Electrospray Ionization Ion-trap Tandem Mass Research Articles

Determination of terminal glycan and total monosaccharide profiles of reelin glycoprotein in SH-SY5Y neuroblastoma cell line by lectin blotting and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry system

Reelin (400 kDa) is an extracellular matrix glycoprotein that is a key regulator of the many significant biological processes including the brain formation, cell aggregation, and dendrite formation. The glycosylation contributes to the nature of the protein through folding, localization and trafficking, solubility, antigenicity, biological activity, and half-life. Although reelin is to be known as a glycoprotein, the knowledge of its glycosylation is very limited. In this study, we aimed to characterize the terminal glycan profile of reelin by lectin blotting and monosaccharide analysis of glycan chains by capillary liquid chromatography electrospray ionization ion trap tandem mass spectrometry (CapLC-ESI-MS/MS) in SH-SY5Y neuroblastoma cell line. According to our results, reelin was detected in different protein fragments (310, 250, and 85 kDa) in addition to full-length form (400 kDa) in the cell line. The reelin glycoprotein was found to carry the β-N-Acetylglucosamine, α-Mannose, β-Galactose, and α-2,3 and α2,6 linked sialic acids by lectin blotting. Nevertheless, these terminal monosaccharides were found in different intensity according to reelin fragments. Besides, we purified a reelin fragment (250 kDa), and we analyzed it for their monosaccharide by CapLC-ESI-MS/MS. We found that reelin contained five types of monosaccharides, which were consisted of N-Acetylgalactosamine, N-Acetylglucosamine, Galactose, Glucose, Mannose and Sialic acid, from high to low abundance respectively. The present results provide a valuable guide for biochemical, genetic, and glycobiology based further experiments about reelin glycosylation in cancer perspective.

Pomegranate Fruit and Juice (cv. Mollar), Rich in Ellagitannins and Anthocyanins, Also Provide a Significant Content of a Wide Range of Proanthocyanidins.

Proanthocyanidins (PAs) were characterized in husk, membranes, arils, and seeds of 'Mollar de Elche' pomegranate cultivar using liquid chromatography-electrospray ionization-ion-trap tandem mass spectrometry after acid catalysis in the presence of phloroglucinol (phloroglucinolysis). PAs were also evaluated in commercial juice and in the juice made in the laboratory by pressing fresh arils. Pomegranate PAs were composed of a very rich mixture of flavan-3-ols: catechin, epicatechin, gallocatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate. Catechins and gallocatechins were the most abundant units, while galloylated flavanols were detected at lower concentrations. The highest PA content was found in husk (1840.5 μg/g), followed by seeds (316.2 μg/g) and membranes (51.4 μg/g). With regard to the pomegranate edible parts, arils showed concentrations of 34.4 μg/g and pressed aril juice of 21.3 mg/L, lower than those found in other traditional PA-containing food and beverages, such as apple, grapes, chocolate, red wine, or tea. Higher concentrations similar to those found in red wine were observed in commercial whole fruit pressed pomegranate juice (146.9 mg/L). These PA concentrations represented between 0.1 and 7% of the total polyphenol content in the different pomegranate samples, which were richer in ellagitannins and anthocyanins. The mean degree of polymerization (1.2-2.1) revealed that PAs in Mollar pomegranate samples are constituted mainly of monomers and dimers, which may influence in better absorption and bioavailability of these compounds.

Profiling N-glycans of the egg jelly coat of the sea urchin Paracentrotus lividus by MALDI-TOF mass spectrometry and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry systems.

Sea urchin eggs are surrounded by a carbohydrate-rich layer, termed the jelly coat, that consists of polysaccharides and glycoproteins. In the present study, we describe two mass spectrometric strategies to characterize the N-glycosylation of the Paracentrotus lividus egg jelly coat, which has an alecithal-type extracellular matrix like mammalian eggs. Egg jelly was isolated, lyophilized, and dialyzed, followed by peptide N-glycosidase F (PNGase-F) treatment to release N-glycans from their protein chain. These N-glycans were then derivatized by permethylation reaction, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectroscopy (CapLC ESI-Ion trap-MS/MS). N-glycans in the egg jelly coat glycoproteins were indicated by sodiated molecules at m/z 1579.8, 1783.9, 1988.0, 2192.0, and 2397.1 for permethylated oligosaccharides on MALDI-TOF MS. Fragmentation and structural characterization of these oligosaccharides were performed by ESI-Ion trap MS/MS. Then, MALDI-TOF-MS and ESI-Ion trap-MS/MS spectra were interpreted using the GlycoWorkbench software suite, a tool for building, displaying, and profiling glycan masses, to identify the original oligosaccharide structures. The oligosaccharides of the isolated egg jelly coat were mainly of the high mannose type.

