27 research outputs found
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 (MS<sup>2</sup>) to form strong a<sub>2</sub> fragment
ion peaks. Process optimization and determination of reactive functional
groups enabled <i>a priori</i> prediction of MS<sup>2</sup> 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 <sup>13</sup>C‑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 <sup>13</sup>C-diazomethane”
(<sup>13</sup>C-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 <sup>13</sup>C 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 <sup>13</sup>C-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 <sup>13</sup>C-TrEnDi-modified phospholipids was also found to yield lipid class
information without having to perform CID in positive mode. The combination
of <sup>13</sup>C-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
Trimethylation Enhancement Using <sup>13</sup>C‑Diazomethane (<sup>13</sup>C-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 <sup>13</sup>C-TrEnDi. <sup>13</sup>C-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. <sup>13</sup>C-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. <sup>13</sup>C-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. <sup>13</sup>C-TrEnDi also
pushed 24 PE and 6 PC lipids over the limit of quantitation (LoQ)
that prior to modification were above the LoD only
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<sub>i</sub>, f<sub>i</sub></i> values indicated in the table.</p><p>Parameters of neural network.</p
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% O<sub>2</sub>), and the right diagrams for hypercapnia (8% CO<sub>2</sub>, balance O<sub>2</sub>). Continuous RTN stimulation (23 Hz, 3 ms pulses, 30 s total duration, blue bar at the top) raised tidal volume, V<sub>T</sub>, and breathing frequency, fR. During hyperoxic hypercapnia (right), RTN photostimulation produced a small increase in V<sub>T</sub> 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 CO<sub>2</sub> level (Δ<i>p<sub>c</sub></i>  = 30 mmHg, blue bar at the top) was applied in the normocapnic case (left) and on the background of simulated hypercapnia (<i>f<sub>cm</sub></i> = 8%) (right).</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><sub><i>ChR</i></sub> = 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><sub><i>ChR</i></sub> = 0.14) and higher intensity (B, <i>stim</i><sub><i>ChR</i></sub> = 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