Preparative scale mass spectrometry: A brief history of the calutron.

Calutrons were developed in the laboratory of E. O. Lawrence at the University of California at Berkeley. They were a modification of the cyclotrons he had invented and used in his Nobel Prize-winning investigations of the atomic nucleus. At the time their construction was undertaken, calutrons represented the only certain means of preparing enriched uranium isotopes for the construction of a fission bomb. The effort was successful enough that every atom of the 42 kg of 235 U used in the first uranium bomb had passed through at least one stage of calutron separation. At peak production, the first stage separators, α tanks, yielded an aggregate 258-g/day 235 U enriched to about 10 at. % from its natural abundance level of 0.72 at. %. The second stage separators, ß tanks, used the 10 at. % material as feedstock and produced a total 204-g/day 235 U enriched to at least 80 at. %. The latter, weapons grade, material was used in fission bombs. Under typical operating conditions, each α tank operated at a uranium beam intensity at the collectors of approximately 20 mA and each ß tank at a beam intensity of approximately 215 mA at the collectors. Bulk separation of isotopes for bomb production ceased in 1945. Since that time calutrons have been used to separate stable isotopes, but on a more limited scale than wartime weapons production. Stable isotope separations since 1960 have taken place using one modified ß tank.

Taurine Is Covalently Incorporated into Alpha-Tubulin.

Taurine is the most abundant free amino acid in the human body. It is found in relatively high concentrations (1-10 mM) in many animal tissues but not in plants. It has been studied since the early 1800s but has not been found to be covalently incorporated into proteins in any animal tissue. Taurine has been found in only one macromolecular complex as a post-transcriptional modification to mitochondrial tRNA. Tubulin is the subunit of microtubules found in all eukaryotic species and almost all eukaryotic cells and subject to numerous post-translational modifications (PTMs). An important PTM on α-tubulin is the removal and re-ligation of the final carboxyl residue, tyrosine. We here demonstrate that taurine can be covalently incorporated at the C-terminal end of alpha-tubulin in avian erythrocytes in a reaction that requires the de-tyrosination PTM and prevents the re-tyrosination PTM. Further, this is, to our knowledge, the first instance of taurine incorporation into a large protein.

An efficient solution for resolving iTRAQ and TMT channel cross-talk.

Isobaric tagging reagents such as isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass tag (TMT) typically have isotopic impurities that cause significant cross-talk between channels. Here, we present an efficient solution to compensate for channel cross-talk using linear algebra and find that it is between 20× and 120× faster than previous methods. We also find that the effects of channel cross-talk are as important to manage as the effects of ratio compression because of precursor impurities, and we have released an open-source tool to perform both types of calculations.

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion.

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.

Cyclodextrin and malto-dextrose collision cross sections determined in a drift tube ion mobility mass spectrometer using nitrogen bath gas.

In this study, we have evaluated a low field limit drift tube ion mobility device for ion mobility-mass spectrometry (IM-MS) measurements that uses nitrogen as a bath gas with electrospray ionization on a modified Q-TOF instrument. We have determined reduced mobility (K0) and collision cross section (CCS) values for a group of analyte ions that have been characterized previously in other drift tube IM-MS instruments. Our determinations of CCS for this set of ions as well as for standards are in agreement with published values. Because of their importance in biophysics and pharmaceuticals, we expanded our analysis to investigate the properties of cyclodextrins in this system. We present CCS data for both positively and negatively charged cyclodextrins and, for purposes of comparison, maltodextrose ions. Our results are the first reports of these materials as negative ions.

Correction: Characterization of hydroxypropyl-beta-cyclodextrins used in the treatment of Niemann-Pick Disease type C1.

[This corrects the article DOI: 10.1371/journal.pone.0175478.].

Editorial for Special Issue: Approaches to Top-Down Proteomics: In Honour of Prof. Patrick H. O'Farrell.

Presaging the current discipline of Proteomics, Prof Patrick H. O'Farrell recognized the critical need for detailed protein analyses to dissect and thereby understand molecular mechanisms. [...].

Characterization of hydroxypropyl-beta-cyclodextrins used in the treatment of Niemann-Pick Disease type C1.

