trap-Fourier Transform Ion Cyclotron Resonance Research Articles

Cryo kinetics of N2 adsorption onto bimetallic rhodium-iron clusters in isolation.

We report the N2 cryo adsorption kinetics of selected gas phase mixed rhodium-iron clusters [RhiFej]+ in the range of i = 3-8 and j = 3-8 in 26K He buffer gas by the use of a cryo tandem RF-hexapole trap-Fourier transform ion cyclotron resonance mass spectrometer. From kinetic data and fits, we extract relative rate constants for each N2 adsorption step and possible desorption steps. We find significant trends in adsorption behavior, which reveal adsorption limits, intermittent adsorption limits, and equilibrium reactions. For those steps, which are in equilibrium, we determine the Gibbs free energies. We conclude on likely ligand shell reorganization and some weakly bound N2 ligands for clusters where multiple N2 adsorbates are in equilibrium. The relative rate constants are transferred to absolute rate constants, which are slightly smaller than the collision rate constants calculated by the average dipole orientation (Langevin) theory. The calculated sticking probabilities increase, in general, with the size of the clusters and decrease with the level of N2 adsorption, in particular, when reaching an adsorption/desorption equilibrium. We receive further evidence on cluster size dependent properties, such as cluster geometries and metal atom distributions within the clusters through the accompanying spectroscopic and computational study on the equiatomic i = j clusters [Klein et al., J. Chem. Phys. 156, 014302 (2022)].

Characterization of liquid–liquid extraction fractions from lignocellulosic biomass by high performance liquid chromatography hyphenated to tandem high-resolution mass spectrometry

The conversion of lignocellulosic biomass is a major challenge in the field of renewable energies and bio-based chemicals. The diversity of biomasses and processes leads to complex products having a wide range of polarities and molecular weights. Nowadays, the molecular description of these oxygenated matrices is still largely incomplete and new analytical strategies are required to have a better understanding of biomass products properties. The present study proposes a reliable protocol based on successive liquid–liquid extractions prior to high performance liquid chromatography hyphenated to high-resolution tandem mass spectrometry (HPLC/MSn) using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ/FT-ICR). The protocol allowed to fractionate an industrial sample coming from the sulfuric acid-based pretreatment of a wheat straw into four key chemical families: carbohydrates, organic acids, phenols and neutral compounds. Each fraction was separately analyzed, which limited matrix effects during mass spectrometry ionization step. Electrospray and atmospheric pressure chemical ionization sources were used in both positive and negative modes in order to ionize and detect a maximum of compounds. Thanks to HPLC/MSn, structures of heavy lignin-carbohydrate complexes (LCC) were elucidated (up to 600 g/mol) as well as carbohydrate oligomers having acid functionalities. Mono, di, tri and tetra-aromatic compounds coming from lignin were also detected. The results reported in this paper demonstrate the complexity of pretreated biomass samples and propose an analytical approach from sample simplification to data treatment in order to describe the biomass composition.

Quantitative proteomic analysis of mitochondria from human ovarian cancer cells and their paclitaxel-resistant sublines.

Paclitaxel resistance is a major obstacle for the treatment of ovarian cancer. The chemoresistance mechanisms are partly related to the mitochondria. Identification of the relevant proteins in mitochondria will help in clarifying the possible mechanisms and in selecting effective chemotherapy for patients with paclitaxel resistance. In the present study, mitochondria from two paclitaxel-sensitive human ovarian cancer cell lines (SKOV3 and A2780) and their corresponding resistant cell lines (SKOV3-TR and A2780-TR) were isolated. Guanidine-modified acetyl-stable isotope labeling and liquid chromatography-hybrid linear ion trap Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) were performed to find the expressed differential proteins. Comparative proteomic analysis revealed eight differentially expressed proteins in the ovarian cancer cells and their paclitaxel-resistant sublines. Among them, mimitin and 14-3-3 ζ/δ were selected for further research. The effects of mimitin and 14-3-3 ζ/δ were explored using specific siRNA interference in ovarian cancer cell lines and immunohistochemistry in human tissue specimens. The downregulation of mimitin and 14-3-3 ζ/δ using specific siRNA in paclitaxel-resistant ovarian cancer cells led to an increase in the resistance index to paclitaxel. Multivariate analyses demonstrated that lower expression levels of the mimitin and 14-3-3 ζ/δ proteins were positively associated with shorter progression-free survival (PFS) and overall survival (OS) in patients with primary ovarian cancer (mimitin: PFS: P = 0.041, OS: P = 0.003; 14-3-3 ζ/δ: PFS: P = 0.031, OS: P = 0.011). Mimitin and 14-3-3 protein ζ/δ are potential markers of paclitaxel resistance and prognostic factors in ovarian cancer.

