Fragmentation Of Precursor Ions Research Articles

Analyzing DNA barcoding and identifying toxins caused by neurotoxic mushroom poisoning using liquid chromatography tandem mass spectrometry

Background: Neurotoxic mushroom poisoning often exhibits rapid symptom onset, typically attributed to compounds such as Ibotenic acid, which affect the central nervous system. This study addresses a new case of mushroom-related food poisoning in southern Thailand. Objective: The objectives are to determine the presence of ibotenic acid in cases of mushroom-related food poisoning utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to identify toxic Amanita species implicated in these cases. Materials and methods: Remnant mushroom samples obtained from three clinically reported cases were used. Nucleotide similarity was compared against the rRNA/ ITS databases using NCBI BLAST search. Phylogenetic analyses were conducted using maximum likelihood (ML) and FastTree approaches. LC-MS/MS was employed to separate of Ibotenic acid, determine its molecular weight and perform precursor ion fragmentation. Results: Analysis of the rRNA/ITS databases revealed a high nucleotide similarity between suspected mushroom samples and Amanita digitosa. Detailed phylogenetic analysis confirmed that mushroom samples from the three poisoning cases clustered with A. digitosa. LC-MS/MS analysis showed the presence of ibotenic acid, with precursor ion (m/z 159) and product ion (m/z 113.1) as the major toxic substances. Clinically, patients poisoned by ibotenic acid-containing mushrooms exhibited a short latent period with symptoms of nausea, vomiting, vertigo, delirium, confusion, and fatigue. Conclusion: The genus Amanita comprises both edible and inedible species that produce several lethal toxins. The report of ibotenic acid in A. digitosa is a novel finding, valuable for food safety monitoring and healthcare decision-maker. This is especially notable due to the accuracy and rapidity of the analytical process.

Transformer-based de novo peptide sequencing for data-independent acquisition mass spectrometry.

Tandem mass spectrometry (MS/MS) stands as the predominant high-throughput technique for comprehensively analyzing protein content within biological samples. This methodology is a cornerstone driving the advancement of proteomics. In recent years, substantial strides have been made in Data-Independent Acquisition (DIA) strategies, facilitating impartial and non-targeted fragmentation of precursor ions. The DIA-generated MS/MS spectra present a formidable obstacle due to their inherent high multiplexing nature. Each spectrum encapsulates fragmented product ions originating from multiple precursor peptides. This intricacy poses a particularly acute challenge in de novo peptide/protein sequencing, where current methods are ill-equipped to address the multiplexing conundrum. In this paper, we introduce Casanovo-DIA, a deep-learning model based on transformer architecture. It deciphers peptide sequences from DIA mass spectrometry data. Our results show significant improvements over existing STOA methods, including DeepNovo-DIA and PepNet. Casanovo-DIA enhances precision by 15.14% to 34.8%, recall by 11.62% to 31.94% at the amino acid level, and boosts precision by 59% to 81.36% at the peptide level. Integrating DIA data and our Casanovo-DIA model holds considerable promise to uncover novel peptides and more comprehensive profiling of biological samples. Casanovo-DIA is freely available under the GNU GPL license at https://github.com/Biocomputing-Research-Group/Casanovo-DIA.

Study of protonated dimers of cytosine, cytidine, and deoxycytidine using survival yield method and quantum mechanics calculations