2-Hydroxypropyl-beta-cyclodextrin (HPßCD) has gained recent attention as a potential therapeutic intervention in the treatment of the rare autosomal-recessive, neurodegenerative lysosomal storage disorder Niemann-Pick Disease Type C1 (NPC1). Notably, HPßCD formulations are not comprised of a single molecular species, but instead are complex mixtures of species with differing degrees of hydroxypropylation of the cyclodextrin ring. The degree of substitution is a critical aspect of the complex mixture as it influences binding to other molecules and thus could potentially modulate biological effects. VTS-270 (Kleptose HPB) and Trappsol® Cyclo™ are HPßCD products under investigation as novel treatments for NPC1. The purpose of the present work is to compare these two different products; analyses were based on ion distribution and abundance profiles using mass spectrometry methodology as a means for assessing key molecular distinctions between products. The method incorporated electrospray ionization and analysis with a linear low-field ion mobility quadrupole time-of-flight instrument. We observed that the number of hydroxypropyl groups (the degrees of substitution) are substantially different between the two products and greater in Trappsol Cyclo than in VTS-270. The principal ions of both samples are ammonium adducts. Isotope clusters for each of the major ions show doubly charged homodimers of the ammonium adducts. In addition, both products show doubly charged homodimers from adduction of both a proton and ammonium. Doubly charged heterodimers are also present, but are more intense in Trappsol Cyclo than in VTS-270. Based on the analytical differences observed between VTS-270 and Trappsol Cyclo with respect to the degree of substitution, the composition and fingerprint of the complex mixture, and the impurity profiles, these products cannot be considered to be the same; the potential biological and clinical implications of these differences are not presently known.

ProteinProcessor: A probabilistic analysis using mass accuracy and the MS spectrum.

Current approaches to protein identification rely heavily on database matching of fragmentation spectra or precursor peptide ions. We have developed a method for MALDI TOF-TOF instrumentation that uses peptide masses and their measurement errors to confirm protein identifications from a first pass MS/MS database search. The method uses MS1-level spectral data that have heretofore been ignored by most search engines. This approach uses the distribution of mass errors of peptide matches in the MS1 spectrum to develop a probability model that is independent of the MS/MS database search identifications. Peptide mass matches can come from both precursor ions that have been fragmented as well as those that are tentatively identified by accurate mass alone. This additional corroboration enables us to confirm protein identifications to MS/MS-based scores that are otherwise considered to be only of moderate quality. Straightforward and easily applicable to current proteomic analyses, this tool termed "ProteinProcessor" provides a robust and invaluable addition to current protein identification tools.

Morphologically compatible mass spectrometric analysis of lipids in cytological specimens.

INTRODUCTION: Modern lipid analysis requires mass spectrometric techniques, though to date these have been developed and applied primarily to histological serial sections. As such, there has been little emphasis on using mass spectrometry in such a way that the same specimen can yield both chemical and morphological information. Here, we present a mass spectrometric method that enables measurement of lipids from cells on cytospin slides in a way that preserves the cells for subsequent cytomorphologic evaluation. MATERIALS AND METHODS: Standardized cultures of MDA-MB-231, a breast cancer cell line, were prepared as cytospins and subjected to analysis using a Prosolia Flowprobe sampling and ionization source attached to a Thermo Scientific Quadrupole-Orbitrap mass spectrometer. Chemical compositions were deduced with accurate mass measurements and fragmentation of high intensity peaks to further determine chemical structure. After mass spectrometry, the slides were stained and cover-slipped, and the cells were reviewed for cytomorphologic features of breast cancer. These were compared to control slides of the same cellular concentration that had not been subjected to this analysis. RESULTS: Spectra from samples of all cellular concentrations demonstrated characteristic qualitative features that were discovered to represent phosphatidylcholines, phosphatidylglycerols, and phosphatidylserines with fragmentation and accurate mass matching. Cytomorphologic analysis demonstrated excellent preservation of the cells subjected to the Flowprobe analysis. CONCLUSION: Direct extraction, ionization, and identification of lipids is possible from cytologic preparations in such a way that the analyzed material is preserved and useful for subsequent microscopic analysis.

An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags.

Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis.

Proteomics Is Analytical Chemistry: Fitness-for-Purpose in the Application of Top-Down and Bottom-Up Analyses.

Molecular mechanisms underlying health and disease function at least in part based on the flexibility and fine-tuning afforded by protein isoforms and post-translational modifications. The ability to effectively and consistently resolve these protein species or proteoforms, as well as assess quantitative changes is therefore central to proteomic analyses. Here we discuss the pros and cons of currently available and developing analytical techniques from the perspective of the full spectrum of available tools and their current applications, emphasizing the concept of fitness-for-purpose in experimental design based on consideration of sample size and complexity; this necessarily also addresses analytical reproducibility and its variance. Data quality is considered the primary criterion, and we thus emphasize that the standards of Analytical Chemistry must apply throughout any proteomic analysis.

