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.
Significant sensitivity enhancements in the tandem mass spectrometry-based analysis of complex mixtures of several phospholipid classes has been achieved via (13)C-TrEnDi. (13)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. (13)C-TrEnDi created a PE-exclusive PIS of m/z 202.1, two PS-exclusive PISs of m/z 148.1 and m/z 261.1, and a PIS of m/z 199.1 for PC species (observed at odd m/z values) and SM species (observed at even m/z 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. (13)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. (13)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.
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(2)) to form strong a2 fragment ion peaks. Process optimization and determination of reactive functional groups enabled a priori prediction of MS(2) 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) is a derivatization
technique that significantly enhances the signal intensity of glycerophospholipid
species in mass spectrometry (MS) and tandem mass spectrometry (MS/MS)
analyses. Here, we describe a novel apparatus that is able to conduct
in situ TrEnDi (iTrEnDi) by generating and immediately reacting small
amounts of gaseous diazoalkane with analyte molecules. iTrEnDi allows
complete and rapid methylation of phosphatidylcholine (PC), phosphatidylethanolamine
(PE), phosphatidic acid (PA), and sphingomyelin (SM) in a safe manner
by removing any need for direct handling of dangerous diazoalkane
solutions. iTrEnDi-modified PC ([PCTr]+) and
PE ([PETr]+) showed similar sensitivity enhancements
and fragmentation patterns compared to our previously reported methodology.
iTrEnDi yielded dimethylated PA ([PATr]), which exhibited
dramatically improved chromatographic behavior and a 14-fold increase
in liquid chromatography MS (LCMS) sensitivity compared to unmodified
PA. In comparison to in-solution-based TrEnDi, iTrEnDi demonstrated
a modest decrease in sensitivity, likely due to analyte losses during
handling. However, the enhanced safety benefits of iTrEnDi coupled
with its ease of use and capacity for automation, as well as its accommodation
of more-reactive diazoalkane species, vastly improve the accessibility
and utility of this derivatization technique. Finally, as a proof
of concept, iTrEnDi was used to produce diazoethane (DZE), a more-reactive
diazoalkane than diazomethane. Reaction between DZE and PC yielded
ethylated [PCTr]+, which fragmented via MS/MS
to produce a high-intensity characteristic fragment ion, enabling
a novel and highly sensitive precursor ion scan.
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