High-resolution Mass Analysis Research Articles

229 RAPID, UNTARGETED LIPID DETERMINATION IN INDIVIDUAL BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS BY HIGH-RESOLUTION DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY

Lipid structural analysis in individual pre-implantation mammalian embryos is hampered by the small amount of biological material, such that most studies use staining methods or gas chromatography analysis generate information only on the fatty acyl residues. Recent developments in high-resolution desorption electrospray ionization mass spectrometry (DESI-MS) allow the analysis of free fatty acids (FA) and glycerophospholipids (PL) in individual bovine embryos. Here, we report on the use of DESI-MS for the sensitive analysis of triacylglycerol (TAG) species, profiles of FA and PL in individual bovine oocytes and embryos. Bovine oocytes (n = 40) and blastocysts (n = 42) were frozen in a minimal volume of PBS (2 to 5 µL). Samples were directly deposited on glass slides after thawing. After drying, a volume of 500 µL of methanol : water (1 : 1, vol/vol) was carefully deposited on the surface of the glass slide and removed by orienting the glass slide vertically to eliminate PBS salts. An Orbitrap mass spectrometer was used for the experiments. Parameters for the positive ion mode were as follows: acetonitrile (ACN) supplemented with 3 µL mL–1 of AgNO3 at a 5 µL min–1 flow rate, injection time of 1000 ms, and a mass-to-charge range of m/z 400 to 1500. For the negative ion mode, the solvent combination used was acetonitrile + dimethylformamide (1 : 1, vol/vol) at a 1.0 µL min–1 flow rate, a maximum injection time of 1000 ms, and a mass-to-charge range of m/z 150 to 1000. Positive ion mode data for the detection of TAG species were acquired first, followed by acquisition of FA and PL in the negative ion mode. Detection of TAG by DESI, which is extremely useful for bovine embryo cryopreservation and metabolism research, has been performed by adding AgNO3 in the DESI spray to obtain silver adducts, which are easily recognised by the characteristic 1 : 1 abundance ratio of the 107 : 109 Ag isotopes. The most abundant fatty acyl residues present in TAG species were palmitic (P), linoleic (L), oleic (O), and stearic (S) acids, such as TAG of m/z 937, PPL (50 : 2); m/z 965, POO (52 : 1); m/z 967, POS (52 : 2); m/z 989, OOL/LLS (54 : 4); and m/z 991, OOO, SOL (54 : 3). Free FA and PL profiles collected from the same samples in the negative ion mode were similar to those in our recent report (2012 J. Mass Spectrom. 47, 29–33). Lipid attribution has been performed based on high-resolution mass analysis. Multivariate statistics from this data set will allow visualisation of differences observed in the lipid profiles among samples. In conclusion, we report the use of DESI-MS for the sensitive analysis of TAG in individual bovine oocytes and embryos and the creation of profiles of FA, PL, and TAG species in the same sample by DESI-MS.

Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.

Automated Platform for High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

An automated platform has been developed for acquisition and visualization of mass spectrometry imaging (MSI) data using nanospray desorption electrospray ionization (nano-DESI). The new system enables robust operation of the nano-DESI imaging source over many hours by precisely controlling the distance between the sample and the nano-DESI probe. This is achieved by mounting the sample holder onto an automated XYZ stage, defining the tilt of the sample plane, and recalculating the vertical position of the stage at each point. This approach is useful for imaging of relatively flat samples such as thin tissue sections. Custom software called MSI QuickView was developed for visualization of large data sets generated in imaging experiments. MSI QuickView enables fast visualization of the imaging data during data acquisition and detailed processing after the entire image is acquired. The performance of the system is demonstrated by imaging rat brain tissue sections. Low background noise enables simultaneous detection of lipids and metabolites in the tissue section. High-resolution mass analysis combined with tandem mass spectometry (MS/MS) experiments enabled identification of the observed species. In addition, the high dynamic range (>2000) of the technique allowed us to generate ion images of low-abundance isobaric lipids. A high-spatial resolution image was acquired over a small region of the tissue section revealing the distribution of an abundant brain metabolite, creatine, on the boundary between the white and gray matter. The observed distribution is consistent with the literature data obtained using magnetic resonance spectroscopy.