SILAC‐based quantitative proteomic analysis of secretome between activated and reverted hepatic stellate cells

Activated hepatic stellate cell (HSC) is the main myofibroblast cell in the liver fibrosis (LF). An important characteristic of the recovery of LF is not only the apoptosis of activated HSCs but also reversal of myofibroblast-like phenotype to a quiescent-like phenotype. Understanding the changes of secreted proteins in the reversion of activated HSCs may provide the broader view of cellular regulatory networks and discover candidate markers or targets for therapeutic strategies of LF. In this study, stable isotope labeling with amino acids (SILAC) combined with linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT MS) was performed on in vitro activated HSCs and reverted HSCs to obtain a proteomic view of secretory proteins. In total, 330 proteins showed significant differences in reverted HSCs. Among these, 109 upregulated proteins were mainly involved in amino acid metabolism pathway and glucose metabolism pathway using GeneGO/MetaCore software, while 221 downregulated proteins are closely associated with HSCs activation, such as cytoskeleton remodeling, chemokines, and cell adhesion. Additionally, a set of novel proteins associated with HSCs activation and reversion were validated by Western blotting in the cell secretion and in the sera of LF, including vitronectin, laminin beta 1, and ubiquitin conjugation factor E4B. Our study provided the valuable insight into the mechanisms in the reversion of activated HSCs and identified some potential biomarkers of LF in clinical studies. All MS data have been deposited in the ProteomeXchange with identifier PXD000773 (http://proteomecentral.proteomexchange.org/dataset/PXD000773).

Performance evaluation of a dual linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer for proteomics research

A novel dual cell linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) and its performance characteristics are reported. A linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer has been modified to incorporate a LTQ-Velos mass spectrometer. This modified instrument features efficient ion accumulation and fast MS/MS acquisition capabilities of dual cell linear RF ion trap instruments coupled to the high mass accuracy, resolution, and dynamic range of a FT-ICR for improved proteomic coverage. The ion accumulation efficiency is demonstrated to be an order of magnitude greater than that observed with LTQ-FT Ultra instrumentation. The proteome coverage with yeast was shown to increase over the previous instrument generation by 50% (100% increase on the peptide level). In addition, many lower abundance level yeast proteins were only detected with this modified instrument. This novel configuration also enables beam type CID fragmentation using a dual cell RF ion trap mass spectrometer. This technique involves accelerating ions between traps while applying an elevated DC offset to one of the traps to accelerate ions and induce fragmentation. This instrument design may serve as a useful option for labs currently considering purchasing new instrumentation or upgrading existing instruments. A novel hybrid mass spectrometer that allows increased MS/MS acquisition rates with high mass measurement accuracy and new ion fragmentation methods greatly improves the number of proteins, posttranslational modifications and protein-protein interactions that can be identified from cells.

Ambient ion/molecule reactions in low‐temperature plasmas (LTP): reactive LTP mass spectrometry

Ion/molecule reactions are commonly used to characterize structures due to their high specificity. Herein, we present ambient ion/molecule reactions performed in the course of low-temperature plasma (LTP) ionization of condensed-phase analytes in order to increase the specificity of LTP-based ambient analysis. The ion population of the cold plasma is modified by addition of a reagent to the plasma before it is directed at a surface bearing the analyte. Desorbed ions were analyzed using linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). Acylium ions generated from tetramethylurea react with 1,3-dioxane analyte to afford distinctive Eberlin product ions. Reactions of alkylamines, such as n-hexylamine and n-octylamine, with benzaldehyde produce the corresponding imines. Reaction of ruthenocene with trifluoroacetic anhydride forms the unusual trifluoroacetate ruthenocene. A LTP source can be used to generate reagent ions that can undergo ion/molecule reactions in the ambient environment with an analyte at condensed phase on a surface. The experiment is a 'reactive' version of the standard low-temperature plasma (LTP) ambient ionization experiment. This approach provides additional information by combining ion/molecule chemistry with conventional MS and MS/MS data to characterize particular analytes.