RationaleCytosine and its conjugates are prone to form protonated, triply‐bonded dimers. Therefore, the nucleic‐acid cytosine‐rich sequence forms the four‐stranded noncanonical secondary structure known as the intercalated motif (i‐motif). This process has resulted in studies on cytosine protonated dimers. This communication focuses on the protonated dimers of cytosine and its nucleoside using the survival yield (SY) method and quantum mechanics calculations.MethodsTo obtain the precursor ion fragmentation curve, the plot of SY against Ecomδ, the product ion spectra of the protonated dimers were obtained using a Waters/Micromass Q‐TOF Premier mass spectrometer. Quantum mechanics calculations were performed using GAUSSIAN 16, and full geometry optimizations and energy calculations were performed within the density functional theory framework at B3LYP/6‐31G(d,p).ResultsThe precursor ion fragmentation curve allowed the rating of the gas‐phase stabilities of the analyzed protonated dimers. Substitution of sugar moiety at N1 cytosine atom decreased the gas‐phase stabilities of the protonated dimers. The deoxycytidine dimer was found to be more stable than the cytidine dimer and cytidine–deoxycytidine dimer. Quantum chemical calculations indicated that cytosine aminohydroxy tautomer may be involved in the formation of protonated cytosine–cytosine nucleoside dimers but not in the formation of cytosine dimers.ConclusionsThe results obtained for nucleoside dimers indicated that the SY method may reflect the i‐motif stabilities observed under physiological conditions. Therefore, the analysis of other protonated dimers of variously substituted cytosine–cytosine nucleoside using the SY method may be important to study the effect of cytosine substitution on the i‐motif stabilities. Cytosine tautomer containing C2‐OH… N(2H)‐C4 moiety may be involved in the formation of protonated cytosine–cytosine nucleoside dimers.

Structural Analysis of Monoclonal Antibodies with Top-down and Middle-down Electron Transfer Dissociation Mass Spectrometry: The First Decade

Monoclonal antibodies (mAbs) are protein biotherapeutics with a proven efficacy toward fighting life-threatening diseases. Their exceptional healing potential drives the annual increase in the number of novel mAbs and other antibody-like molecules entering clinical trials and the number of approved mAb-based drugs. Mass spectrometry (MS) offers high selectivity and specificity for the potentially unambiguous identification and comprehensive structural characterization of proteins, including at the proteoform level. It is thus not surprising that MS-based approaches are playing a central role in the biopharma laboratories, complementing and advancing traditional biotherapeutics characterization workflows. A combination of MS approaches is required to comprehensively characterize mAbs’ structures: the commonly employed bottom-up MS approaches are efficiently complemented with mass measurements at the intact and subunit (middle-up) levels, together with product ion analysis following gas-phase fragmentation of precursor ions performed at the intact (top-down) and subunit (middle-down) levels. Here we overview our group’s contribution to increasing the efficiency of these approaches and the development of the novel strategies over the past decade. Our particular focus has been on the top-down and middle-down MS methods that utilize electron transfer dissociation (ETD) for gas-phase protein ion fragmentation. Several approaches pioneered by our group, particularly an ETD-based middle-down approach, constitute a part of commercial software solutions for the mAb’s characterization workflows.

Bioanalysis of lanreotide in dog plasma using liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study of beagle dogs after single subcutaneous injection

Lanreotide is similar to a naturally occurring hormone, somatostatin; thus, it may be used to treat acromegaly or metastatic gastroenteropancreatic neuroendocrine tumours. Here, a bioanalytical method coupling ultra-performance liquid chromatography with tandem mass spectrometry to quantify lanreotide and an internal standard (IS) was developed and validated in dog plasma. The plasma samples were extracted using typical protein precipitation processes. The analyte and internal standard were separated on Phenomenex Kinetex® C18 with 0.1% formic acid and acetonitrile in the mobile phase at a flow rate of 0.4 mL/min. The fragmentation of precursor ions to product ions was optimized at m/z 548.8 → 170.0 for lanreotide [M + 2H]2+ and 472.2 → 436.2 for IS [M + H]+. The peak retention times of lanreotide and IS were 1.09 min and 1.22 min, respectively. The calibration curve samples in dog plasma ranged from 0.3 to 1000 ng/mL and showed good linearity, with a correlation coefficient of r2=0.9996. The lower limit of quantitation was 0.3 ng/mL. The intra- and inter-day precision (relative standard deviation) values for each quality control level were < 9.7 % and < 9.3 %, respectively; intra- and inter-day accuracy were < 109.3% and < 110.4%, respectively. Lanreotide in dog plasma was stable in various conditions. The maximum plasma concentration of lanreotide in male beagle dogs after subcutaneous injection of Somatuline® (lanreotide) Autogel 120 mg was 88.1 ng/mL. The half-life (T1/2) of lanreotide in beagle dogs was long, approximately 198.6 h; the area under the plasma-concentration curve from 0 to 840 h (day 35) was 6,995 ng⋅h/mL. This novel quantification method using UPLC-MS/MS was successfully applied to the pharmacokinetic analysis of lanreotide in dog plasma. The results will assist future studies of drug formulation and repurposing.