High‐resolution ultra‐high mass spectrometry: Increasing the m/z range of protein analysis

The proof of principle for high-resolution TOF mass analysis spanning the entire range of intact singly charged proteins has recently been demonstrated. The centers of the isotope distributions of individual proteins in a complex distribution can be defined to within 0.5 Da or better up to 200 kDa with internal calibration. This achievement will have an enormous effect on the process of routine protein analysis over the next few years as the technology mainstreams. The greatest obstacle to high-resolution in the ultra-high mass range (m/z > 20000) is the expansion-induced kinetic energy (KE) and its spread. The solution to this problem is to trap the ions in a buffer gas so that the motion of the ions can be completely defined by the applied fields. If this can accomplished without mass dependence, then any ion, regardless of size, can be mass analyzed with high resolution. This article discusses the methodology that we used to capture atmosphere sampled singly charged proteins in vacuum at a point just before they enter the mass analyzer to completely eliminate the expansion-induced KE. We then used digitally created quadrupole waveforms to inject the ions into the mass analyzer in a well-collimated plug with a controlled amount of KE. Trapping the ions to remove the expansion-induced KE and then electrodynamically manipulating the ions are the key steps to high-resolution mass analysis at any value of m/z. The impact of this technology will be discussed.

Increasing the trapping mass range to m/z = 109—A major step toward high resolution mass analysis of intact RNA, DNA and viruses

This work demonstrates sampling of singly charged particles up to 200nm in diameter at atmospheric pressure into vacuum and trapping large numbers (>106) at a point in front of the end cap electrode of a linear quadrupole ion guide/trap for on-demand injection into the acceleration region of a time-of-flight mass spectrometer in a well-collimated ion packet. This procedure was shown to yield trapping efficiencies that ranged from 4 to 5% for 10nm diameter urea particles (∼400kDa) to 1% for 200nm urea particles (∼3×109Da). Analysis of the inlet optimization procedure suggests that the inlet can be adapted to sample and trap beyond the 200nm range. Review of the most likely places for ion loss in the sampling process suggests that the sampling and trapping efficiencies can be improved well beyond the 4–5% shown. Moreover, it suggests that sampling of smaller than 10nm ions could achieve efficiencies in the 10s of percent range thereby suggesting new levels of sensitivity can be achieved for small ions (<200kDa). Finally, demonstration of trapping large numbers of 200nm (3×109Da) ions for on-demand ejection in well collimated temporally discrete ion packets is a prelude to resolved mass analysis in that range.

Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics

Selected reaction monitoring on a triple quadrupole mass spectrometer is currently experiencing a renaissance within the proteomics community for its, as yet, unparalleled ability to characterize and quantify a set of proteins reproducibly, completely, and with high sensitivity. Given the immense benefit that high resolution and accurate mass instruments have brought to the discovery proteomics field, we wondered if highly accurate mass measurement capabilities could be leveraged to provide benefits in the targeted proteomics domain as well. Here, we propose a new targeted proteomics paradigm centered on the use of next generation, quadrupole-equipped high resolution and accurate mass instruments: parallel reaction monitoring (PRM). In PRM, the third quadrupole of a triple quadrupole is substituted with a high resolution and accurate mass mass analyzer to permit the parallel detection of all target product ions in one, concerted high resolution mass analysis. We detail the analytical performance of the PRM method, using a quadrupole-equipped bench-top Orbitrap MS, and draw a performance comparison to selected reaction monitoring in terms of run-to-run reproducibility, dynamic range, and measurement accuracy. In addition to requiring minimal upfront method development and facilitating automated data analysis, PRM yielded quantitative data over a wider dynamic range than selected reaction monitoring in the presence of a yeast background matrix because of PRM's high selectivity in the mass-to-charge domain. With achievable linearity over the quantifiable dynamic range found to be statistically equal between the two methods, our investigation suggests that PRM will be a promising new addition to the quantitative proteomics toolbox.

