Multiple Stage MS Research Articles

Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis.

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) is commonly used to extract Tibetan medicine; however, many unpredictable unknown compounds remain after using the spectrum database. The present article developed a universal method for identifying components in Tibetan medicine using ion trap mass spectrometry (IT-MS). The method includes standardized and programmed protocols for sample preparation, MS setting, LC prerun, method establishment, MS acquisition, multiple-stage MS operation, and manual data analysis. Two representative compounds in the Tibetan medicine Abelmoschus manihot seeds were identified using multiple-stage fragmentation, with a detailed analysis of typical compound structures. In addition, the article discusses aspects such as ion mode selection, mobile phase adjustment, scanning range optimization, collision energy control, collision mode switchover, fragmentation factors, and limitations of the method. The developed standardized analysis method is universal and can be applied to unknown compounds in Tibetan medicine.

A Novel Triple Stage Ion Trap MS method validated for curcumin pharmacokinetics application: A comparison summary of the latest validated curcumin LC/MS methods

Even through more sensitive methods for quantifying trace levels of curcumin in plasma were urgently needed, beyond tandem mass spectrometry, the Paul trap MS/MS/MS has never been tested for curcumin quantification. Because of its unique trap function to accumulate target compounds selectively by optimizing multiple stage MS experiments, it showed great potential to remove background issues reported at low concentration ranges. A novel Triple stage Ion Trap Mass Spectrometry coupled with High Performance Liquid Chromatography (HPLC–ITMS/MS/MS) method has been developed and validated as a curcumin quantification method, for the first time. The use of isotope labeled curcumin-d6 as a novel internal standard (IS) is suggested and tested according to FDA validation procedure. A simplified sample preparation is introduced and validated by coupling a novel acetonitrile precipitation with molecular weight cut-off size exclusion method. This method demonstrated excellent recovery rate of 96.69%–109.26% and minimum matrix effect of 95.40%–110.98%. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for curcumin in rat plasma were 0.1 ng/ml and 1 ng/ml respectively. The linear calibration curve for quantifying curcumin in rat plasma was 1–3000 ng/ml (r2 > 0.99) with intra-day and inter-day RSD and accuracy within ±5.11%. Its application in a Pharmacokinetics (PK) study demonstrated detection of curcumin at a very low plasma level (1.0 ng/ml) and it would be applied to larger sample size animal and clinical pharmacokinetic studies. The presented ITMS/MS/MS quantification method has shown its advantages, including better sensitivity, accuracy, precision, wider calibration range and simplicity in sample preparation, when comparing with other curcumin LC/MS analysis methods in the latest 4 years.

Glycosphingolipidomics of donkey milk by hydrophilic interaction liquid chromatography coupled to ESI and multistage MS.

Hydrophilic interaction liquid chromatography coupled to negative-ion electrospray linear ion-trap multiple-stage MS (HILIC-ESI-MSn , n=2,3) was used to characterize polar lipids occurring in donkey milk. Besides the detection of abundant phospholipids, the structural characterization and content evaluation of minor glycosphingolipids (GSLs) were assessed. We report an unprecedented characterization of 11 hexosyl-ceramides (HexCer), 10 Hex2 Cer, and 4 Hex3 Cer. CID-MS/MS spectra in negative ion mode mainly afford information on the monosaccharide number and ceramide constitution (i.e., N-acyl residue and long-chain base), whereas MS/MS/MS spectra on the ceramide anions allow to recognize for each GSL the sphingoid base. The occurrence of sphingosine (S), sphinganine (DS), and phytosphingosine (P) was inferred from the fragmentation patterns. The milk samples exhibit a relatively high number of phytosphingosine substitutes, perhaps because of the feeding of donkeys, mainly based on pasture grass. However, the incidence of hydroxylated species on the α-carbon of the acyl chain was also revealed. The fatty acid composition of N-acyl chains showed high values of long-chain saturated fatty acids such as 20:0, 22:0, 23:0, and 24:0. An average content of GSL is also provided and three representative mono-, di-, and tri-HexCer in donkey milk are the following: HexCer 18:0/24:1 phytosphingosine nonhydroxylated [PN] at m/z 862.6 as chloride adduct [M+Cl]- , and content 225.9±2.8μg 100mL-1 ; Hex2 Cer 18:0/16:0 sphinganine nonhydroxylated [DSN] at m/z 862.7 as deprotonated adduct [M-H]- , and content 70.8±1.4μg 100mL-1 ; and Hex3 Cer 18:1/24:1 [SN] at m/z 1132.8 as [M-H]- , and content 38.5±0.7μg 100mL-1 .