Alternative calibration strategies for the clinical laboratory: application to nortriptyline therapeutic drug monitoring.

BACKGROUND: The addition of a calibration curve with every run is both time-consuming and expensive for clinical mass spectrometry assays. We present alternative calibration strategies that eliminate the need for a calibration curve except as required by laboratory regulations. METHODS: We measured serum nortriptyline concentrations prospectively in 68 patients on 16 days over a 2-month period using a method employing calibration schemes that relied on the measurement of the response ratio (RR) corrected by the response factor (RF), i.e., a measurement of the RR for an equimolar mixture of the analyte and internal standard. Measurements were taken with contemporaneous RF (cRF) measurements as well as sporadic RF (sRF) measurements. The measurements with these alternative calibration schemes were compared against the clinical results obtained by interpolation on a calibration curve, and those differences were evaluated for analytical and clinical significance. RESULTS: The differences between the values measured by cRF and sRF calibration and interpolation on a calibration curve were not significant (P = 0.088 and P = 0.091, respectively). Both the cRF- and sRF-based calibration results demonstrated a low mean bias against the calibration curve interpolations of 3.69% (95% CI, -15.8% to 23.2%) and 3.11% (95% CI, -16.4% to 22.6%), respectively. When these results were classified as subtherapeutic, therapeutic, or supratherapeutic, there was categorical agreement in 95.6% of the cRF calibration results and 94.1% of the sRF results. CONCLUSIONS: cRF and sRF calibration in a clinically validated liquid chromatography-tandem mass spectrometry assay yields results that are analytically and clinically commensurate to those produced by interpolation with a calibration curve.

Comprehensive analysis of LC/MS data using pseudocolor plots.

We have developed new applications of the pseudocolor plot for the analysis of LC/MS data. These applications include spectral averaging, analysis of variance, differential comparison of spectra, and qualitative filtering by compound class. These applications have been motivated by the need to better understand LC/MS data generated from analysis of human biofluids. The examples presented use data generated to profile steroid hormones in urine extracts from a Cushing's disease patient relative to a healthy control, but are general to any discovery-based scanning mass spectrometry technique. In addition to new visualization techniques, we introduce a new metric of variance: the relative maximum difference from the mean. We also introduce the concept of substructure-dependent analysis of steroid hormones using precursor ion scans. These new analytical techniques provide an alternative approach to traditional untargeted metabolomics workflow. We present an approach to discovery using MS that essentially eliminates alignment or preprocessing of spectra. Moreover, we demonstrate the concept that untargeted metabolomics can be achieved using low mass resolution instrumentation.

Quantitative proteomic analysis of Niemann-Pick disease, type C1 cerebellum identifies protein biomarkers and provides pathological insight.

Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials.

Warburg effect's manifestation in aggressive pheochromocytomas and paragangliomas: insights from a mouse cell model applied to human tumor tissue.

A glycolytic profile unifies a group of pheochromocytomas and paragangliomas (PHEOs/PGLs) with distinct underlying gene defects, including von Hippel-Lindau (VHL) and succinate dehydrogenase B (SDHB) mutations. Nevertheless, their tumor aggressiveness is distinct: PHEOs/PGLs metastasize rarely in VHL-, but frequently in SDHB-patients. To date, the molecular mechanisms causing the more aggressive phenotype in SDHB-PHEOs/PGLs remain largely unknown. Recently, however, an excellent model to study aggressive PHEOs (mouse tumor tissue (MTT) cells) has been developed from mouse PHEO cells (MPC). We employed this model for a proteomics based approach to identify changes characteristic for tumor aggressiveness, which we then explored in a homogeneous set of human SDHB- and VHL-PHEOs/PGLs. The increase of glucose transporter 1 in VHL, and of hexokinase 2 in VHL and SDHB, confirmed their glycolytic profile. In agreement with the cell model and in support of decoupling of glycolysis, the Krebs cycle and oxidative phosphorylation (OXPHOS), SDHB tumors showed increased lactate dehydrogenase levels. In SDHB-PGLs OXPHOS complex activity was increased at complex III and, as expected, decreased at complex II. Moreover, protein and mRNA expression of all tested OXPHOS-related genes were higher in SDHB- than in VHL-derived tumors. Although there was no direct evidence for increased reactive oxygen species production, elevated superoxide dismutase 2 expression may reflect elevated oxidative stress in SDHB-derived PHEOs/PGLs. For the first time, we show that despite dysfunction in complex II and evidence for a glycolytic phenotype, the Warburg effect does not seem to fully apply to SDHB-PHEOs/PGLs with respect to decreased OXPHOS. In addition, we present evidence for increased LDHA and SOD2 expression in SDHB-PHEOs/PGLs, proteins that have been proposed as promising therapeutic targets in other cancers. This study provides new insight into pathogenic mechanisms in aggressive human PHEOs/PGLs, which may lead to identifying new diagnostic and prognostic markers in the near future.