Abstract 4781: Analysis of a new chemopreventative agent SHetA2 and its metabolites in Beagle dog liver and plasma

Abstract Flexible heteroarotinoid SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino] methanethione]} is a new candidate anticancer compound which is effective against a number of different types of tumors. We conducted preclinical safety and toxicity testing of SHetA2 in Beagle dogs with emphasis on determination of SHetA2 and its metabolites (especially GSH-conjugates) in liver. SHetA2 (in 30% aqueous Solutol® HS 15 to improve bioavailability) was administered orally to male and female Beagle dogs for 29 consecutive days. The dose levels of SHetA2 were 0 (control group 1), 100 (group 2), 400 (group 3), and 1500 (group 4) mg/kg/day. The liver and plasma samples were collected on day 29 at 2 hours after drug administration. SHetA2 and its metabolites levels were measured by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS) on Micromass Quattro micro API triple quadrupole mass-spectrometer. Metabolites formation was also confirmed by high-resolution accurate mass analysis on Waters SYNAPT time-of-flight (TOF) mass spectrometer. The following known metabolites of SHetA2 were quantified in liver (using the parent compound calibration curve): monohydroxy SHetA2 [SHetA2(O1)], dihydroxy SHetA2 [SHetA2(O2)], and GSH-conjugates of SHetA2 [SHetA2-GSH], monohydroxy SHetA2 [SHetA2(O1)-GSH], and dihydroxy SHetA2 [SHetA2(O2)-GSH]. Trihydroxy SHetA2 and its GSH-conjugate were below quantification level or not detected. The GSH-metabolites were of particular interest since conjugation with GSH may serve as a potential indicator of a compound toxicity. However, despite the relatively high levels of SHetA2-GSH-conjugate in dog liver, no drug-related mortality or clinical signs of toxicity were seen in any dose groups. We also found significant levels of several previously unknown metabolites - glucuronide conjugates of SHetA2, SHetA2(O1), and SHetA2(O2). These glucuronides were not reported in mouse and rat liver, in contrast to our dog study. The exact position of glucuronidation was not determined. It may represent a common N- or a rare case of S-glucuronidation for SHetA2, and, in addition, O-glucuronidation for oxidation products of SHetA2. The most abundant metabolites in liver were: glucuronide of SHetA2, SHetA2(O1) and its glucuronide, and GSH-conjugate of SHetA2 whose levels were at 29-80%, 16-31%, 14-39%, and 9-20%, respectively, of SHetA2 levels (2,600-14,000 ng/g; based on average values). Dihydroxy SHetA2 showed two major peaks: “SHetA2(O2)a” (mixture of SHetA2(O2) and its glucuronide conjugate) and “SHetA2(O2)b” (identity was not confirmed; requires additional experiments for its structure elucidation). In plasma, SHetA2(O1) was a major metabolite - 52-86 % of SHetA2 level (600-1,600 ng/mL). Glucuronide of SHetA2 was only at 7-13% of the parent compound. All GSH-conjugates were below quantification level or not detected in plasma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4781. doi:1538-7445.AM2012-4781

Antineoplastic Agents. 587. Isolation and Structure of 3-Epipancratistatin from Narcissus cv. Ice Follies

Bioassay-guided (cancer cell line) separation of an extract prepared from Narcissus cv. Ice Follies (from The Netherlands) led to the isolation of a new Amaryllidaceae isocarbostiryl, 3-epipancratistatin (1b), as well as narciclasine (2). This Narcissus cultivar was found to be a good source of narciclasine. The structure of 1b was established by high-resolution mass and high-field 2D NMR spectroscopic analyses. Against a panel of murine and human cancer cell lines, 3-epipancratistatin (1b) led to cell growth inhibition (GI(50) 2.2-0.69 μg/mL) some 100× less than that found for pancratistatin (1a) and narciclasine (2), thereby revealing an important configurational requirement in 1a for strong cancer cell growth inhibition.