Characterization of mercury-containing protein in human plasma

Characterization of mercury binding protein in the human body is very important for understanding the metabolism and the mechanism of toxication of ingested mercuric compounds. In this study, mercury-containing protein in human plasma was separated by on-line heart-cutting two-dimensional high performance liquid chromatography (2D-HPLC). This 2D separation system used size exclusion liquid chromatography (SEC) followed by weak anion exchange liquid chromatography (WAX) and the two LC parts were coupled by a six-port valve equipped with a storage loop and controled by the computer. The WAX effluent was determined by both UV detection and inductively coupled plasma-mass spectrometry (ICP-MS) to locate the mercury-containing protein. A unique mercury-containing protein fraction was obtained by 2D-HPLC separation and subsequently identified by HPLC coupled with linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry (HPLC-LTQ-FT). The database search confirmed that the mercury-containing protein in the human plasma is human serum albumin (HSA). The stoichiometry and thermodyamics interaction of inorganic mercury (Hg(2+)) with HSA was studied by isothermal titration calorimetry (ITC) and two binding types were observed. Mercury-containing protein in human plasma was separated and identified in the present study and it is important for understanding the metabolism of mercury in the human body.

Changes in physiology and protein abundance in salt-stressed wheat chloroplasts

Leaves are the final site of salinity perception through the roots. To better understand how wheat chloroplasts proteins respond to salt stress, the study aimed to the physiochemical and comparative proteomics analysis. Seedlings (12-days-old) were exposed to 150 mM NaCl for 1, 2, or 3 days. Na(+) ions were rapid and excessively increase in roots, stems and leaves. Photosynthesis and transpiration rate, stomatal conductance, and relative water content decreased whereas the level of proline increased. Statistically significant positive correlations were found among the content of hydrogen peroxide, activity of catalase, and superoxide dismutase under salt stress in wheat. Protein abundance within the chloroplasts was examined by two-dimensional electrophoresis. More than 100 protein spots were reproducibly detected on each gel, 21 protein spots were differentially expressed during salt treatment. Using linear quadruple trap-Fourier transform ion cyclotron resonance (LTQ-FTICR) hybrid mass spectrometry, 65 unique proteins assigned in the differentially abundant spots. Most proteins were up-regulated at 2 and 3 days after being down-regulated at 1 day. Others showed only slight responses after 3 days of treatment, including Rubisco, glutamate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase, photosystem I, and pyridoxal biosynthesis protein PDX1.2 and PDX1.3. The ATP synthase (α, β, and γ) and V-type proton ATPase subunits were down-regulated resulting showed negative impact by Na(+) on the photosynthetic machinery. This ephemeral increase and subsequent decrease in protein contents may demonstrate a counterbalancing influence of identified proteins. Several proteins such as cytochrome b6-f (Cyt b6-f), germin-like-protein, the γ-subunit of ATP synthase, glutamine synthetase, fructose-bisphosphate aldolase, S-adenosylmethionine synthase, carbonic anhydrase were gradually up-regulated during the period of treatment, which can be identified as marker proteins.

