Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers

Sphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS³ protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. JLR LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations.     

Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of post-translational modifications

Chromatin is regulated at many different levels, from higher-order packing to individual nucleosome placement. Recent studies have shown that individual histone modifications, and combinations thereof, play a key role in modulating chromatin structure and gene activity. Reported here is an analysis of Arabidopsis histone H3 modifications by nanoflow-HPLC coupled to electrospray ionization on a hybrid linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS). We find that the sites of acetylation and methylation, in general, correlate well with other plants and animals. Two well-studied modifications, dimethylation of Lys-9 (correlated with silencing) and acetylation of Lys-14 (correlated with active chromatin) while abundant by themselves were rarely found on the same histone H3 tail. In contrast, dimethylation at Lys-27 and monomethylation at Lys-36 were commonly found together. Interestingly, acetylation at Lys-9 was found only in a low percentage of histones while acetylation of Lys-14 was very abundant. The two histone H3 variants, H3.1 and H3.2, also differ in the abundance of silencing and activating marks confirming other studies showing that the replication-independent histone H3 is enriched in active chromatin.     

Top-down protein sequencing and MS3 on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

Top-down proteomics, the analysis of intact proteins (instead of first digesting them to peptides), has the potential to become a powerful tool for mass spectrometric protein characterization. Requirements for extremely high mass resolution, accuracy, and ability to efficiently fragment large ions have often limited top-down analyses to custom built FT-ICR mass analyzers. Here we explore the hybrid linear ion trap (LTQ)-Orbitrap, a novel, high performance, and compact mass spectrometric analyzer, for top-down proteomics. Protein standards from 10 to 25 kDa were electrosprayed into the instrument using a nanoelectrospray chip. Resolving power of 60,000 was ample for isotope resolution of all protein charge states. We achieved absolute mass accuracies for intact proteins between 0.92 and 2.8 ppm using the "lock mass" mode of operation. Fifty femtomole of cytochrome c applied to the chip resulted in spectra with excellent signal-to-noise ratio and only low attomole sample consumption. Different protein charge states were dissociated in the LTQ, and the sensitivity of the orbitrap allowed routine, high resolution, and high mass accuracy fragment detection. This resulted in unambiguous charge state determination of fragment ions and identification of unmodified and modified proteins by database searching. Protein fragments were further isolated and fragmented in the LTQ followed by analysis of MS(3) fragments in the orbitrap, localizing modifications to part of the sequence and helping to identify the protein with these small peptide-like fragments. Given the ready availability and ease of operation of the LTQ-Orbitrap, it may have significant impact on top-down proteomics.     

Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer

Complete phosphorylation mapping of protein kinases was successfully undertaken using an automated LC/MS/MS approach. This method uses the direct combination of triple quadrupole and ion trapping capabilities in a hybrid triple quadrupole linear ion trap to selectively identify and sequence phosphorylated peptides. In particular, the use of a precursor ion scan of m/z -79 in negative ion mode followed by an ion trap high resolution scan (an enhanced resolution scan) and a high sensitivity MS/MS scan (enhanced product ion scan) in positive mode is a very effective method for identifying phosphorylation sites in proteins at low femtomole levels. Coupling of this methodology with a stable isotope N-terminal labeling strategy using iTRAQtrade mark reagents enabled phosphorylation mapping and relative protein phosphorylation levels to be determined between the active and inactive forms of the protein kinase MAPKAPK-1 in the same LC/MS run.     

Gas phase hydrogen/deuterium exchange reactions of peptide ions in a quadrupole ion trap mass spectrometer

Hydrogen/deuterium exchange reactions of protonated and sodium cationized peptide molecules have been studied in the gas phase with a MALDI/quadrupole ion trap mass spectrometer. Unit-mass selected precursor ions were allowed to react with deuterated ammonia introduced into the trap cell by a pulsed valve. The reactant gas pressure, reaction time, and degree of the internal excitation of reactant ions were varied to explore the kinetics of the gas phase isotope exchange. Protonated peptide molecules exhibited a high degree of reactivity, some showing complete exchange of all labile hydrogen atoms. On the contrary, peptide molecules cationized with sodium exhibited only very limited reactivity, indicating a vast difference between the gas phase structures of the two ions. © 1997 Wiley-Liss Inc.     

Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications

Background

Functional genomics tools provide researchers with the ability to apply high-throughput techniques to determine the function and interaction of a diverse range of genes. Mutagenised plant populations are one such resource that facilitate gene characterisation. They allow complex physiological responses to be correlated with the expression of single genes in planta, through either reverse genetics where target genes are mutagenised to assay the affect, or through forward genetics where populations of mutant lines are screened to identify those whose phenotype diverges from wild type for a particular trait. One limitation of these types of populations is the prevalence of gene redundancy within plant genomes, which can mask the affect of individual genes. Activation or enhancer populations, which not only provide knock-out but also dominant activation mutations, can facilitate the study of such genes.

Results

We have developed a population of almost 50,000 activation tagged A. thaliana lines that have been archived as individual lines to the T₃ generation. The population is an excellent tool for both reverse and forward genetic screens and has been used successfully to identify a number of novel mutants. Insertion site sequences have been generated and mapped for 15,507 lines to enable further application of the population, while providing a clear distribution of T-DNA insertions across the genome. The population is being screened for a number of biochemical and developmental phenotypes, provisional data identifying novel alleles and genes controlling steps in proanthocyanidin biosynthesis and trichome development is presented.

Conclusion

This publicly available population provides an additional tool for plant researcher's to assist with determining gene function for the many as yet uncharacterised genes annotated within the Arabidopsis genome sequence http://aafc-aac.usask.ca/FST. The presence of enhancer elements on the inserted T-DNA molecule allows both knock-out and dominant activation phenotypes to be identified for traits of interest.     

Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications

Background  

Histone modifications and histone variants are of importance in many biological processes. To understand the biological functions of the global dynamics of histone modifications and histone variants in higher plants, we elucidated the variants and post-translational modifications of histones in soybean, a legume plant with a much bigger genome than that of Arabidopsis thaliana.     

Global mapping of post-translational modifications on histone H3 variants in mouse testes

Mass spectrometry (MS)-based characterization is important in proteomic research for verification of structural features and functional understanding of gene expression. Post-translational modifications (PTMs) such as methylation and acetylation have been reported to be associated with chromatin remodeling during spermatogenesis. Although antibody- and MS-based approaches have been applied for characterization of PTMs on H3 variants during spermatogenesis, variant-specific PTMs are still underexplored. We identified several lysine modifications in H3 variants, including testis-specific histone H3 (H3t), through their successful separation with MS-based strategy, based on differences in masses, retention times, and presence of immonium ions. Besides methylation and acetylation, we detected formylation as a novel PTM on H3 variants in mouse testes. These patterns were also observed in H3t. Our data provide high-throughput structural information about PTMs on H3 variants in mouse testes and show possible applications of this strategy in future proteomic studies on histone PTMs.     

Characterization of histone H2A and H2B variants and their post-translational modifications by mass spectrometry

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. The main histone H2B variant, H2BA, was acetylated at Lys-12, -15, and -20. The analysis of core histone subtypes with their modifications provides a first step toward an understanding of the functional significance of the diversity of histone structures.     

Going global: Novel histone modifications in the globular domain of H3

Post-translational modifications (PTM) of histones are key regulators of chromatin function. New mass spectometrical technologies have revealed that PTMs are not restricted to the histone tails, but can also be found in the globular domains, especially at the DNA-binding surface of the nucleosomes. Recent work on this new group of epigenetic marks showed that these modifications have not only the potential to alter the physical properties of the nucleosome, but may act as signals that regulate the recruitment of effector proteins to chromatin as well.     

