</p

Sociodemographic characteristics of the overall sample and the subsample of lifetime drinkers (people who reported drinking any type of alcoholic beverage at least 12 times during their lifetime).

<p>Sociodemographic characteristics of the overall sample and the subsample of lifetime drinkers (people who reported drinking any type of alcoholic beverage at least 12 times during their lifetime).</p

Current (last month), 12-month and lifetime prevalence of alcohol abuse, alcohol dependence and alcohol abuse and/or dependence disorders (AUD) by cities.

<p>Current (last month), 12-month and lifetime prevalence of alcohol abuse, alcohol dependence and alcohol abuse and/or dependence disorders (AUD) by cities.</p