Neutral loss (NL) spectral data presents a mirror of MS2 data and is a valuable yet largely untapped resource for molecular discovery and similarity analysis. Tandem mass spectrometry (MS2) data is ...effective for the identification of known molecules and the putative identification of novel, previously uncharacterized molecules (unknowns). Yet, MS2 data alone is limited in characterizing structurally related molecules. To facilitate unknown identification and complement the METLIN-MS2 fragment ion database for characterizing structurally related molecules, we have created a MS2 to NL converter as a part of the METLIN platform. The converter has been used to transform METLIN’s MS2 data into a neutral loss database (METLIN-NL) on over 860 000 individual molecular standards. The platform includes both the MS2 to NL converter and a graphical user interface enabling comparative analyses between MS2 and NL data. Examples of NL spectral data are shown with oxylipin analogues and two structurally related statin molecules to demonstrate NL spectra and their ability to help characterize structural similarity. Mirroring MS2 data to generate NL spectral data offers a unique dimension for chemical and metabolite structure characterization.
Single quadrupole mass spectrometry (MS) with enhanced in-source multiple fragment ion monitoring was designed to perform high sensitivity quantitative mass analyses. Enhanced in-source fragmentation ...amplifies fragmentation from traditional soft electrospray ionization producing fragment ions that have been found to be identical to those generated in tandem MS. We have combined enhanced in-source fragmentation data with criteria established by the European Union Commission Directive 2002/657/EC for electron ionization single quadrupole quantitative analysis to perform quantitative analyses. These experiments were performed on multiple types of complex samples that included a mixture of 50 standards, as well as cell and plasma extracts. The dynamic range for these quantitative analyses was comparable to triple quadrupole multiple reaction monitoring (MRM) analyses at up to 5 orders of magnitude with the cell and plasma extracts showing similar matrix effects across both platforms. Amino acid and fatty acid measurements performed from certified NIST 1950 plasma with isotopically labeled standards demonstrated accuracy in the range of 91–110% for the amino acids, 76–129% for the fatty acids, and good precision (coefficient of variation <10%). To enhance specificity, a newly developed correlated ion monitoring algorithm was designed to facilitate these analyses. This algorithm autonomously processes, aligns, filters, and compiles multiple ions within one chromatogram enabling both precursor and in-source fragment ions to be correlated within a single chromatogram, also enabling the detection of coeluting species based on precursor and fragment ion ratios. Single quadrupole instrumentation can provide MRM level quantitative performance by monitoring/correlating precursor and fragment ions facilitating high sensitivity analysis on existing single quadrupole instrumentation that are generally inexpensive, easy to operate, and technically less complex.
Enhanced in-source fragmentation/annotation (EISA) has recently been shown to produce fragment ions that match tandem mass spectrometry data across a wide range of small molecules. EISA has been ...developed to facilitate data-dependent acquisition (DDA), data-independent acquisiton (DIA), and multiple-reaction monitoring (MRM), enabling molecular identifications in untargeted metabolomics and targeted quantitative single-quadrupole MRM (Q-MRM) analyses. Here, EISA has been applied to peptide-based proteomic analysis using optimized in-source fragmentation to generate fragmentation patterns for a mixture of 38 peptides, which were comparable to the b- and y-type fragment ions typically observed in tandem MS experiments. The optimal in-source fragmentation conditions at which high-abundance peptide fragments and precursor ions coexist were compared with automated data-dependent acquisition (DDA) in the same quadrupole time-of-flight (QTOF–MS) mass spectrometer, generating a significantly higher fragment percentage of peptides from both singly and doubly charged b- and y-type fragment (b+, y+, b2+, and y2+) ions. Higher fragment percentages were also observed for these fragment ion series over linear ion trap instrumentation. An XCMS-EISA annotation/deconvolution program was developed, making use of the retention time and peak shape continuity between precursor fragment ions, to perform automated proteomic data analysis on the enhanced in-source fragments. Post-translational modification (PTM) characterization on peptides was demonstrated with EISA, producing fragment ions corresponding to a neutral loss of phosphoric acid with greater intensity than observed with DDA on a QTOF–MS. Moreover, Q-MRM demonstrated the ability to use EISA for peptide quantification. The availability of more sophisticated in-source fragmentation informatics, beyond XCMS-EISA, will further enable EISA for sensitive autonomous identification and Q-MRM quantitative analyses in proteomics.