The impact of novel compound 2'-hydroxyflavanone on proteomic networks of VHL-mutant renal cell carcinoma.

e13557 Background: Von Hippel-Lindau (VHL) tumor suppressor is a major genetic factor that determines hypoxic acclimatization and response to therapy in renal cell carcinoma (RCC). Our initial studies revealed that 2'-Hydroxyflavanone (2HF) effectively inhibits the survival of VHL-mutant RCC. Hence, we performed proteomic analyses to study the signaling networks of clinical significance that are regulated by 2HF in VHL-mutant RCC. Methods: The 786-O cells, an established cell line from a male Caucasian patient, were provided by William Kaelin Jr., MD, Harvard Medical School, Boston, MA. The cells were cultured either in the presence or absence of 50 μM 2HF for 48 h in RPMI medium at 37°C in 5% CO2 and lysed in a buffer containing 6M urea. The lysate (200 µg of protein) was reduced, alkylated and digested with trypsin. Tryptic digests were desalted using a C-18 cartridge (Waters, USA) and subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses on linear ion trap–Fourier transform ion cyclotron resonance tandem mass spectrometer (LTQ-FT, Thermo) coupled to an Eksigent nano-LC system. MS/MS spectra were searched against human protein sequence database using Mascot (Matrix Science), and protein identifications were validated by Scaffold (version 3.0, Proteome Software). Quantifications were performed by extracted peptide intensities (Progenesis LC-MS, Nonlinear Dynamics). Results: 2HF treatment lead to up-regulation of 21 proteins including protein disulfide-isomerase A4 and macrophage migration inhibition factor. 2HF treatment lead to down-regulation of 23 proteins including vimentin, HSP90AB1, 14-3-3 gamma and glutaminase. The network analyses revealed that the identified proteins form critical nodes of regulation of vital tumor proteins like MAP3K14, ERK1/2, Myc and P53. Conclusions: Proteomic analyses of 2HF treated VHL-mutant RCC revealed a striking regulation of proteins of importance in RCC survival, invasion and metastases. The critical nodes of regulation as identified by the network analyses could facilitate further studies on the development of network targeting therapies using 2HF and other anti-cancer compounds for the effective management of VHL-mutant RCC.

Comparative Lipidomic Profiling of S. cerevisiae and Four Other Hemiascomycetous Yeasts.

Glycerophospholipids (GP) are the building blocks of cellular membranes and play essential roles in cell compartmentation, membrane fluidity or apoptosis. In addition, GPs are sources for multifunctional second messengers. Whereas the genome and proteome of the most intensively studied eukaryotic model organism, the baker’s yeast (Saccharomyces cerevisiae), are well characterized, the analysis of its lipid composition is still at the beginning. Moreover, different yeast species can be distinguished on the DNA, RNA and protein level, but it is currently unknown if they can also be differentiated by determination of their GP pattern. Therefore, the GP compositions of five different yeast strains, grown under identical environmental conditions, were elucidated using high performance liquid chromatography coupled to negative electrospray ionization-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry in single and multistage mode. Using this approach, relative quantification of more than 100 molecular species belonging to nine GP classes was achieved. The comparative lipidomic profiling of Saccharomyces cerevisiae, Saccharomyces bayanus, Kluyveromyces thermotolerans, Pichia angusta, and Yarrowia lipolytica revealed characteristic GP profiles for each strain. However, genetically related yeast strains show similarities in their GP compositions, e.g., Saccharomyces cerevisiae and Saccharomyces bayanus.

Proteomic analysis of drug metabolizing networks in renal cell carcinomas with differential drug sensitivity and VHL expression.