Changes in glycosyl inositol phosphoceramides during the cell cycle of the red alga Galdieria sulphuraria

Sphingolipids are significant component of plant-cell plasma membranes, as well as algal membranes, and mediate various biological processes. One of these processes is the change in lipid content during the cell cycle. This change is key to understanding cell viability and proliferation. There are relatively few papers describing highly glycosylated glycosyl inositol phosphorylceramide (GIPC) due to problems associated with the extractability of GIPCs and their analysis, especially in algae.After alkaline hydrolysis of total lipids from the red alga Galdieria sulphuraria, GIPCs were measured by high-resolution tandem mass spectrometry and fragmentation of precursor ions in an Orbitrap mass spectrometer in order to elucidate the structures of molecular species. Fragmentation experiments such as tandem mass spectrometry in the negative ion mode were performed to determine both the ceramide group and polar head structures. Measurement of mass spectra in the negative regime was possible because the phosphate group stabilizes negative molecular ions [M-H]-. Analysisof GIPCs at various stages of the cell cycle provided information on their abundance. It was found that, depending on the phases of the cell cycle, in particular during division, the uptake of all three components of GIPC, i.e., long-chain amino alcohols, fatty acids, and polar heads, changes. Structural modifications of the polar headgroup significantly increased the number of molecular species. Analysis demonstrated a convex characteristic for molecular species with only one saccharide (hexose or hexuronic acid) as the polar head. For two carbohydrates, the course of Hex-HexA was linear, while for HexA-HexA it was concave. The same was true for GIPC with three and four monosaccharides.

Proteomics with Enhanced In-Source Fragmentation/Annotation: Applying XCMS-EISA Informatics and Q-MRM High-Sensitivity Quantification.

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.

Simultaneous Determination of Total Vitamins B1, B2, B3, and B6 in Infant Formula and Related Nutritionals by Enzymatic Digestion and LC-MS/MS-A Multi-Laboratory Testing Study Final Action: AOAC Method 2015.14.

A multi-laboratory study was completed for AOAC First Action Method 2015.14. Ten laboratories from eight countries participated. Each laboratory analyzed (in duplicate) a placebo and 14 fortified nutritionals. Product matrices analyzed included milk, soy, partially hydrolyzed milk, partially hydrolyzed soy, and elemental-based infant formula powders, milk based infant formula ready-to-feed liquids, adult low-fat powders, and adult high fat and high protein ready-to-drink nutritionals. Data was then compared to standard method performance requirements (SMPR). Samples were prepared by enzymatic digestion with papain, α-amylase, and phosphatase to hydrolyze protein and complex carbohydrate and to free phosphorylated vitamin forms respectively. Stable-isotope labeled internal standards were incorporated into the sample preparation to correct for variability in both the sample preparation and instrument response. Prepared samples and working standard solutions were injected onto an ultra-high-pressure liquid chromatograph, interfaced to a triple-quadrupole mass spectrometer (MS/MS). Reverse phase gradient chromatography, using mobile phases of methanol and 20 mM ammonium formate in water on a C-18 column, were used for the analysis. The MS/MS was configured to monitor precursor-fragment ion pairs for each analyte and internal standard. Vitamins B1 (thiamine), B2 (riboflavin), B3 (niacin), and B6 (pyridoxine) were quantified by least squares regression using the response ratio of the analyte to its internal standard. Total vitamins B1, B2, B3, and B6 had average repeatability of 2.3%, 3.9%, 2.7%, and 2.2% RSD, and reproducibility of 8.2%, 6.9%, 6.7%, and 5.8% RSD, respectively. Repeatability and reproducibility SMPR were met for 53/56 and 50/56 fortified-product/analyte pairs analyzed, respectively.

