Trimethylation Enhancement using Diazomethane (TrEnDi): Rapid On-Column Quaternization of Peptide Amino Groups via Reaction with Diazomethane Significantly Enhances Sensitivity in Mass Spectrometry Analyses via a Fixed, Permanent Positive Charge

Defining cellular processes relies heavily on elucidating the temporal dynamics of proteins. To this end, mass spectrometry (MS) is an extremely valuable tool; different MS-based quantitative proteomics strategies have emerged to map protein dynamics over the course of stimuli. Herein, we disclose our novel MS-based quantitative proteomics strategy with unique analytical characteristics. By passing ethereal diazomethane over peptides on strong cation exchange resin within a microfluidic device, peptides react to contain fixed, permanent positive charges. Modified peptides display improved ionization characteristics and dissociate via tandem mass spectrometry (MS²) to form strong a₂ fragment ion peaks. Process optimization and determination of reactive functional groups enabled <i>a priori</i> prediction of MS² fragmentation patterns for modified peptides. The strategy was tested on digested bovine serum albumin (BSA) and successfully quantified a peptide that was not observable prior to modification. Our method ionizes peptides regardless of proton affinity, thus decreasing ion suppression and permitting predictable multiple reaction monitoring (MRM)-based quantitation with improved sensitivity

Trimethylation Enhancement Using Diazomethane (TrEnDi) II: Rapid In-Solution Concomitant Quaternization of Glycerophospholipid Amino Groups and Methylation of Phosphate Groups via Reaction with Diazomethane Significantly Enhances Sensitivity in Mass Spectrometry Analyses via a Fixed, Permanent Positive Charge

A novel mass spectrometry (MS)-based lipidomics strategy that exposes glycerophospholipids to an ethereal solution of diazomethane and acid, derivatizing them to contain a net fixed, permanent positive charge, is described. The sensitivity of modified lipids to MS detection is enhanced via improved ionization characteristics as well as consolidation of ion dissociation to form one or two strong, characteristic polar headgroup fragments. Our strategy has been optimized to enable a priori prediction of ion fragmentation patterns for four subclasses of modified glycerophospholipid species. Our method enables analyte ionization regardless of proton affinity, thereby decreasing ion suppression and permitting predictable precursor ion-based quantitation with improved sensitivity in comparison to MS-based methods that are currently used on unmodified lipid precursors

Trimethylation Enhancement Using ¹³C‑Diazomethane: Gas-Phase Charge Inversion of Modified Phospholipid Cations for Enhanced Structural Characterization

Methylation of phospholipids (PL) leads to increased uniformity in positive electrospray ionization (ESI) efficiencies across the various PL subclasses. This effect is realized in the approach referred to as “trimethylation enhancement using ¹³C-diazomethane” (¹³C-TrEnDi), which results in the methyl esterification of all acidic sites and the conversion of amines to quaternary ammonium sites. Collision-induced dissociation (CID) of these cationic modified lipids enables class identification by forming distinctive headgroup fragments based on the number of ¹³C atoms incorporated during derivatization. However, there are no distinctive fragment ions in positive mode that provide fatty acyl information for any of the modified lipids. Gas-phase ion/ion reactions of ¹³C-TrEnDi-modified phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), and sphingomyelin (SM) cations with dicarboxylate anions are shown to charge-invert the positively charged phospholipids to the negative mode. An electrostatically bound complex anion is shown to fragment predominantly via a novel headgroup dication transfer to the reagent anion. Fragmentation of the resulting anionic product yields fatty acyl information, in the case of the glycerophospholipids (PE, PS, and PC), via ester bond cleavage. Analogous information is obtained from modified SM lipid anions via amide bond cleavage. Fragmentation of the anions generated from charge inversion of the ¹³C-TrEnDi-modified phospholipids was also found to yield lipid class information without having to perform CID in positive mode. The combination of ¹³C-TrEnDi modification of lipid mixtures with charge inversion to the negative-ion mode retains the advantages of uniform ionization efficiency in the positive-ion mode with the additional structural information available in the negative-ion mode without requiring the lipids to be ionized directly in both ionization modes

Perturbations of respiratory neural activity pattern by pontine removal and vagal stimulations.

<p><b>A</b>. Model performance after vagotomy. <b>B</b>. Model performance after vagotomy and subsequent removal of pontine excitatory drive. Note the apneustic breathing pattern characterized by the significant increase in the duration of inspiration and slowing of the respiratory oscillations. <b>C</b>. Simulations of the effects of brief and continuous stimulation of mechanoreceptor afferents. The first stimulus (bottom trace, 7 ml of lung inflation) applied in the middle of the respiratory phase terminated the current inspiration. The second, reduced stimulus (5 ml) applied at the same phase was unable to terminate inspiration. The third stimulus of the same size as the second one (5 ml) applied later in inspiration terminated the inspiratory phase. Finally, continuous linearly increasing stimulation was applied. This stimulation shortened inspiration and prolonged expiration and then produced expiratory “apnea”, when all inspiratory neurons were inhibited by continuously active expiratory neurons.</p

Parameters of neural network.