e13137 Background: Kidney tumors with mutated VHL constitute 75% of renal cancers. The VHL activity dependent therapeutic outcome is the current focus of translational research in renal oncology. We observed that CAKI-2 kidney cancer cells with wt VHL are more sensitive to sorafenib than 786-O cells with mt VHL, which are more sensitive to temsirolimus (Singhal SS et al 2009). Hence, we performed proteomic analysis to investigate the molecular basis of differential drug sensitivity. Methods: The cell cultures were performed in RPMI medium at 37°C in 5% CO2. The cells were lysed in buffer containing 20mM Tris, 50mM Nacl, 6M urea, 10mM NaPP, 1mM NaF, and 1mM Na3VO4. The lysate (200μg of protein) was subjected to reduction and alkylation of cysteins using 2.5mM DTT and 7mM idoacetamide followed by trypsin digestion and solid phase extraction using a C-18 cartridge (Waters, USA). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed on a linear ion trap–Fourier transform ion cyclotron resonance tandem mass spectrometer (LTQ-FT, Thermo) coupled to an Eksigent nano-LC system. MS/MS spectra were searched against a human protein database by the Mascot software (Matrix Science) and Label-free quantification was done by using spectral counts from the Scaffold program (Proteome Software) with previously validated method (Prokai et al 2009). Results: We detected significant differences in the levels of phase I and II drug metabolizing enzymes in two cell types (Table). Carbonyl reduction catalyzed by AKR1C1 and UGDH involved glucorunidation are vital both in the activation and inactivation of drugs. NQO1 mediates the anticancer effects of beta lapachone (Bey et al. 2006). Conclusions: Our proteomic identification of vital differences in the principal enzymes of drug metabolizing networks identify key molecular determinants of drug sensitivity, which may be further investigated and developed as drug sensitivity biomarkers for clinical use. Proteins identified by LC-MS/MS Fold increase in spectra in CAKI-2 cells comparedto 786-O cells Aldo-keto reductase (family 1 member C1, AKRC1) 6.1 NAD(P)H quinone dehydrogenase 1 (NQO1) 2.8 UDP-glucose 6- dehydrogenase (UGHD) 5.2 No significant financial relationships to disclose.

Probing the Phosphoproteome of HeLa Cells Using Nanocast Metal Oxide Microspheres for Phosphopeptide Enrichment

Metal oxide affinity chromatography (MOAC) has become a prominent method to enrich phosphopeptides prior to their analysis by liquid chromatography-mass spectrometry. To overcome limitations in material design, we have previously reported the use of nanocasting as a means to generate metal oxide spheres with tailored properties. Here, we report on the application of two oxides, tin dioxide (stannia) and titanium dioxide (titania), for the analysis of the HeLa phosphoproteome. In combination with nanoflow LC-MS/MS analysis on a linear ion trap-Fourier transform ion cyclotron resonance instrument, we identified 619 phosphopeptides using the new stannia material, and 896 phosphopeptides using titania prepared in house. We also compared the newly developed materials to commercial titania material using an established enrichment protocol. Both titania materials yielded a comparable total number of phosphopeptides, but the overlap of the two data sets was less than one-third. Although fewer peptides were identified using stannia, the complementarity of SnO(2)-based MOAC could be shown as more than 140 phosphopeptides were exclusively identified by this material.

A Tandem Mass Spectrometry Acquisition Strategy Based on Exclusion of Precursor Ions

Abstract Because of the complexity of proteome samples, comprehensive analysis to characterize all proteins is still not possible with present methodologies. It has been shown that replicate runs could increase the number of identified proteins. However, the redundancy of protein identifications was high. High-abundance peptides tended to be repeatedly analyzed in different runs. To reduce the redundancy and improve the efficiency of identification, the tandem mass spectrometry (MS/MS) acquisition method of linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry was studied, and an acquisition strategy based on exclusion of precursor ions was developed. It proved that the strategy could extremely reduce the redundancy of MS/MS acquisition and improve the efficiency of protein identifications.

Glycerophospholipid profiling by high‐performance liquid chromatography/mass spectrometry using exact mass measurements and multi‐stage mass spectrometric fragmentation experiments in parallel

A profiling method for glycerophospholipids (GPs) in biological samples was developed using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry (LIT-FTICRMS) with electrospray ionization (ESI) in the negative ionization mode. The method allowed qualitative (identification and structure elucidation) and relative quantitative determination of various classes of GPs including phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, phosphatidic acids, phosphatidylglycerols, and cardiolipins in a single experiment. Chromatographic separation was optimized by the examination of different buffer systems and special emphasis was paid on the detection by ESI-MS. The hybrid LIT-FTICRMS system was operated in the data-dependent mode, switching automatically between FTICRMS survey scans and LIT-MS/MS experiments. Thereby, exact masses for elemental composition determination and fragmentation data for identification and assignment of fatty acid residues are provided at the same time. The low absolute instrumental limits of detection (0.05 pmol for phosphatidylglycerol to 1 pmol for phosphatidic acid) complemented by a linear dynamic range of 1.5 to 2.5 orders of magnitude facilitated the relative quantification of GP species in a lipid extract from Saccharomyces cerevisiae. The developed method is a valuable tool for in-depth GP profiling of biological systems.