Linear ion-trap MSn with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis

1-O-acylceramide is a new class of epidermal cer-amide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSn) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions were subjected to LIT MS2 to yield predominately the [M + H - H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to yield the [M + H - H2O - (1-O-FA)]+ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS4 on [M + H - H2O - (1-O-FA)]+ ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0- to 30:0-O-acyl) and N-acyl chains (from 14:0- to 34:1-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1-, 17:1-, 18:2-, and 19:1-sphingosine LCBs with odd fatty acyl chain or with monounsaturated N- or O-fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers, are present, underscoring the complexity of this Cer family.

Analytical strategy for determination of known and unknown destruxins using hybrid quadrupole-Orbitrap high-resolution mass spectrometry.

An analytical strategy based on a hybrid quadrupole-Orbitrap mass spectrometry was proposed for the simultaneous screening of known destruxins and characterization of potential members of this class of secondary metabolites, in order to evaluate the metabolite production of entomopathogenic fungi used as biocontrol agents. Initially, the fragmentation pathway of the known and commercially available destruxin A was established combining high resolution mass spectrometry (HRMS) and multiple stage MS data in order to obtain the strategy for the characterization of other destruxins for which reference standards were not available. Nineteen known destruxins including A, B, C, D, Ed, F, A1, B1, Ed1, A2, B2, D2, A3, DesmA, DesmB, DesmC, DesmB2, and two chloro-derivatives (Cl and E2 chlorohydrin) were unequivocally identified in Metarhizium brunneum using the proposed strategy. In addition, four unknown destruxins, namely C1, Ed2, G, and G1, were structurally elucidated and characterized forthe first time in this fungal strain.

Multiple stage MS in analysis of plasma, serum, urine and in vitro samples relevant to clinical and forensic toxicology.

This paper reviews MS approaches applied to metabolism studies, structure elucidation and qualitative or quantitative screening of drugs (of abuse) and/or their metabolites. Applications in clinical and forensic toxicology were included using blood plasma or serum, urine, in vitro samples, liquids, solids or plant material. Techniques covered are liquid chromatography coupled to low-resolution and high-resolution multiple stage mass analyzers. Only PubMed listed studies published in English between January 2008 and January 2015 were considered. Approaches are discussed focusing on sample preparation and mass spectral settings. Comments on advantages and limitations of these techniques complete the review.

Use of UHPLC high-resolution Orbitrap mass spectrometry to investigate the genes involved in the production of secondary metabolites in Aspergillus flavus

The fungus Aspergillus flavus is known for its ability to produce the toxic and carcinogenic aflatoxins in food and feed. While aflatoxins are of most concern, A. flavus is predicted to be capable of producing many more metabolites based on a study of its complete genome sequence. Some of these metabolites could be of great importance in food and feed safety. Here we describe an analytical methodology based on Orbitrap HRMS technology that allows the untargeted determination of fungal metabolites, in support of the study of the function of genes involved in secondary metabolism in fungi. The applied strategy implies the detection and identification of differentially expressed metabolites in extracts of wild-type and mutant fungal strains, using Orbitrap high-resolution mass spectrometry (HRMS) accurate mass data. The suitability of this approach was demonstrated by the confirmation of previously characterised genes involved in the aflatoxin biosynthetic pathway, namely a polyketide synthase (pksA), an oxidoreductase (ordA) and a methyltransferase (omtA) gene. Subsequently, the proposed methodology was applied for the detection and identification of metabolites produced by a yet uncharacterised gene cluster in A. favus, cluster 23. Comparative Orbitrap HRMS analysis of extracts of A. flavus wild-type strain and an over-expression mutant for the transcription factor of gene cluster 23 (lepE) demonstrated that this gene cluster is responsible for the production a set of 2-pyridone derivatives, the leporins. Besides the known derivatives leporin B and leporin B precursor that could be identified by automatic de-replication of the accurate mass data, five other compounds belonging to this class of fungal secondary metabolites were detected and identified for the first time, combining MS and multiple-stage MS data.