Production of reliable MALDI spectra with quality threshold clustering of replicates.

We present the first application of the quality threshold (QT) clustering algorithm to mass spectrometry (MS) data. The unique abilities of QT clustering to yield precision nodes that are commensurate with the mass measurement precision of the instrument are exploited to generate a consensus spectrum out of multiple replicate spectra. The spectral dot product and confidence intervals are used as a tool for evaluating the similarity and reproducibility between the consensus and replicates. The method is equally applicable to high and low resolution measurements. This paper demonstrates applications to linear spectra from a matrix assisted laser desorption ionization (MALDI) time of flight (TOF) instrument as well as peptide fragmentation data obtained from a TOF/TOF after unimolecular decomposition. The advantages of clustering to mitigate the inherent precision the shortcomings of MALDI data are discussed.

De-AMPylation of the small GTPase Rab1 by the pathogen Legionella pneumophila.

The bacterial pathogen Legionella pneumophila exploits host cell vesicle transport by transiently manipulating the activity of the small guanosine triphosphatase (GTPase) Rab1. The effector protein SidM recruits Rab1 to the Legionella-containing vacuole (LCV), where it activates Rab1 and then AMPylates it by covalently adding adenosine monophosphate (AMP). L. pneumophila GTPase-activating protein LepB inactivates Rab1 before its removal from LCVs. Because LepB cannot bind AMPylated Rab1, the molecular events leading to Rab1 inactivation are unknown. We found that the effector protein SidD from L. pneumophila catalyzed AMP release from Rab1, generating de-AMPylated Rab1 accessible for inactivation by LepB. L. pneumophila mutants lacking SidD were defective for Rab1 removal from LCVs, identifying SidD as the missing link connecting the processes of early Rab1 accumulation and subsequent Rab1 removal during infection.

Serpinin: a novel chromogranin A-derived, secreted peptide up-regulates protease nexin-1 expression and granule biogenesis in endocrine cells.

Previously we demonstrated that chromogranin A (CgA) promoted secretory granule biogenesis in endocrine cells by stabilizing and preventing granule protein degradation in the Golgi, through up-regulation of expression of the protease inhibitor, protease nexin-1 (PN-1). However, the mechanism by which CgA signals the increase of PN-1 expression is unknown. Here we identified a 2.9-kDa CgA-C-terminus peptide, which we named serpinin, in conditioned media from AtT-20 cells, a corticotroph cell line, which up-regulated PN-1 mRNA expression. Serpinin was secreted from AtT-20 cells upon high potassium stimulation and increased PN-1 mRNA transcription in these cells, in an actinomycin D-inhibitable manner. CgA itself and other CgA-derived peptides, when added to AtT-20 cell media, had no effect on PN-1 expression. Treatment of AtT-20 cells with 10 nm serpinin elevated cAMP levels and PN-1 mRNA expression, and this effect was inhibited by a protein kinase A inhibitor, 6-22 amide. Serpinin and a cAMP analog, 8-bromo-cAMP, promoted the translocation of the transcription factor Sp1 into the nucleus, which is known to drive PN-1 expression. Additionally, an Sp1 inhibitor, mithramycin A inhibited the serpinin-induced PN-1 mRNA up-regulation. Furthermore, a luciferase reporter assay demonstrated serpinin-induced up-regulation of PN-1 promoter activity in an Sp1-dependent manner. When added to CgB-transfected 6T3 cells, a mutant AtT20 cell line, serpinin induced granule biogenesis as evidenced by the presence of CgB puncta accumulation in the processes and tips. Our findings taken together show that serpinin, a novel CgA-derived peptide, is secreted upon stimulation of corticotrophs and plays an important autocrine role in up-regulating PN-1-dependent granule biogenesis via a cAMP-protein kinase A-Sp1 pathway to replenish released granules.

Quantitative proteomics analysis of inborn errors of cholesterol synthesis: identification of altered metabolic pathways in DHCR7 and SC5D deficiency.

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.