Targets for covalent protein modification by 4‐hydroxynonenal/4‐hydroxyhexenal‐mediated carbonyl stress in the mitochondria

Protein carbonylation is associated with various neurodegenerative diseases. Characterization of selectivity of 4‐hydroxy‐2‐nonenal (HNE) and 4‐hydroxy‐2‐hexenal (HHE) modifications, major components of carbonyl stress, helps to understand its impact on protein function and/or activity, as well as its effects on downstream targets or other interacting proteins. Rat brain mitochondrial proteins modified by reactive carbonyl species representing end‐products of lipid peroxidation such as HNE and HHE were investigated using high performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS). Shotgun‐based approach involving conventional data‐dependent acquisition on a hybrid linear ion trap‐Fourier transform ion cyclotron resonance mass spectrometer was employed after prior enrichment of HNE‐/HHE‐modified peptides by solid‐phase hydrazide strategy. Selective enrichment of substoichiometric levels of modified peptides from complex mixtures followed by LC‐MS/MS identified several peptides that were modified with HNE or HHE with amino acid resolution. Additionally, bioinformatics (Ingenuity Pathway Analysis) was used to identify biological pathways, networks, and functions significantly altered by such modifications. This research was supported by the grant AG025384 from the National Institutes of Health.

On the inter‐instrument and inter‐laboratory transferability of a tandem mass spectral reference library: 1. Results of an Austrian multicenter study

The inter-instrument and inter-laboratory transferability of a tandem mass spectral reference library originally built on a quadrupole-quadrupole-time-of-flight instrument was examined. The library consisted of 3759 MS/MS spectra collected from 402 reference compounds applying several different collision-energy values for fragmentation. In the course of the multicenter study, 22 test compounds were sent to three different laboratories, where 418 tandem mass spectra were acquired using four different instruments from two manufacturers. The study covered the following types of tandem mass spectrometers: quadrupole-quadrupole-time-of-flight, quadrupole-quadrupole-linear ion trap, quadrupole-quadrupole-quadrupole, and linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. In each participating laboratory, optimized instrumental parameters were gathered solely from routinely applied workflows. No standardization procedure was applied to increase the inter-instrument comparability of MS/MS spectra. The acquired tandem mass spectra were matched against the established reference library using a sophisticated matching algorithm, which is presented in detail in a companion paper. Correct answers, meaning that the correct compound was retrieved as top hit, were obtained in 98.1% of cases. For the remaining 1.9% of spectra, the correct compound was matched at second rank. The observed high percentage of correct assignments clearly suggests that the developed mass spectral library search approach is to a large extent platform independent.

On the inter‐instrument and the inter‐laboratory transferability of a tandem mass spectral reference library: 2. Optimization and characterization of the search algorithm

A sophisticated matching algorithm developed for highly efficient identity search within tandem mass spectral libraries is presented. For the optimization of the search procedure a collection of 410 tandem mass spectra corresponding to 22 compounds was used. The spectra were acquired in three different laboratories on four different instruments. The following types of tandem mass spectrometric instruments were used: quadrupole-quadrupole-time-of-flight (QqTOF), quadrupole-quadrupole-linear ion trap (QqLIT), quadrupole-quadrupole-quadrupole (QqQ), and linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LIT-FTICR). The obtained spectra were matched to an established MS/MS-spectral library that contained 3759 MS/MS-spectra corresponding to 402 different reference compounds. All 22 test compounds were part of the library. A dynamic intensity cut-off, the search for neutral losses, and optimization of the formula used to calculate the match probability were shown to significantly enhance the performance of the presented library search approach. With the aid of these features the average number of correct assignments was increased to 98%. For statistical evaluation of the match reliability the set of fragment ion spectra was extended with 300 spectra corresponding to 100 compounds not included in the reference library. Performance was checked with the aid of receiver operating characteristic (ROC) curves. Using the magnitude of the match probability as well as the precursor ion mass as benchmarks to rate the obtained top hit, overall correct classification of a compound being included or not included in the mass spectrometric library, was obtained in more than 95% of cases clearly indicating a high predictive accuracy of the established matching procedure.