Recent advances in mass spectrometry analysis of histone post-translational modifications: potential clinical impact of the PAT-H-MS approach

Histone post-translational modifications (hPTMs) contribute to the regulation of gene expression and increasing evidence links them to the development of various pathologies, highlighting their potential as biomarkers for prognostic, diagnostic and therapeutic applications. Mass spectrometry (MS) has emerged as a powerful analytical tool for hPTM analysis, which has also been applied to the analysis of epigenetic aberrations in diseases. However, the potential offered by the MS-based hPTM analysis of clinical samples for epigenetic biomarker discovery has been left largely unexploited. This article summarizes the contribution of MS-based approaches to clinical epigenetics, with a special focus on the PAThology tissue analysis of Histones by Mass Spectrometry (PAT-H-MS) approach – which represents the first application of MS-based hPTM analysis to formalin-fixed paraffin-embedded clinical samples – discussing its strengths and limitations, as well as possible implementations.     

Aurora1 phosphorylation activity on histone H3 and its cross-talk with other post-translational histone modifications in Arabidopsis

The enzymological properties of AtAurora1, a kinase responsible for the cell cycle-dependent phosphorylation of histone H3 at S10, and its cross-talk with other post-translational histone modifications, were determined. In vitro phosphorylation of H3S10 by AtAurora1 is strongly increased by K9 acetylation, and decreased by K14 acetylation and T11 phosphorylation. However, S10 phosphorylation activity is unaltered by mono-, di- or trimethylation of K9. An interference of H3K9 dimethylation by SUVR4 occurs by a pre-existing phosphorylation at S10. Hence, cross-talk in plants exists between phosphorylation of H3S10 and methylation, acetylation or phosphorylation of neighbouring amino acid residues. AtAurora1 undergoes autophosphorylation in vivo regardless of the presence of substrate, and forms dimers in planta . Of the three ATP-competitive Aurora inhibitors tested, Hesperadin was most effective in reducing the in vivo kinase activity of AtAurora1. Hesperadin consistently inhibited histone H3S10 phosphorylation during mitosis in Arabidopsis cells, but did not affect other H3 post-translational modifications, suggesting a specific inhibition of AtAurora in vivo . Inactivation of AtAurora also caused lagging chromosomes in a number of anaphase cells, but, unlike the situation in mammalian cells, Hesperadin did not influence the microtubule dynamics in dividing cells.     

Application of mass spectrometry to the identification and quantification of histone post-translational modifications

The core histones are the primary protein component of chromatin, which is responsible for the packaging of eukaryotic DNA. The NH(2)-terminal tail domains of the core histones are the sites of numerous post-translational modifications that have been shown to play an important role in the regulation of chromatin structure. In this study, we discuss the recent application of modern analytical techniques to the study of histone modifications. Through the use of mass spectrometry, a large number of new sites of histone modification have been identified, many of which reside outside of the NH(2)-terminal tail domains. In addition, techniques have been developed that allow mass spectrometry to be effective for the quantitation of histone post-translational modifications. Hence, the use of mass spectrometry promises to dramatically alter our view of histone post-translational modifications.     

Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation

Coupling of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) to a sensitive linear ion trap (LTQ) mass spectrometer (MS) is a challenging, but highly promising approach for quantitative high-throughput proteomic profiling. Integration of the advantages of pulsed-Q dissociation (PQD) and collision-activated dissociation (CAD) fragmentation methods into a PQD-CAD hybrid mode, together with PQD optimization and data manipulation with a bioinformatics algorithm, resulted in a robust, sensitive and accurate iTRAQ quantitative proteomic workflow. The workflow was superior to the default PQD setting when profiling the proteome of a gastric cancer cell line, SNU5. Taken together, we established an optimized PQD-CAD hybrid workflow in LTQ-MS for iTRAQ quantitative proteomic profiling that may have wide applications in biological and biomedical research.     