Approximating Isotope Distributions of Biomolecule Fragments.

In mass spectrometry (MS)-based proteomics, protein and peptide sequences are determined by the isolation and subsequent fragmentation of precursor ions. When an isolation window captures only part of a precursor’s isotopic distribution, the isotope distributions of the fragments depend on the subset of isolated precursor isotopes. Approximation of the expected isotope distributions of these fragments prior to sequence determination enables MS2 deisotoping, monoisotopic mass calculation, charge assignment of fragment peaks, and deconvolution of chimeric spectra. However, currently such methods do not exist, and precursor isotope distributions are often used as a proxy. Here, we present methods that approximate the isotope distribution of a biomolecule’s fragment given its monoisotopic mass, the monoisotopic mass of its precursor, the set of isolated precursor isotopes, and optionally sulfur atom content. Our methods use either the Averagine model or splines, the latter of which have similar accuracy to the Averagine approach, but are 20 times faster to compute. Theoretical and approximated isotope distributions are consistent for fragments of in silico digested peptides. Furthermore, mass spectrometry experiments with the angiotensin I peptide and HeLa cell lysate demonstrate that the fragment methods match isotope peaks in MS2 spectra more accurately than the precursor Averagine approach. The algorithms for the approximation of fragment isotope distributions have been added to the OpenMS software library. By providing the means for analyzing fragment isotopic distributions, these methods will improve MS2 spectra interpretation.

Large-Scale Differentiation and Site Specific Discrimination of Hydroxyproline Isomers by Electron Transfer/Higher-Energy Collision Dissociation (EThcD) Mass Spectrometry.

3- and 4-Hydroxyprolines (HyP) are regioisomers that play different roles in various species and organs. Despite their distinct functions inside cells, they are generally considered indistinguishable using mass spectrometry due to their identical masses. Here, we demonstrate, for the first time, that characteristic w ions can be produced by electron-transfer/higher energy collision dissociation (EThcD) dual fragmentation technique to confidently discriminate 3-HyP/4-HyP isomers. An integrated and high throughput strategy was developed which combined online LC separation with EThcD for large-scale differentiation of 3-HyP/4-HyP in complex samples. An automated algorithm was developed for charge state dependent characterization of 3-HyP/4-HyP isomers. Using this combined discrimination approach, we identified 108 3-HyP sites and 530 4-HyP sites from decellularized pancreas, allowing more than 5-fold increase of both 3-HyP and 4-HyP identifications compared to previous reports. This approach outperformed ETD and HCD in the analysis of HyP-containing peptides with unique capacity to generate w ions for HyP discrimination, improved fragmentation of precursor ions, as well as unambiguous localization of modifications. A high content of 3-HyP was observed in the C-terminal (GPP)n domain of human CO1A1, which was previously only identified in vertebrate fibrillar collagens from tendon. Unexpectedly, some unusual HyP sites at Xaa position in Gly-HyP-Ala, Gly-HyP-Val, Gly-HyP-Gln, Gly-HyP-Ser, and Gly-HyP-Arg were also confirmed to be 3-hydroxylated, whose functions and enzymes are yet to be discovered. Overall, this novel discrimination strategy can be readily implemented into de novo sequencing or other proteomic search engines.

Customized Consensus Spectral Library Building for Untargeted Quantitative Metabolomics Analysis with Data Independent Acquisition Mass Spectrometry and MetaboDIA Workflow.