Rapid and simultaneous analysis of three molecular sea surface temperature proxies and application to sediments from the Sea of Marmara

Reconstructing ocean temperature values is a major target in paleoceanography and climate research. However, most temperature proxies are organism-based and thus suffer from an “ecological bias”. Multiproxy approaches can potentially overcome this bias but typically require more investment in time and resources, while being susceptible to errors induced by sample preparation steps necessary before analysis. Three lipid-based temperature proxies are widely used: UK′37 (based on long chain alkenones from phytoplanktonic haptophytes), TEX86 [based on glycerol dialkyl glycerol tetraethers (GDGTs) from pelagic archaea] and LDI (based on long chain diols from phytoplanktonic eustigmatophytes). So far, separate analytical methods, including gas chromatography (GC) and liquid chromatography (LC), have been used to determine these proxies. Here we present a sensitive method for determining all three in a single normal phase high performance LC–atmospheric pressure chemical ionization mass spectrometry (NP-HPLC–APCI-MS) analysis. Each of the long chain alkenones and long chain diols was separated and unambiguously identified from the accurate masses and characteristic fragmentation during multiple stage MS analysis (MS2). Comparison of conventional GC and HPLC–MS methods showed similar results for UK′37 and LDI, respectively, using diverse environmental samples and an Emiliania huxleyi culture. Including the three sea surface temperature (SST) proxies; the NP-HPLC–APCI-MS method in fact allows simultaneous determination of nine paleoenvironmental proxies. The extent to which the ecology of the source organisms (ecological bias) influences lipid composition and thereby the reconstructed temperature values was demonstrated by applying the new method to a sediment core from the Sea of Marmara, covering the last 21kyr BP. Reconstructed SST values differed considerably between the proxies for the Last Glacial Maximum (LGM) and the period of Sapropel S1 formation at ca. 10kyr BP, whereas the trends during the late Holocene were similar. Changes in the composition of alkenone-producing species at the transition from the LGM to the Bølling/Allerød (B/A) were inferred from unreasonably high UK′37-derived SST values (ca. 20°C) during the LGM. We ascribe discrepancies between the reconstructed temperature records during S1 deposition to habitat change, e.g. a different depth due to changes in nutrient availability.

Fragment ion diagnostic strategies for the comprehensive identification of chemical profile of Gui-Zhi-Tang by integrating high-resolution MS, multiple-stage MS and UV information

Gui-Zhi-Tang was considered to be the most famous formula in Shang-Han-Lun, treating 32 symptoms of disease after adding up or cutting down crude drugs. The chemical constituents in Gui-Zhi-Tang were comprehensively studied by rapid resolution liquid chromatography quadrupole time-of-flight mass spectrometry (RRLC-Q-TOF-MS) combined with rapid resolution liquid chromatography – diode array detector – ion trap mass spectrometry (RRLC-DAD-IT-MS) in this work, and a total number of 187 compounds were detected. Systematic diagnostic ion filtering strategies were recommended for flavonoids and oleanane-type triterpenoids, respectively. On the basis of the summarized strategies, compounds in Glycyrrhizae could be easily classified into flavones, isoflavones, flavanones or chalcones with special structures, and triterpene saponins with different sugar moieties. Consequently, among 187 compounds, 144 ones were confirmed or assumed tentatively. In addition, nine potential novel compounds were reported for the first time. This approach provided a rapid method for characterizing the constituents in Gui-Zhi-Tang, and would be also helpful in other TCM formulae analysis.

In vitro phase I metabolism of the depsipeptide enniatin B

The enniatins are a group of more than 20 cyclic depsipeptides from fungi with numerous biological effects. Enniatin B is commonly one of the principal analogues in species of the genus Fusarium, known to have ionophoric, antibiotic and insecticidal activity. In the present study, enniatin B was incubated with rat, dog and human liver microsomes. The compound was extensively metabolised, and 12 biotransformation products (M1-M12) were detected and their structures tentatively identified using a combination of mass spectrometric techniques and chemical derivatisation. Ion trap mass spectrometry, multiple-stage MS(n) fragmentation and high-resolution mass spectrometry were the instrumental backbone for structural determination, while acetylation, methylation and Jones oxidation were useful derivatisation techniques for the localisation of the site of biotransformation. Comparison of mass spectrometric data of the metabolism products with that of enniatin B suggested that M1-M5 are monohydroxylated species, while M8-M12 are the result of multiple oxidations (oxygenation and dehydrogenation). Metabolites M6 and M7 appeared to be enniatin B homologues and are the result of N-demethylation. Our findings show that oxidation and N-demethylation are the principal metabolic pathways in enniatin B phase I metabolism.