Microdose Clinical Trial: Quantitative Determination of Nicardipine and Prediction of Metabolites in Human Plasma

A sample treatment procedure and high-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantitative determination of nicardipine in human plasma were developed for a microdose clinical trial with nicardipine, a non-radioisotope labeled drug. The calibration curve was linear in the range of 1-500 pg/mL using 1 mL of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 0.2-100 ng/mL using 20 microL of plasma, was also conducted. Each method was successfully applied to making determinations in plasma using LC/MS/MS after administration of a microdose (100 microg) and clinical dose (20 mg) to each of six healthy volunteers. We tested new approaches in the search for metabolites in plasma after microdosing. In vitro metabolites of nicardipine were characterized using linear ion trap-fourier transform ion cyclotron resonance mass spectrometry (LIT-FTICRMS) and the nine metabolites predicted to be in plasma were analyzed using LC/MS/MS. There is a strong possibility that analysis of metabolites by LC/MS/MS may advance to utilization in microdose clinical trials with non-radioisotope labeled drugs.

Characterization of 4-Hydroxy-2-nonenal-Modified Peptides by Liquid Chromatography−Tandem Mass Spectrometry Using Data-Dependent Acquisition: Neutral Loss-Driven MS3 versus Neutral Loss-Driven Electron Capture Dissociation

Reactive oxygen species generated during oxidative stress can lead to unfavorable cellular consequences, predominantly due to formation of 4-hydroxy-2-nonenal (HNE) during lipid peroxidation. Data-dependent and neutral loss (NL)-driven MS(3) acquisition have been reported for the identification of HNE adducts by mass spectrometry-based proteomics. However, the limitation associated with this method is the ambiguity in correct assignment of the HNE modification site when more than one candidate site is present as MS(3) is triggered on the neutral loss ion. We introduce NL-triggered electron capture dissociation tandem mass spectrometry (NL-ECD-MS/MS) for the characterization of HNE-modification sites in peptides. With this method performed using a hybrid linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, ECD in the FTICR unit of the instrument is initiated on precursor ions of peptides showing the neutral loss of 156 Da corresponding to an HNE molecule in the prescan acquired via collision-induced dissociation tandem mass spectrometry in the linear ion trap. In addition to manifold advantages associated with the ECD method of backbone fragmentation, including extensive sequence fragments, ECD tends to retain the HNE group during MS/MS of the precursor ion, facilitating the correct localization of the modification site. The results also suggest that predisposition of a peptide molecular ion to lose HNE during collision-induced dissociation-based fragmentation is independent of its charge state (2+ or 3+). In addition, we have demonstrated that coupling of solid-phase enrichment of HNE-modified peptides facilitates the detection of this posttranslational modification by NL-driven strategies for low-abundance proteins that are susceptible to substoichiometric carbonylation during oxidative stress.

The Impact of Peptide Abundance and Dynamic Range on Stable-Isotope-Based Quantitative Proteomic Analyses

Recently, mass spectrometry has been employed in many studies to provide unbiased, reproducible, and quantitative protein abundance information on a proteome-wide scale. However, how instruments' limited dynamic ranges impact the accuracy of such measurements has remained largely unexplored, especially in the context of complex mixtures. Here, we examined the distribution of peptide signal versus background noise (S/N) and its correlation with quantitative accuracy. With the use of metabolically labeled Jurkat cell lysate, over half of all confidently identified peptides had S/N ratios less than 10 when examined using both hybrid linear ion trap-Fourier transform ion cyclotron resonance and Orbitrap mass spectrometers. Quantification accuracy was also highly correlated with S/N. We developed a mass precision algorithm that significantly reduced measurement variance at low S/N beyond the use of highly accurate mass information alone and expanded it into a new software suite, Vista. We also evaluated the interplay between mass measurement accuracy and S/N; finding a balance between both parameters produced the greatest identification and quantification rates. Finally, we demonstrate that S/N can be a useful surrogate for relative abundance ratios when only a single species is detected.