Evaluation of ion activation strategies and mechanisms for the gas-phase fragmentation of sulfoquinovosyldiacylglycerol lipids from Rhodobacter sphaeroides

Sulfoquinovosyldiacylglycerol (SQDG) lipids, found in plants and photosynthetic bacteria, can substitute for phospholipids under phosphate limiting conditions. Here, various low-energy ion activation strategies have been evaluated for the identification and characterization of deprotonated SQDG lipids from a crude membrane lipid extract of Rhodobacter sphaeroides, using collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) in either a triple quadrupole mass spectrometer or in a hybrid quadrupole ion trap-multipole mass spectrometer coupled with high resolution/accurate mass analysis capabilities. In the triple quadrupole instrument, using energy resolved CID-MS/MS experiments, the SQDG head group specific product ion at m/z 225 (C6H9O7S−), rather than m/z 81 (SO3H−), was determined to provide the greatest sensitivity for SQDG lipid detection, and is therefore the preferred ‘fingerprint’ ion for the identification of this lipid class from within complex lipid mixtures when using precursor ion scan mode MS/MS experiments. A comparison of conventional ion trap CID-MS/MS and -MSn, with ‘low Q’ CID-MS/MS, pulsed Q dissociation (PQD)-MS/MS and higher energy collision induced dissociation (HCD)-MS/MS performed in an LTQ Orbitrap Velos mass spectrometer, revealed that HCD-MS/MS coupled with high resolution/accurate mass analysis represents the most sensitive, and perhaps most importantly the most specific strategy, for ion trap based identification and characterization of SQDG lipids, due to the ability to readily distinguish the SQDG head group specific product ion at m/z 225.0069 from other products that may be present at the same nominal m/z value. Finally, the mechanisms responsible for formation of each of the major product ions observed by low-energy CID-MS/MS of deprotonated SQDG lipids were elucidated using uniform H/D exchange, HCD-MS/MS and high resolution mass analysis. Formation of the m/z 225 ‘fingerprint’ ion occurs via a charge-remote cis-elimination reaction, likely involving transfer of a hydrogen from the hydroxyl group located on the C2 position of the sugar ring.

Ultra High-Mass Resolution Paper Spray by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Paper Spray Ionization is an atmospheric pressure ionization technique that utilizes an offline electro-osmotic flow to generate ions off a paper medium. This technique can be performed on a Bruker SolariX Fourier transform ion cyclotron resonance mass spectrometer by modifying the existing nanospray source. High-resolution paper spray spectra were obtained for both organic and biological samples to demonstrate the benefit of linking the technique with a high-resolution mass analyzer. Error values in the range 0.23 to 2.14 ppm were obtained for calf lung surfactant extract with broadband mass resolving power (m/Δm50%) above 60,000 utilizing an external calibration standard.

Structural characterization of wax esters by electron ionization mass spectrometry

The interpretation of the electron ionization mass spectra of straight-chain and methyl-branched saturated and unsaturated wax esters (WEs) is discussed in this study based on the spectra of 154 standards. The most important fragments indicative of the structure of the acid and alcohol chains are identified and summarized for WEs with various number of double bonds in the chains. Briefly, most WEs provide acylium ions allowing structural characterization of the acid part, whereas the alcohol part gives corresponding alkyl radical cations. The elemental composition of selected important fragments is established from a high-resolution accurate mass analysis. The ion abundances are discussed with respect to the length and unsaturation of the aliphatic chains. The interpretation of the spectra of branched or unsaturated WEs requires the recognition of small but important peaks that are difficult to discern among the other fragments. We demonstrate that such fragments are easily detected in differential mass spectra. This approach requires spectra of WE standards (e.g., straight-chain analogs in the case of branched WEs) recorded under the same experimental conditions. The WEs mass spectral database provided in the supplemental data can be used as a reference for the analysis of the GC/EI-MS data.