Phosphoproteome analysis of human liver tissue by long‐gradient nanoflow LC coupled with multiple stage MS analysis

This article was originally published in Electrophoresis 2010, 31, 1080–1089, DOI 10.1002/elps.200900493

Advances in structure elucidation of small molecules using mass spectrometry.

The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successive chapter discusses mass spectral fragmentation pathways, biotransformation reactions and drug metabolism studies, the mass spectral simulation and generation of in silico mass spectra, expert systems for mass spectral interpretation, and the use of computational chemistry to explain gas-phase phenomena. A single chapter discusses data handling for hyphenated approaches including mass spectral deconvolution for clean mass spectra, cheminformatics approaches and structure retention relationships, and retention index predictions for gas and liquid chromatography. The last section reviews the current state of electronic data sharing of mass spectra and discusses the importance of software development for the advancement of structure elucidation of small molecules.Electronic supplementary materialThe online version of this article (doi:10.1007/s12566-010-0015-9) contains supplementary material, which is available to authorized users.

Identification of rat urinary metabolites of rifabutin using LC–MSn and LC–HR-MS

Rifabutin, an anti-mycobacterial agent, is reported to be extensively metabolized in vivo into more than 20 biotransformation products, with similar profile both in humans and rats. Among the metabolites formed, only seven have been characterized, the remaining are unknown. Hence, the purpose of the present study was to fill this gap by using modern in silico tools combined with advanced liquid chromatography–mass spectrometry (LC–MS) techniques. Initially a comprehensive mass fragmentation pattern for rifabutin was established using Frontier™ 5.1 software coupled with the data collected from multiple-stage MS (MSn), high resolution MS (HR-MS) and hydrogen/deuterium exchange MS (HDE-MS) experiments. The metabolites were then predicted in silico by using different software like MetaSite™, Metabolite Predict™ and MetWorks™. The in silico results were verified through in vivo studies by administration of 20mg/kg drug to rats followed by LC–MS analyses. The urine was collected post-dose at different time intervals, and subjected to sample preparation involving sequentially protein-precipitation, liquid-freeze separation, and solid-phase extraction. The drug and metabolites were separated on an HPLC column followed by LC–MS studies. The difference of accurate masses of the drug and metabolites, and differences in their mass fragmentation pattern helped to assign structures to the metabolites and define the site of change. Here also, in silico detection tools were used, which provided complementary information. Using this strategy, 23 metabolites were detected and identified in rat urine without their isolation. The new sixteen metabolites were monohydroxy (05), dihydroxy (04), N-dealkyl (01), 25-O-desacetyl-27-O-demethyl (01), 25-O-desacetyl-23-O-acetyl (01), 25-O-desacetyl-monohydroxy (01), 27-O-demethyl-monohydroxy (01) and dehydrogenated (02) rifabutin.

Mapping of phosphorylation sites in human MSK1 activated by a novel interaction with MRK‐β

The desire to map reliable phosphorylation signaling network has motivated the development of high-performance techniques. Targeted biochemical studies and updated methods employing MS techniques are most used in mapping the phosphorylation sites and verifying novel interactions of kinases. Previously, we have established a novel method to efficiently facilitate more comprehensive, accurate phosphorylation site mapping of individual phosphoproteins by using combination of multiple stage MS analysis with target-decoy database search against the much smaller targeted database. In this study, by applying this method, we have identified the phosphorylation sites in human MSK1 mitogen- and stress-activated protein kinase 1, which has been proved to be a multi-phosphorylated kinase that plays key roles in various cell functions, activated by a novel interaction with MRK-beta. The results show that this method can find out not only those previously identified active sites in MSK1, but also some novel phosphorylated sites, which correlates with biochemical evidence that, besides p38 and extracellular signal-regulated kinase, MRK-beta could also activate MSK1 through direct interaction. Hence, we conclude this method is sensitive and reliable as expected and it can be further combined with automated screening and biochemical study in efficiently building up a more comprehensive phosphoprotein network.