<p>Note that <i>PSR</i> is the peripheral feedback provided by the lung stretch receptors. In our model, we use , the inspired lung volume (excess of the lung volume above the basal volume, see (25)), as the <i>PSR</i> signal that is multiplied by the <i>e_i, f_i</i> values indicated in the table.</p><p>Parameters of neural network.</p

Trimethylation Enhancement Using ¹³C‑Diazomethane (¹³C-TrEnDi): Increased Sensitivity and Selectivity of Phosphatidylethanolamine, Phosphatidylcholine, and Phosphatidylserine Lipids Derived from Complex Biological Samples

Significant sensitivity enhancements in the tandem mass spectrometry-based analysis of complex mixtures of several phospholipid classes has been achieved via ¹³C-TrEnDi. ¹³C-TrEnDi-modified phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) lipids extracted from HeLa cells demonstrated greater sensitivity via precursor ion scans (PISs) than their unmodified counterparts. Sphingomyelin (SM) species exhibited neither an increased nor decreased sensitivity following modification. The use of isotopically labeled diazomethane enabled the distinction of modified PE and modified PC species that would yield isobaric species with unlabeled diazomethane. ¹³C-TrEnDi created a PE-exclusive PIS of <i>m</i>/<i>z</i> 202.1, two PS-exclusive PISs of <i>m</i>/<i>z</i> 148.1 and <i>m</i>/<i>z</i> 261.1, and a PIS of <i>m</i>/<i>z</i> 199.1 for PC species (observed at odd <i>m</i>/<i>z</i> values) and SM species (observed at even <i>m</i>/<i>z</i> values). The standardized average area increase after TrEnDi modification was 10.72-fold for PE species, 2.36-fold for PC, and 1.05-fold for SM species. The sensitivity increase of PS species was not quantifiable, as there were no unmodified PS species identified prior to derivatization. ¹³C-TrEnDi allowed for the identification of 4 PE and 7 PS species as well as the identification and quantitation of an additional 4 PE and 4 PS species that were below the limit of detection (LoD) prior to modification. ¹³C-TrEnDi also pushed 24 PE and 6 PC lipids over the limit of quantitation (LoQ) that prior to modification were above the LoD only

Comparison of model simulations with experimental data.

<p><b>A</b>. Breathing stimulation elicited in conscious adult rat in a flow-through, whole-body plethysmography chamber by photostimulation via channelrodopsin genetically engineered in RTN Phox2b-expressing glutamatergic neurons. The left diagrams were constructed for hyperoxic normocapnia (100% O₂), and the right diagrams for hypercapnia (8% CO₂, balance O₂). Continuous RTN stimulation (23 Hz, 3 ms pulses, 30 s total duration, blue bar at the top) raised tidal volume, V_T, and breathing frequency, fR. During hyperoxic hypercapnia (right), RTN photostimulation produced a small increase in V_T but no increase in fR. Adapted from Abbott et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109894#pone.0109894-Abbott1" target="_blank">[52]</a>, Fig. 1B, with permission. <b>B</b>. The results of our simulations. An additional 30s-duration increase in the blood CO₂ level (Δ<i>p_c</i>  = 30 mmHg, blue bar at the top) was applied in the normocapnic case (left) and on the background of simulated hypercapnia (<i>f_cm</i> = 8%) (right).</p

Parameters of the model.

<p>Parameters of the model.</p

Data-model comparisons of respiratory phase dependent photostimulation of pre-BötC neurons.

<p>(<b>A</b>) Five respiratory airflow traces illustrating the response to stimulation of pre-BötC neurons at different respiratory phases. The orange trace indicates a lack of response to photostimulation when it occurs during the post-inspiratory phase (refractory period). The green trace shows a slight prolongation of inspiration (green arrow) when the stimulus is delivered during the inspiratory phase and the black traces show initiation of inspiration during the stimulus after a short latency (based on data from the study of Alsahafi et al. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006148#pcbi.1006148.ref047" target="_blank">47</a>], <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006148#pcbi.1006148.g007" target="_blank">Fig 7B</a>, used with authors’ permission). (<b>B</b>) The model output traces of pre-I/I and post-I activities show a similar behavior to the experimental data when all pre-BötC populations are stimulated with a 300 ms simulated light pulse. The applied stimulations are shown at the bottom (<i>stim</i>_<i>ChR</i> = 0.2). The timing of the laser stimulus is indicated with blue shading and dashed red lines.</p

Model performance vs. experimental data for responses to sustained stimulations of inhibitory neurons within the BötC.

<p>(<b>A</b> and <b>B</b>) Model responses to sustained stimulation of inhibitory neurons within the pre-BötC with low-intensity (A; <i>stim</i>_<i>ChR</i> = 0.14) and higher intensity (B, <i>stim</i>_<i>ChR</i> = 0.18) stimulation. The timing of the stimulus is indicated with blue shading and dashed red lines. Output activity of all neuron populations in the model are shown. The oscillation frequency decreases maximally in A, and is fully suppressed in B, similar to our experimental results (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006148#pcbi.1006148.g007" target="_blank">Fig 7</a>) (<b>C</b>). Increases of stimulation intensity reduced oscillation frequency and terminated inspiratory activity at the highest intensities examined, directionally similar to the experimental results shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006148#pcbi.1006148.g007" target="_blank">Fig 7C</a>.</p