High-resolution ion mobility as an alternative to quadrupole-based precursor isolation for reducing chimeric fragmentation spectra in bottom-up proteomics
Ontology highlight
ABSTRACT:
INSTRUMENT(S): 6546 Q-TOF LC/MS (Agilent) with MOBIE platform (MOBILion)
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Daniel DeBord
PROVIDER: MSV000098195 | MassIVE | Fri Jun 13 19:20:00 BST 2025
REPOSITORIES: MassIVE
Similar Datasets
Project description:Quantitative bottom-up shotgun lipidomics relies on molecular species-specific "signature" fragments consistently detectable in tandem mass spectra of analytes and standards. Molecular species of glycerophospholipids are typically quantified using carboxylate fragments of their fatty acid moieties produced by higher-energy collisional dissociation of their molecular anions. However, employing standards whose fatty acids moieties are similar, yet not identical, to the target lipids could severely compromise their quantification. We developed a generic and portable fragmentation model implemented in the open-source LipidXte software that harmonizes the abundances of carboxylate anion fragments originating from fatty acid moieties having different sn-1/2 positions at the glycerol backbone, length of the hydrocarbon chain, and number and location of double bonds. The postacquisition adjustment enables unbiased absolute (molar) quantification of glycerophospholipid species independent of instrument settings, collision energy, and employed internal standards.
| S-EPMC6751524 | biostudies-literature
Project description:Shotgun lipidomics has evolved into a myriad of multi-dimensional strategies for molecular lipid characterization, including bioinformatics tools for mass spectrum interpretation and quantitative measurements to study systems-lipidomics in complex biological extracts. Taking advantage of spectral mass accuracy, scan speed and sensitivity of improved quadrupole linked time-of-flight mass analyzers, we developed a bias-free global lipid profiling acquisition technique of sequential precursor ion fragmentation called MS/MSALL. This generic information-independent tandem mass spectrometry (MS) technique consists of a Q1 stepped mass isolation window through a set mass range in small increments, fragmenting and recording all product ions and neutral losses. Through the accurate MS and MS/MS information, the molecular lipid species are resolved, including distinction of isobaric and isomeric species, and composed into more precise lipidomic outputs. The method demonstrates good reproducibility and at least 3 orders of dynamic quantification range for isomeric ceramides in human plasma. More than 400 molecular lipids in human plasma were uncovered and quantified in less than 12 min, including acquisitions in both positive and negative polarity modes. We anticipate that the performance of sequential precursor ion fragmentation both in quality and throughput will lead to the uncovering of new avenues throughout the biomedical research community, enhance biomarker discovery and provide novel information target discovery programs as it will prospectively shed new insight into affected metabolic and signaling pathways.
| S-EPMC3901199 | biostudies-literature
Project description:Conventional TopN data-dependent acquisition (DDA) LC-MS/MS analysis identifies only a limited fraction of all detectable precursors because the ion-sampling rate of contemporary mass spectrometers is insufficient to target each precursor in a complex sample. TopN DDA preferentially targets high-abundance precursors with limited sampling of low-abundance precursors and repeated analyses only marginally improve sample coverage due to redundant precursor sampling. In this work, advanced precursor ion selection algorithms were developed and applied in the bottom-up analysis of HeLa cell lysate to overcome the above deficiencies. Precursors fragmented in previous runs were efficiently excluded using an automatically aligned exclusion list, which reduced overlap of identified peptides to ∼10% between replicates. Exclusion of previously fragmented high-abundance peptides allowed deeper probing of the HeLa proteome over replicate LC-MS runs, resulting in the identification of 29% more peptides beyond the saturation level achievable using conventional TopN DDA. The gain in peptide identifications using the developed approach translated to the identification of several hundred low-abundance protein groups, which were not detected by conventional TopN DDA. Exclusion of only identified peptides compared with the exclusion of all previously fragmented precursors resulted in an increase of 1000 (∼10%) additional peptide identifications over four runs, suggesting the potential for further improvement in the depth of proteomic profiling using advanced precursor ion selection algorithms.
| S-EPMC5557716 | biostudies-literature
Project description:Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) started with spatial mapping of peptides and proteins. Since then, numerous bottom-up protocols have been developed. However, achievable spatial resolution and sample preparation with many wet steps hindered the development of single cell-level workflows for bottom-up spatial proteomics. This study presents a protocol optimized for MALDI-MSI measurements of single cells within the context of their 2D culture. Sublimation of CHCA, followed by a dip in ice-cold ammonium phosphate monobasic (AmP), produced peptide-rich mass spectra while maintaining matrix crystal sizes around 400 nm. This enables MALDI-MSI imaging of proteins in single cells grown on an ITO slide with a throughput of approximately 7800 cells per day. 89 peptide-like features corresponding to a single MDA-MB-231 breast cancer cell were detected. Furthermore, by combining the MALDI-MSI data with LC-MS/MS data obtained on cell pellets, we have successfully identified 24 peptides corresponding to 17 proteins, including actin, vimentin, and transgelin-2.