Phosphoproteome analysis of human liver tissue by long‐gradient nanoflow LC coupled with multiple stage MS analysis

Reversible protein phosphorylation plays a critical role in liver development and function. Comprehensively cataloging the phosphoproteins and their phosphorylation sites in human liver tissue will facilitate the understanding of physiological and pathological mechanisms of liver. Owing to lacking of efficient approach to fractionate phosphopeptides, nanoflow-RPLC with long-gradient elution was applied to reduce the complexity of the phosphopeptides in this study. Two approaches were performed to further improve the coverage of phosphoproteome analysis of human liver tissue. In one approach, ten-replicated long-gradient LC-MS/MS runs were performed to analyze the enriched phosphopeptides, which resulted in the localization of 1080 phosphorylation sites from 495 proteins. In another approach, proteins from liver tissue were first fractionated by SDS-PAGE and then long-gradient LC-MS/MS analysis was performed to analyze the phosphopeptides derived from each fraction, which resulted in the localization of 1786 phosphorylation sites from 911 proteins. The two approaches showed the complementation in phosphoproteome analysis of human liver tissue. Combining the results of the two approaches, identification of 2225 nonredundant phosphorylation sites from 1023 proteins was obtained. The confidence of phosphopeptide identifications was strictly controlled with false discovery rate (FDR) < or = 1% by a MS(2)/MS(3) target-decoy database search approach. Among the localized 2225 phosphorylated sites, as many as 70.07% (1559 phosphorylated sites) were also reported by others, which confirmed the high confidence of the sites determined in this study. Considering the data acquired from low accuracy mass spectrometer and processed by a conservative MS(2)/MS(3) target-decoy approach, the number of localized phosphorylation sites obtained for human liver tissue in this study is quite impressive.

Analysis of novel over‐ and under‐sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MS(n)). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di- and hexasaccharides were analyzed by ESI MS(n). MS(2) on bisulfated 4,5-Delta-HexAGalNAc revealed an additional sulfate ester group at 4,5-Delta-HexA. MS(2) data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5-Delta-HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS(1) mode indicated direct correlation between the sulfate distribution and HexA epimerization. MS(n) performed on ions that, according to mass calculation, correspond to pentasulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)], trisulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)] with IdoA-derived 4,5-Delta-HexA at the nonreducing end, tetrasulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] and monosulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] with GlcA-derived 4,5-Delta-HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over-, regular, and under-sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.

In vitro and in vivo investigation of metabolic fate of rifampicin using an optimized sample preparation approach and modern tools of liquid chromatography–mass spectrometry

Biotransformation products of rifampicin formed in vitro and in vivo were identified using modern tools of liquid chromatography–mass spectrometry (LC–MS). For the same, first the mass fragmentation pattern of rifampicin was delineated by conducting multiple-stage MS (MS n ), high resolution MS (HR-MS) and hydrogen/deuterium exchange MS (HDE-MS) experiments in an electro-spray ionization (ESI) mode. Then in vitro metabolism was investigated by incubating the drug with rat liver S9 fraction and microsomes at pH 7.4 in the presence of NADPH for 2 h at 37 °C. In vivo metabolites were generated by administering the drug to Sprague–Dawley (SD) rats ( n = 5) at a dose of 50 mg kg −1 and collecting blood, urine and feaces at different time points up to 72 h. The samples were processed using an optimized sample preparation approach, involving protein-precipitation using acetonitrile; followed by liquid-freeze separation at −20 °C; and solid-phase extraction of aqueous layer. HPLC conditions were optimized using known standards of rifampicin metabolites or degradants. The extracted and concentrated samples were then subjected to LC-DAD–MS/time-of-flight (LC-DAD–MS/TOF) analyses in both ESI positive and negative modes. The structures of metabolites were elucidated by comparing their accurate mass values and fragmentation patterns with those of rifampicin, use of in silico identification tools and relative retention time matching with the standards. The nature of the metabolites and their relative amounts varied. In total, 21 metabolites were identified, of which many structures were new, not reported till date.