Data independent acquisition-mass spectrometry (DIA-MS) coupled with liquid chromatography is a promising approach for rapid, automatic sampling of MS/MS data in untargeted metabolomics. However, wide isolation windows in DIA-MS generate MS/MS spectra containing a mixed population of fragment ions together with their precursor ions. This precursor-fragment ion map in a comprehensive MS/MS spectral library is crucial for relative quantification of fragment ions uniquely representative of each precursor ion. However, existing reference libraries are not sufficient for this purpose since the fragmentation patterns of small molecules can vary in different instrument setups. Here we developed a bioinformatics workflow called MetaboDIA to build customized MS/MS spectral libraries using a user's own data dependent acquisition (DDA) data and to perform MS/MS-based quantification with DIA data, thus complementing conventional MS1-based quantification. MetaboDIA also allows users to build a spectral library directly from DIA data in studies of a large sample size. Using a marine algae data set, we show that quantification of fragment ions extracted with a customized MS/MS library can provide as reliable quantitative data as the direct quantification of precursor ions based on MS1 data. To test its applicability in complex samples, we applied MetaboDIA to a clinical serum metabolomics data set, where we built a DDA-based spectral library containing consensus spectra for 1829 compounds. We performed fragment ion quantification using DIA data using this library, yielding sensitive differential expression analysis.

Ion mobility spectrometry–mass spectrometry (IMS–MS) for on- and offline analysis of atmospheric gas and aerosol species

Abstract. Measurement techniques that provide molecular-level information are needed to elucidate the multiphase processes that produce secondary organic aerosol (SOA) species in the atmosphere. Here we demonstrate the application of ion mobility spectrometry-mass spectrometry (IMS–MS) to the simultaneous characterization of the elemental composition and molecular structures of organic species in the gas and particulate phases. Molecular ions of gas-phase organic species are measured online with IMS–MS after ionization with a custom-built nitrate chemical ionization (CI) source. This CI–IMS–MS technique is used to obtain time-resolved measurements (5 min) of highly oxidized organic molecules during the 2013 Southern Oxidant and Aerosol Study (SOAS) ambient field campaign in the forested SE US. The ambient IMS–MS signals are consistent with laboratory IMS–MS spectra obtained from single-component carboxylic acids and multicomponent mixtures of isoprene and monoterpene oxidation products. Mass-mobility correlations in the 2-D IMS–MS space provide a means of identifying ions with similar molecular structures within complex mass spectra and are used to separate and identify monoterpene oxidation products in the ambient data that are produced from different chemical pathways. Water-soluble organic carbon (WSOC) constituents of fine aerosol particles that are not resolvable with standard analytical separation methods, such as liquid chromatography (LC), are shown to be separable with IMS–MS coupled to an electrospray ionization (ESI) source. The capability to use ion mobility to differentiate between isomers is demonstrated for organosulfates derived from the reactive uptake of isomers of isoprene epoxydiols (IEPOX) onto wet acidic sulfate aerosol. Controlled fragmentation of precursor ions by collisionally induced dissociation (CID) in the transfer region between the IMS and the MS is used to validate MS peak assignments, elucidate structures of oligomers, and confirm the presence of the organosulfate functional group.

Lipid Profiling of In Vitro Cell Models of Adipogenic Differentiation: Relationships With Mouse Adipose Tissues.