Schistosoma mansoni is a parasitic helminth that infects millions of people mostly in tropical parts of the world. Different life cycle stages of S.mansoni, that infect or develop in the human host, ...promote distinct immune responses and are known for their ability to modulate host immune responses. However, the molecular mechanisms through which the parasites interact with, and modulate the host immune system remain incompletely understood. Despite the well-known ability of various lipids to modulate immune responses, a comprehensive analysis of the lipidome of the different life cycle stages has not been performed. Using three complementary MS-based platforms to detect and quantify around 350 lipid species, we here characterized the lipid profiles of S. mansoni cercariae, worms and eggs, as well as extracts and excretory/secretory (ES) products of different life cycle stages of S. mansoni. We identified life cycle stage specific signatures of lipid classes of which cercariae were found to have the most distinct profile. Moreover, we detected several immunolomodulatory oxylipids in the different life cycle stages. Specifically, prostaglandins were found to be most highly enriched in egg preparations, while resolvins were specifically detected in cercariae. Together, the generation of this detailed lipid database of the different life cycle stages of S. mansoni will not only be important for a better understanding of the biology of the parasite itself but also of host-parasite interactions and how that could result in immunomodulation.
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•Comprehensive Lipidomics study of Schistosoma mansoni and its life stages.•Description of a Schistosoma mansoni lipid database.•Identification of several important immunomodulatory lipids, leads for further research.
High-Throughput Venomics Slagboom, Julien; Derks, Rico J. E.; Sadighi, Raya ...
Journal of proteome research,
06/2023, Letnik:
22, Številka:
6
Journal Article
Recenzirano
Odprti dostop
In this study, we present high-throughput (HT) venomics, a novel analytical strategy capable of performing a full proteomic analysis of a snake venom within 3 days. This methodology comprises a ...combination of RP-HPLC-nanofractionation analytics, mass spectrometry analysis, automated in-solution tryptic digestion, and high-throughput proteomics. In-house written scripts were developed to process all the obtained proteomics data by first compiling all Mascot search results for a single venom into a single Excel sheet. Then, a second script plots each of the identified toxins in so-called Protein Score Chromatograms (PSCs). For this, for each toxin, identified protein scores are plotted on the y-axis versus retention times of adjacent series of wells in which a toxin was fractionated on the x-axis. These PSCs allow correlation with parallel acquired intact toxin MS data. This same script integrates the PSC peaks from these chromatograms for semiquantitation purposes. This new HT venomics strategy was performed on venoms from diverse medically important biting species; Calloselasma rhodostoma, Echis ocellatus, Naja pallida, Bothrops asper, Bungarus multicinctus, Crotalus atrox, Daboia russelii, Naja naja, Naja nigricollis, Naja mossambica, and Ophiophagus hannah. Our data suggest that high-throughput venomics represents a valuable new analytical tool for increasing the throughput by which we can define venom variation and should greatly aid in the future development of new snakebite treatments by defining toxin composition.
Imbalances in the amounts of amyloid-β peptides (Aβ) generated by the membrane proteases β- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase ...have shown that increasing membrane thickness modulates Aβ generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aβ37 and/or Aβ38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aβ secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies.
Tandem Mass Spectrometry across Platforms Hoang, Corey; Uritboonthai, Winnie; Hoang, Linh ...
Analytical chemistry (Washington),
04/2024, Letnik:
96, Številka:
14
Journal Article
Recenzirano
Odprti dostop
PubChem serves as a comprehensive repository, housing over 100 million unique chemical structures representing the breadth of our chemical knowledge across numerous fields including metabolism, ...pharmaceuticals, toxicology, cosmetics, agriculture, and many more. Rapid identification of these small molecules increasingly relies on electrospray ionization (ESI) paired with tandem mass spectrometry (MS/MS), particularly by comparison to genuine standard MS/MS data sets. Despite its widespread application, achieving consistency in MS/MS data across various analytical platforms remains an unaddressed concern. This study evaluated MS/MS data derived from one hundred molecular standards utilizing instruments from five manufacturers, inclusive of quadrupole time-of-flight (QTOF) and quadrupole orbitrap “exactive” (QE) mass spectrometers by Agilent (QTOF), Bruker (QTOF), SCIEX (QTOF), Waters (QTOF), and Thermo QE. We assessed fragment ion variations at multiple collisional energies (0, 10, 20, and 40 eV) using the cosine scoring algorithm for comparisons and the number of fragments observed. A parallel visual analysis of the MS/MS spectra across instruments was conducted, consistent with a standard procedure that is used to circumvent the still prevalent issue of mischaracterizations as shown for dimethyl sphingosine and C20 sphingosine. Our analysis revealed a notable consistency in MS/MS data and identifications, with fragment ions’ m/z values exhibiting the highest concordance between instrument platforms at 20 eV, the other collisional energies (0, 10, and 40 eV) were significantly lower. While moving toward a standardized ESI MS/MS protocol is required for dependable molecular characterization, our results also underscore the continued importance of corroborating MS/MS data against standards to ensure accurate identifications. Our findings suggest that ESI MS/MS manufacturers, akin to the established norms for gas chromatography mass spectrometry instruments, should standardize the collision energy at 20 eV across different instrument platforms.