Chemical Fingerprinting of Medicinal Plants “Gui-jiu” by LC-ESI Multiple-Stage MS

The rhizomes or roots of Dysosma versipellis (Hance) M. Cheng, Dysosma pleiantha (Hance) Woodson, and Sinopodophyllum emodi (Wall. Ex Royle) Ying under the same name “Gui-jiu”, were widely used for medicinal purposes. Valid quality control of Gui-jiu is desirable due to the fact that they are highly toxic. In the present paper, an accurate fingerprint method was developed to identify the three species. Liquid chromatography coupled with photodiode array detection and electrospray ionization-multiple stage mass spectrometry were developed to identify the fingerprint components. According to the characteristic fragmentation behavior of known lignans and flavonoids isolated from D. versipellis, a total of 15 constituents in the crude extracts were structurally characterized on the basis of retention time, UV and tandem mass spectrometric analysis. Eight batches of D. versipellis, three batches of D. pleiantha, and two batches of S. emodi were collected from different locations and these representative samples reflected the similar chemical constituent properties. The extracts were separated by a C18 reversed phase LC column, with a gradient solvent system. The proposed method provides a scientific and technical platform to the herbal industry for quality control and safety assurance of herbal preparations that contain this class of poisonous podophyllotoxin-type lignans.

LC–high-resolution multiple stage spectrometric analysis of diuretic compounds: Unusual mass fragmentation pathways

The analysis of diuretic compounds has become of great concern because of their extensive use both in therapy and in illicit treatments (such as masking agents in sport doping and drug abuse). The variety of chemical structures of this class of drugs encouraged the development of new methods and techniques of analysis, especially as regards to acidic compounds. LC/MS has so grown to be the reference technique for this kind of analysis in forensic and anti-doping confirmation purposes. Multiple stage MS permits identification of single drugs with high selectivity, but some unexpected pathways could weaken the entire process. In this work we aim to explain some unusual fragmentation steps using high-resolution MS n . For example, in the case of amiloride an intense product ion in MS 3 analysis generates an apparent loss of 10 Da. Water adduct formation and successive carbon monoxide elimination can explain this uncommon behavior, which was studied using different ion traps. Bendroflumethiazide MS n spectra show instead three successive HF losses, in spite of the presence of a radical site in the parent structure. Homolytic cleavages with radical ion production occur also in the case of protonated positive ion of ethacrynic acid (loss of chlorine radical) showing that such fragmentation behavior is not so rare as generally reported. Different ionization modes were studied and a tentative correlation with acidic-base properties was done. Multiple stage high-resolution mass spectra of positive and negative ions were discussed.

Cell-cycle-dependent phosphorylation of the nuclear pore Nup107-160 subcomplex.

The nuclear pore complex (NPC) mediates macromolecular transport between the nucleus and the cytoplasm. Many NPC proteins (nucleoporins, Nups) are modified by phosphorylation. It is believed that phosphorylation regulates the breakdown of the nuclear envelope at mitosis and the disassembly of the NPC into different subcomplexes. In this study, we examined the cell-cycle-dependent phosphorylation of the Nup107-160 subcomplex, a core building block of the NPC. Using in vivo (32)P labeling in HeLa cells, we found that Nup107, Nup96, and Nup133 are phosphorylated during mitosis. To precisely map the phosphorylation sites within the complex, we used a comprehensive multiple-stage MS approach (MS, MS(2), and MS(3)), establishing that Nup160, Nup133, Nup96, and Nup107 are all targets of phosphorylation. We determined that the phosphorylation sites are clustered mainly at the N-terminal regions of these proteins, which are predicted to be natively disordered. In addition, we determined the cell-cycle dependence of the phosphorylation of these sites by using stable isotope labeling and MS(2) analysis. Measurement of the site-specific phosphorylation ratios between mitotic and G(1) cells led us to conclude that several phosphorylation events of the subcomplex are mainly mitotic. Based on these results and our finding that the entire Nup107-160 subcomplex is stable throughout the cell cycle, we propose that phosphorylation does not affect interactions within the Nup107-160 subcomplex, but regulates the association of the subcomplex with the NPC and other proteins.