Quantitative estimation of circulating metabolites without synthetic standards by ultra‐high‐performance liquid chromatography/high resolution accurate mass spectrometry in combination with UV correction

An early assessment of metabolite exposure in preclinical species can provide quantitative estimation on possible active or toxic metabolites. Frequently, synthetic metabolite standards are not available at the preclinical stage, precluding the quantitation of metabolites by means of calibration curves and quality control (QC) samples. We present here an approach to determine the extent of circulating metabolites using 'metabolite standards' generated by in vitro incubations in combination with the correction for mass spectrometry response based on UV response. The study was done by coupling ultra-high-performance liquid chromatography (UHPLC) to LTQ-Orbitrap high-resolution mass spectrometry, and the quantitation was based on full scan high-resolution accurate mass analysis in combination with retention time. First, we investigated the separation capacity of a 10.5 min UHPLC method and the quantitative capability of an LTQ-Orbitrap for full scan accurate mass quantitation by spiking chemical standards of buspirone and its six metabolites in blank plasma. Then we demonstrated the use of a UV correction approach to quantitatively estimate buspirone and its metabolites in plasma samples from a rat pharmacokinetics study. We compared the concentration versus time profiles of buspirone and its six metabolites in rat plasma samples obtained using three different approaches, including using UV correction, using individual standard curves for each metabolite prepared from the synthetic standard, and using a calibration curve of the parent compound buspirone. We demonstrated the estimated metabolite exposure of buspirone using this UV correction approach resulted in rank ordering of metabolite exposure within three-fold of the value obtained with metabolite standards, in contrast to eight-fold without UV correction. The approach presented in this paper provides a practical solution to an unmet bioanalytical need for quantitative information on metabolites without standards in preclinical in vivo studies.

Limitation of Time-of-Flight Resolution in the Ultra High Mass Range

In this work, we have examined the reason for the deterioration of resolution and mass accuracy of time-of-flight mass analyzers with increasing mass after the expansion-induced kinetic energy has been eliminated by collisional cooling in an ion guide. Theoretically, removing the expansion-induced kinetic energy by collisional cooling permits the ions to travel along the ion guide axes without significant deviation so that they can be injected into the analyzer in a well-collimated ion beam with well-defined kinetic energy. If the ions can be injected into an orthogonal acceleration time-of-flight mass analyzer (oa-TOF) in this manner, high-resolution mass analysis can be obtained regardless of mass or m/z. Unfortunately, high resolution did not result. It is our contention that the effusive expansion out of the first ion guide yields dispersive axial ejection that reduces TOF resolving power with increasing mass not m/z.

Negative electron transfer dissociation Fourier transform mass spectrometry of glycosaminoglycan carbohydrates.

Electron transfer through gas phase ion-ion reactions has led to the widespread application of electron- based techniques once only capable in ion trapping mass spectrometers. Although any mass analyzer can in theory be coupled to an ion-ion reaction device (typically a 3-D ion trap), some systems of interest exceed the capabilities of most mass spectrometers. This case is particularly true in the structural characterization of glycosaminoglycan (GAG) oligosaccharides. To adequately characterize highly sulfated GAGs or oligosaccharides above the tetrasaccharide level, a high resolution mass analyzer is required. To extend previous efforts on an ion trap mass spectrometer, negative electron transfer dissociation coupled with a Fourier transform ion cyclotron resonance mass spectrometer has been applied to increasingly sulfated heparan sulfate and heparin tetrasaccharides as well as a dermatan sulfate octasaccharide. Results similar to those obtained by electron detachment dissociation are observed.

Mass spectrometry of atmospheric aerosols—Recent developments and applications. Part I: Off‐line mass spectrometry techniques

Many of the significant advances in our understanding of atmospheric particles can be attributed to the application of mass spectrometry. Mass spectrometry provides high sensitivity with a fast response time to probe chemically complex particles. This review focuses on recent developments and applications in the field of mass spectrometry of atmospheric aerosols. In Part I of this two-part review, we concentrate on off-line mass spectrometry techniques, which require sample collection on filters but can provide detailed molecular speciation. In particular, off-line mass spectrometry techniques utilizing tandem mass spectrometry experiments and high resolution mass analyzers provide improved insight into secondary organic aerosol formation and heterogeneous reaction pathways through detailed structural elucidation at the molecular level.