Systematic comparison of a two-dimensional ion trap and a three-dimensional ion trap mass spectrometer in proteomics

The utility and advantages of the recently introduced two-dimensional quadrupole ion trap mass spectrometer in proteomics over the traditional three-dimensional ion trap mass spectrometer have not been systematically characterized. Here we rigorously compared the performance of these two platforms by using over 100,000 tandem mass spectra acquired with identical complex peptide mixtures and acquisition parameters. Specifically we compared four factors that are critical for a successful proteomic study: 1) the number of proteins identified, 2) sequence coverage or the number of peptides identified for every protein, 3) the data base matching SEQUEST X(corr) and S(p) score, and 4) the quality of the fragment ion series of peptides. We found a 4-6-fold increase in the number of peptides and proteins identified on the two-dimensional ion trap mass spectrometer as a direct result of improvement in all the other parameters examined. Interestingly more than 70% of the doubly and triply charged peptides, but not the singly charged peptides, showed better quality of fragmentation spectra on the two-dimensional ion trap. These results highlight specific advantages of the two-dimensional ion trap over the conventional three-dimensional ion traps for protein identification in proteomic experiments.     

Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.     

The use of a hybrid linear trap/FT-ICR mass spectrometer for on-line high resolution/high mass accuracy bottom-up sequencing.

In this work we present a hybrid linear trap/Fourier transform ion cyclotron resonance (ICR) mass spectrometer to perform protein sequencing using the bottom-up approach. We demonstrate that incorporation of the linear trap greatly enhances the overall performance of the hybrid system for the study of complex peptide mixtures separated by fast high-performance liquid chromatography gradients. The ability to detect in the linear trap enables employment of automatic gain control to greatly reduce space charging in the ICR cell irregardless of ion flux. Resulting accurate mass measurements of 2 ppm or better using external calibration are achieved for the base peak as well as ions at 2% relative abundance. The linear trap is used to perform ion accumulation and activation prior to detection in the ICR cell which increases the scan rate. The increased duty cycle allows for data-dependent mass analysis of coeluting peptides to be acquired increasing protein sequence coverage without increasing the gradient length. In addition, the linear trap could be used as an ion detection device to perform simultaneous detection of tandem mass spectra with full scan mass spectral detection in the ICR cell resulting in the fastest scan cycles for performing bottom-up sequencing of protein digests. Comparisons of protein sequence coverage are presented for product ion detection in the linear trap and ICR cell.     

Characterization of phosphorylation sites on Tpl2 using IMAC enrichment and a linear ion trap mass spectrometer

Advances in analytical techniques, specifically in mass spectrometry, have allowed for both facile protein identification and routine sequencing of proteins at increased sensitivity levels. Protein modifications present additional challenges because they occur at low stoichiometries and often change the analytical behavior of the molecule. For example, characterization of protein phosphorylation provides crucial information to signaling processes that are often associated with disease. Research into protein phosphorylation requires inter-disciplinary co-operation involving multiple investigators with expertise in diverse scientific fields. As such, techniques must be simple, effective, and incorporate multiple checkpoints that confirm the sample contains a phosphorylated protein in order to ensure resources are conserved. In this study, tumor progression locus 2 (Tpl2), which has been implicated in cell cycle regulation and has been shown to play a significant role in critical signal transduction pathways, was transfected into 293T cells, overexpressed and isolated from the cell lysate. Isolated proteins were separated via 1D gel electrophoresis, and their phosphorylation was confirmed using phosphospecific staining. The bands were excised and subjected to tryptic digestion and immobilized metal affinity chromatography (IMAC) prior to analysis by capillary-LC-MS/MS. Three phosphorylation sites were detected on Tpl2. One site had previously been reported in the literature but had not been characterized by mass spectrometric methods until this time; two additional novel sites of phosphorylation were detected.     

Comparison of the Paul ion trap to the linear ion trap for use in global proteomics

A critical evaluation of the performance of a 2-D linear ion trap (IT) instrument to two 3-D quadrupole IT instruments with emphasis on identification of rat serum proteins by bottom-up LC-MS/MS is presented. The speed and sensitivity of each of the instruments were investigated, and the effects that each of these have on the bottom-up proteomics identification approach are discussed.