Our objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo. A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MS(ALL) . Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3-L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose-derived BAT-C1 cells were also characterized. Over 3000 unique lipid species were quantified. Principal component analysis showed that perirenal versus inguinal white adipose tissues varied in lipid composition of triacyl- and diacylglycerols, sphingomyelins, glycerophospholipids and, notably, cardiolipin CL 72:3. In contrast, hexosylceramides and sphingomyelins distinguished brown from white adipose. Adipocyte differentiation models showed broad differences in lipid composition among themselves, upon adipogenic differentiation, and with adipose tissues. Palmitoyl triacylglycerides predominate in 3T3-L1 differentiation models, whereas cardiolipin CL 72:1 and SM 45:4 were abundant in brown adipose-derived cell differentiation models, respectively. MS/MS(ALL) data suggest new lipid biomarkers for tissue-specific lipid contributions to adipogenesis, thus providing a foundation for using in vitro models of adipogenesis to reflect potential changes in adipose tissues in vivo. J. Cell. Biochem. 117: 2182-2193, 2016. © 2016 Wiley Periodicals, Inc.

Molecular formula analysis of fragment ions by isotope-selective collision-induced dissociation tandem mass spectrometry of pharmacologically active compounds.

The purpose of this work is to explore the mass fragment characterization of commonly used drugs through a novel approach, which involves isotope-selective tandem mass spectrometry (MS/MS). Collision-induced dissociation (CID) was performed with a low-resolution linear ion trap mass spectrometer in positive electrospray ionization. Three pharmacologically active ingredients, i.e. omeprazole, meloxicam and brinzolamide, selected as model compounds in their own formulation, were investigated as a sodiated adduct [C17 H19 N3 O3 S + Na](+) (omeprazole) and as protonated adducts, [C14 H13 N3 O4 S2 + H](+) and [C12 H21 N3 O5 S3 + H](+) , meloxicam and brinzolamide, respectively. Selecting a narrow window of ±0.5 m/z units, precursor ion fragmentation by CID-MS/MS of isotopologues A + 0, A + 1 and A + 2 was found very useful to confirm the chemical formula of product ions, thus aiding the establishment of characteristic fragmentation pathways of all three examined compounds. The correctness of putative molecular formula of product ions was easily demonstrated by exploiting the isotope peak abundance ratios (i.e. IF+0 /IF+1 and IF+0 /IF+2 ) as simple constraints in low-resolution MS instrumentations.

Pharmacokinetic determination of ephedrine in Herba Ephedrae and Wu Tou Tang decoctions in rats using ultra performance liquid chromatography tandem mass spectrometry

A rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and validated for the determination and quantification of ephedrine in rat plasma samples. An Acquity UPLC BEH C18 column (1.7 μm, 2.1 mm × 50 mm) was used for chromatographic separation. Electrospray ionization in the positive mode was used, and the precursor-fragment ion pairs of m/z 166/148 and m/z 289/97 were adopted to characterize ephedrine and testosterone (internal standard), respectively. The method was validated using 10, 100 and 500 ng/mL of ephedrine. It demonstrated adequate levels of precision and accuracy, matrix effect, extraction recovery and stability. Linearity over the concentration range of 0.5–2000 ng/mL was acceptable with a correlation coefficient (r2) better than 0.990.To determine the pharmacokinetic behaviour of this sympathomimetic compound in the Sprague-Dawley rats, ephedrine hydrochloride, Herba Ephedrae single-herb and Wu Tou Tang decoctions were administered orally, and ephedrine hydrochloride was also administered by intravenous injection, and blood samples were collected over 24 h. Ephedrine was measured in plasma and pharmacokinetic parameters were determined by using the standard non-compartmental method and calculated by using Practical Pharmacokinetic Program-Version 87/97.The AUC(0–t) and Tmax values were significantly different (p < 0.05). Ephedrine AUC(0–t) values were significantly lower following the Wu Tou Tang decoction compared to the other oral treatments, suggesting that some components in the decoction may reduce the bioavailability of ephedrine

Shotgun Lipidomics by Sequential Precursor Ion Fragmentation on a Hybrid Quadrupole Time-of-Flight Mass Spectrometer.

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.

Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis

Most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify the peptides generated by the proteolysis of a biological sample. However, with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify large fractions of proteomes across multiple samples. Here we present a new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest. It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using a data-independent acquisition method. For this study, the data were acquired on a fast, high resolution quadrupole-quadrupole time-of-flight (TOF) instrument by repeatedly cycling through 32 consecutive 25-Da precursor isolation windows (swaths). This SWATH MS acquisition setup generates, in a single sample injection, time-resolved fragment ion spectra for all the analytes detectable within the 400-1200 m/z precursor range and the user-defined retention time window. We show that suitable combinations of fragment ions extracted from these data sets are sufficiently specific to confidently identify query peptides over a dynamic range of 4 orders of magnitude, even if the precursors of the queried peptides are not detectable in the survey scans. We also show that queried peptides are quantified with a consistency and accuracy comparable with that of selected reaction monitoring, the gold standard proteomic quantification method. Moreover, targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re-mining of the once-and-forever acquired data sets. This combination of unbiased, broad range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic methods and should be equally applicable to the comprehensive analysis of other classes of analytes, beyond proteomics.

ITRAQ Labeling is Superior to mTRAQ for Quantitative Global Proteomics and Phosphoproteomics

Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Migration of sulfate groups between hydroxyl groups was identified after collision-induced dissociation (CID) of sulfated oligosaccharides in an ion trap mass spectrometer in negative ion mode. Analysis of various sulfated oligosaccharides showed that this was a common phenomenon and was particularly prominent in sulfated oligosaccharides also containing sialic acid. It was also shown that the level of migration was increased when the sulfate was positioned on the flexible areas of the oligosaccharides not involved in the pyranose ring, such as the extra-cyclic C-6 carbon of hexoses or N-acetylhexosamines, or on reduced oligosaccharide. This suggested that migration is dependent on the spatial availability of the sulfate in the ion trap during collision. It is proposed that the migration is initiated when the negatively charged -SO3 (-) residue attached to the oligosaccharide precursor becomes protonated by a CID-induced proton transfer. This is supported by the CID fragmentation of precursor ions depleted of acidic protons such as doubly charged [M - 2H](2-) ions or the sodiated [M + Na - 2H](-) ions of oligosaccharides containing one sulfate and one sialic acid in the same molecule. Compared to the CID fragmentation of their monocharged [M - H](-) ions, no migration was observed in CID of proton depleted precursors. Alternative fragmentation parameters to suppress migration of sulfated oligosaccharides also showed that it was not present when sulfated oligosaccharides were fragmented by HCD (High-Energy C-trap Dissociation) in an Orbitrap mass spectrometer.

An experimental approach to enhance precursor ion fragmentation for metabolite identification studies: application of dual collision cells in an orbital trap

Recent advancements in mass spectrometry including data-dependent scanning and high-resolution mass spectrometry have aided metabolite profiling for non-radiolabeled xenobiotics. However, narrowing down a site of metabolism is often limited by the quality of the collision-induced dissociation (CID)-based precursor ion fragmentation. An alternative dissociation technique, higher energy collisional dissociation (HCD), enriches compound fragmentation and yields 'triple-quadrupole-like fragmentation'. Applying HCD along with CID and data-dependent scanning could enhance structural elucidation for small molecules. Liquid chromatography/multi-stage mass spectrometry (LC/MS(n) ) experiments with CID and HCD fragmentation were carried out for commercially available compounds on a hybrid linear ion trap orbital trap mass spectrometer equipped with accurate mass measurement capability. The developed method included stepped normalized collision energy (SNCE) parameters to enhance MS fragmentation without tuning for individual compounds. All the evaluated compounds demonstrated improved fragmentation under HCD as compared with CID. The results suggest that an LC/MS(n) method that incorporated both SNCE HCD- and CID-enabled precursor ion fragmentation afforded comprehensive structural information for the compounds under investigation. A dual collision cell approach was remarkably better than one with only CID MS(n) in an orbital trap. It is evident that such an acquisition method can augment the identification of unknown metabolites in drug discovery by improving fragmentation efficiency of both the parent compound and its putative metabolite(s).