Synthesis, spectral, theoretical, and antimicrobial screening of some heterocyclic oximes

In a wide research program toward new and efficient antimicrobial agents, a series of substituted N-nitroso-3-alkyl piperidone oximes were synthesized and their antibacterial activity against Staphylococcus aureus, Pseudomonas aerugonisa, Escherichia coli, and Klbesiella pneumoniae and antifungal activity against Aspergillus niger, Aspergillus flavus, and Rhizopus were evaluated. Their structure and stereochemistry were characterized by high-resolution 1H NMR, mass and elemental analysis. The spectra of all N-nitroso oximes reveal the presence of two isomers labeled as E (–NOH group is anti to N–N=O moiety) and Z (–NOH group is syn to N–N=O moiety) in solution and the coupling constants ruled out the possibility of normal chair conformation. From the theoretical studies and coupling constant values, it was found that all N-nitroso oximes exist as an equilibrium mixture of CA boat conformation (B 1) and the major isomer was found to be E isomer. The molecular structure of N-nitroso oximes were also determined by semiempirical and Gaussian-03 calculation and the results are in agreement with the experimental studies.

Computing H/D-Exchange rates of single residues from data of proteolytic fragments

BackgroundProtein conformation and protein/protein interaction can be elucidated by solution-phase Hydrogen/Deuterium exchange (sHDX) coupled to high-resolution mass analysis of the digested protein or protein complex. In sHDX experiments mutant proteins are compared to wild-type proteins or a ligand is added to the protein and compared to the wild-type protein (or mutant). The number of deuteriums incorporated into the polypeptides generated from the protease digest of the protein is related to the solvent accessibility of amide protons within the original protein construct.ResultsIn this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based on combinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX data of overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residue resolution.ConclusionsWith our new method it is possible to automatically determine deuterium exchange with higher spatial resolution than the level of digested fragments.

High resolution mass analysis of N- and C-terminal negative ions resulting from resonance electron capture by aliphatic amino acids

High mass resolving power was applied to study resonance electron capture by glycine, alanine, and valine, and accurate mass measurements helped to distinguish between some negative ions having the same nominal masses. It was established that the C- and N-terminal negative ions of the same nominal masses were formed at different electron energies from different resonance states. The typical fragmentation pathways in deprotonated amino acids via loss of water initiated by collisional activation were not observed upon resonant electron capture by aliphatic amino acids. Instead, [M-18](-) negative ions in the vicinity of 5 eV were found to be associated with simultaneous loss of either ammonia and a hydrogen atom or an amino group and a hydrogen molecule.

High Resolution Electron Transfer Dissociation Studies of Unfractionated Intact Histones from Murine Embryonic Stem Cells Using On-line Capillary LC Separation: DETERMINATION OF ABUNDANT HISTONE ISOFORMS AND POST-TRANSLATIONAL MODIFICATIONS

Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed. From a single LC-electron transfer dissociation run, this strategy permits the identification of the most abundant intact proteins, determination of the isoforms present, and the localization of post-translational modifications.

Imaging of Lipids in Spinal Cord Using Intermediate Pressure Matrix-Assisted Laser Desorption-Linear Ion Trap/Orbitrap MS

A hybrid linear ion trap/Orbitrap mass spectrometer was used to perform tandem mass spectrometry (MS/MS) experiments and high-resolution mass analysis of lipids desorbed from nerve tissue. A dramatic improvement in mass spectral resolution and a decrease in background are observed in the spectra collected from the Orbitrap mass analyzer, which allows generation of more accurate mass spectrometric images of the distribution of lipids within nerve tissue. Employment of both mass analyzers provides a rapid and reliable means of compound identification based on MS/MS fragmentation and high-resolution mass spectrometry (HRMS) accurate mass.