The potential benefits of ultra-low flow electrospray ionization (ESI) for the analysis of phosphopeptides in proteomics was investigated. First, the relative flow dependent ionization efficiency of ...nonphosphorylated vs multiplyphosphorylated peptides was characterized by infusion of a five synthetic peptide mix with zero to four phophorylation sites at flow rates ranging from 4.5 to 500 nL/min. Most importantly, similar to what was found earlier by Schmidt et al., it has been verified that at flow rates below 20 nL/min the relative peak intensities for the various peptides show a trend toward an equimolar response, which would be highly beneficial in phosphoproteomic analysis. As the technology to achieve liquid chromatography separation at flow rates below 20 nL/min is not readily available, a sheathless capillary electrophoresis–electrospray ionization-mass spectrometry (CE–ESI-MS) strategy based on the use of a neutrally coated separation capillary was used to develop an analytical strategy at flow rates as low as 6.6 nL/min. An in-line preconcentration technique, namely, transient isotachophoresis (t-ITP), to achieve efficient separation while using larger volume injections (37% of capillary thus 250 nL) was incorporated to achieve even greater sample concentration sensitivities. The developed t-ITP-ESI-MS strategy was then used in a direct comparison with nano-LC–MS for the detection of phosphopeptides. The comparison showed significantly improved phosphopeptide sensitivity in equal sample load and equal sample concentration conditions for CE–MS while providing complementary data to LC–MS, demonstrating the potential of ultra-low flow ESI for the analysis of phosphopeptides in liquid based separation techniques.
Biological activities of immunoglobulin G such as effector functions via Fc receptor interactions are influenced by Fc-linked N-glycans. Here we describe a fast, robust and sensitive nano-LC-ESI-MS ...method for detailed subclass specific analysis of IgG Fc N-glycosylation. A sheath-flow ESI sprayer was used as a sensitive zero dead volume plug-and-play interface for online MS coupling, generating a very constant spray and ionization over the entire LC gradient. The propionic acid containing sheath-liquid effectively suppressed TFA gas-phase ion-pairing, enabling the use of TFA containing mobile phases. The fixed position of the sheath-flow ESI sprayer, far away from the glass capillary inlet, reduced MS contamination as compared to conventional nano-ESI. The method was found to be suitable for fast and detailed subclass specific IgG Fc N-glycosylation profiling in human plasma. The obtained subclass specific IgG Fc N-glycosylation profiles were processed automatically using in house developed software tools. For each of the IgG subclasses the 8 major glycoforms showed an interday analytical variation below 5%. The method was used to profile the IgG Fc N-glycosylation of 26 women at several time points during pregnancy and after delivery, revealing pregnancy-associated changes in IgG galactosylation, sialylation and incidence of bisecting N-acetylglucosamine.
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► We perform nanoLC-MS in a very robust manner by employing a sheath-flow ESI sprayer. ► IgG Fc glycosylation profiling is achieved in a sub-class specific manner. ► The method is fast with a total analysis time of 16
min. ► Changes in IgG Fc N-glycans during pregnancy are described.
The biophysical properties and biological functions of membranes are highly dependent on lipid composition. Supplementing cellular membranes with very long chain fatty acids (vlcFAs) is notoriously ...difficult given the extreme insolubility of vlcFAs in aqueous solution. Herein, we report a solvent-free, photochemical approach to enrich target membranes with vlcFA. To prevent aggregation of vlcFA, we created light-sensitive micelles composed exclusively of poly-ethylene-glycol-nervonic acid amphiphiles (NA-PEG), which spontaneously disassemble in the presence of lipid bilayers. Once embedded within a membrane, UV light is used to cleave off PEG, leaving free nervonic acid (NA, i.e. FA24:1) in the target membrane. When applied to living cells, free NA was processed by the cell to generate various species of membrane and other lipids with incorporated vlcFAs. In this way, we were able to alter the membrane lipid composition of cellular membranes and modulate the enzymatic activity of γ-secretase, an intramembrane protease whose dysfunction has been implicated in the onset and progression of Alzheimer's disease.
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•Delivery of very long chain fatty acids to cell membranes•Light induced cleavage of fatty acid-PEG conjugates results in membrane modulation.•The enzymatic activity of γ-secretase is modulated by modifying the cell membrane.•The dysfunction of an intramembrane protease has been implicated in the onset and progression of Alzheimer disease.
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