| S-EPMC11629377 | biostudies-literature
Project description:Layered alkali titanates of the lepidocrocite type are gaining enormous interest in various fields owing to their unique properties. These materials are mainly synthesized through a hydrothermal alkali treatment. However, this method uses a highly concentrated alkali solution, which has high environmental impacts and is therefore unsuitable for mass synthesis. Herein, we propose an efficient method for the large-scale synthesis of layered sodium titanate structures (Na2-x H x Ti2O5) using a recently reported bottom-up chemical process. The effects of the Na:Ti molar ratio in the peroxo-titanium complex ion precursor on the products are investigated through stoichiometric calculations for a molar ratio range of 10:1-1:1. The optimal ratio for the complete ionization of TiH2 (which is the starting material) to form the peroxo-titanium complex ion is found to be 1.1:1. The amount of alkali raw material required is 99.6% lower than that required in the traditional hydrothermal method. The crystal structures and morphologies of the samples are almost identical regardless of the Na:Ti molar ratio. The precursor-derived peroxo bonds narrow the energy band gaps of the layered titanates even when the amount of titanium ions dissolved in the precursor increases. The proposed method is not only an efficient synthetic route for mass production but also has potential applications in the development of photofunctional materials.
| S-EPMC11292821 | biostudies-literature
Project description:A nonclassical multistep pathway involving bottom-up and break-down stages for the evolution of calcite nanograins ∼50 nm in size was demonstrated in a basic aqueous system. Calcite nanofibrils ∼10 nm wide were produced as the initial crystalline phase via amorphous calcium carbonate through ion-by-ion assembly by the carbonation of Ca(OH)2 at a high pH of ∼13. Bundles ∼50 nm in diameter were then formed by the subsequent oriented attachment of the nanofibrils. Monodispersed calcite nanograins were finally obtained through spontaneous fragmentation of the fibrous forms via a decrease in pH by further carbonation.
| S-EPMC6645547 | biostudies-literature
Project description:Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.
| S-EPMC3033671 | biostudies-literature
Project description:Milk is a complex fluid whose proteome displays a diverse set of proteins of high abundance such as caseins and medium to low abundance whey proteins such as ß-lactoglobulin, lactoferrin, immunoglobulins, glycoproteins, peptide hormones, and enzymes. A sample preparation method that enables high reproducibility and throughput is key in reliably identifying proteins present or proteins responding to conditions such as a diet, health or genetics. Using skim milk samples from Jersey and Holstein-Friesian cows, we compared three extraction procedures which have not previously been applied to samples of cows' milk. Method A (urea) involved a simple dilution of the milk in a urea-based buffer, method B (TCA/acetone) involved a trichloroacetic acid (TCA)/acetone precipitation, and method C (methanol/chloroform) involved a tri-phasic partition method in chloroform/methanol solution. Protein assays, SDS-PAGE profiling, and trypsin digestion followed by nanoHPLC-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) analyses were performed to assess their efficiency. Replicates were used at each analytical step (extraction, digestion, injection) to assess reproducibility. Mass spectrometry (MS) data are available via ProteomeXchange with identifier PXD002529. Overall 186 unique accessions, major and minor proteins, were identified with a combination of methods. Method C (methanol/chloroform) yielded the best resolved SDS-patterns and highest protein recovery rates, method A (urea) yielded the greatest number of accessions, and, of the three procedures, method B (TCA/acetone) was the least compatible of all with a wide range of downstream analytical procedures. Our results also highlighted breed differences between the proteins in milk of Jersey and Holstein-Friesian cows.
| S-EPMC4707256 | biostudies-literature
Project description:Extending chip performance beyond current limits of miniaturisation requires new materials and functionalities that integrate well with the silicon platform. Germanium fits these requirements and has been proposed as a high-mobility channel material, a light emitting medium in silicon-integrated lasers, and a plasmonic conductor for bio-sensing. Common to these diverse applications is the need for homogeneous, high electron densities in three-dimensions (3D). Here we use a bottom-up approach to demonstrate the 3D assembly of atomically sharp doping profiles in germanium by a repeated stacking of two-dimensional (2D) high-density phosphorus layers. This produces high-density (10(19) to 10(20) cm(-3)) low-resistivity (10(-4)Ω · cm) metallic germanium of precisely defined thickness, beyond the capabilities of diffusion-based doping technologies. We demonstrate that free electrons from distinct 2D dopant layers coalesce into a homogeneous 3D conductor using anisotropic quantum interference measurements, atom probe tomography, and density functional theory.
| S-EPMC4530340 | biostudies-literature
Project description:Several recent studies have demonstrated that the bottom-up signaling of a visual stimulus is subserved by interareal gamma-band synchronization, whereas top-down influences are mediated by alpha-beta band synchronization. These processes may implement top-down control of stimulus processing if top-down and bottom-up mediating rhythms are coupled via cross-frequency interaction. To test this possibility, we investigated Granger-causal influences among awake macaque primary visual area V1, higher visual area V4, and parietal control area 7a during attentional task performance. Top-down 7a-to-V1 beta-band influences enhanced visually driven V1-to-V4 gamma-band influences. This enhancement was spatially specific and largest when beta-band activity preceded gamma-band activity by ∼0.1 s, suggesting a causal effect of top-down processes on bottom-up processes. We propose that this cross-frequency interaction mechanistically subserves the attentional control of stimulus selection.SIGNIFICANCE STATEMENT Contemporary research indicates that the alpha-beta frequency band underlies top-down control, whereas the gamma-band mediates bottom-up stimulus processing. This arrangement inspires an attractive hypothesis, which posits that top-down beta-band influences directly modulate bottom-up gamma band influences via cross-frequency interaction. We evaluate this hypothesis determining that beta-band top-down influences from parietal area 7a to visual area V1 are correlated with bottom-up gamma frequency influences from V1 to area V4, in a spatially specific manner, and that this correlation is maximal when top-down activity precedes bottom-up activity. These results show that for top-down processes such as spatial attention, elevated top-down beta-band influences directly enhance feedforward stimulus-induced gamma-band processing, leading to enhancement of the selected stimulus.
| S-EPMC5508256 | biostudies-other