“Aroeira” (Myracrodruon urundeuva) methanol extract: the relationship between chemical compounds and cellular effects

Context: “Aroeira” [Myracrodruon urundeuva Allemão (Anacardiaceae)] is a tree whose leaves have been studied for therapeutic purposes in medicine and dentistry.Objective: The study chemically identifies the leaf extract of aroeira and determines its effect on human gingival fibroblasts.Materials and methods: An 80% methanol leave extract was obtained by maceration and chemically identified through flow-injection analysis–electrospray ionization–ion trap–tandem mass spectrometry (FIA–ESI–IT–MSn). Cytotoxicity of the aroeira’s methanol extract was evaluated in lineage of fibroblasts. Adherent cells were treated with different concentrations of aroeira’s methanol extract in the medium: 0.1, 1, 10, 100 and 1000 μg/mL. Control cells were cultivated in the medium only. Analyses were done at 24, 48, 72 and 96 h of culture by neutral red assay; and at 24, 48 and 96 h by crystal violet assay.Results: FIA–ESI–IT–MS analysis determined the presence of compounds, for the first time in the species: quercetin-O-glucuronide and quercetin-O-deoxyhexose-O-glucose in the extract. On one hand, neutral red and crystal violet assay showed a reduction (to 50% up until 100%) of cellular viability of groups of 100 and 1000 μg/mL compared with control at 96 h (p < 0.05). On the other hand, lower concentrations (0.1; 1 and 10 μg/mL) of the extract were similar to that of the control at 96 h (p < 0.05), in general.Conclusions: In view of the results, we can conclude that the extract of aroeira presents tannins and flavonoids. Furthermore, the extract is capable of modulating the viability of human gingival fibroblasts according to its concentration.

Solid Phase Extraction of Phospholipids from Brazil Nut (Bertholletia excelsa) and Their Characterization by Mass Spectrometry Analysis

The Brazil nut (Bertholletia excelsa - Lecythidaceae) is considered a product with high economic value, being a food widely appreciated for its nutritional qualities. Although previous studies have reported the biochemical composition of Brazil nut oil, the knowledge regarding the phospholipid composition exhibits a disagreement: the composition of fatty acids present in the structures of phospholipids is reported as being different from the composition of the free fatty acids present in the oil. In this work, solid phase extraction (SPE) was employed to provide a fast extraction of the phospholipids from Brazil nuts, in order to compare the phospholipid profile of the in nature nuts and their fatty acids precursor present in the oil. The major phospholipids were characterized by mass spectrometry approach. Their fragmentation pattern through direct infusion electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MS2) proved to be useful to unequivocal characterization of these substances. High resolution (HR) experiments through ESI using a quadruple time of flight mass spectrometry (QTOF) system were performed to reinforce the identifications.

Collision-Induced Dissociation of 40 Flavonoid Aglycones and Differentiation of the Common Flavonoid Subtypes Using Electrospray Ionization Ion-Trap Tandem Mass Spectrometry and Quadrupole Time-of-Flight Mass Spectrometry

Much Literature is available with respect to the collision-induced dissociation (CID) of different subtypes of flavonoid aglycones. However, a simultaneous comparison study of the common flavonoid subtypes under unified conditions is rarely reported, which is of crucial significance for their rapid structural elucidation. This study depicted the negative CID of 40 free flavonoids (comprising nine different subtypes) utilizing electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MSn). High-accuracy quadrupole time-of-flight mass spectrometry was employed to confirm the chemical formula of proposed key fragments. Specific fragmentation features were discovered among different flavonoid subtypes in terms of the essential collision energy, base peak, RDA fragmentation, and diversity of fragmentation pathways: CID of flavanones, chalcones and flavanonols was in need of lower Levels of collision energy [<40%) and exhibited simplified fragmentation pathways; overriding neutral loss of CO2 was detected for flavones, aurones and xanthones, while isoflavones preferred neutral loss of CO; 1,2A- and 1,3A- fragments were respectively dominant for flavonols and flavanones/chalcones, white homoisoflavones underwent specific radical elimination of ring B or methylene-ring B. These differentiated fragmentation characteristics integrating UV absorption resulted in the establishment of a decision tree to characterize and distinguish the common flavonoid subtypes. This study firstly revealed remarkable differentiations in the negative CID behavior of nine common flavonoid aglycone subtypes and the proposed determination strategy would much benefit the screening and rapid characterization of flavonoids from complicated matrixes by tandem mass spectrometry.

Electrospray ionization tandem mass spectrometry of protonated and alkali‐cationized Boc‐N‐protected hybrid peptides containing repeats of D‐Ala‐APyC and APyC‐D‐Ala: Formation of [bn–1 + OCH3 + Na]+ and [bn–1 + OH + Na]+ ions

Differentiation and structural characterization of positional isomers of non-natural amino acid hybrid peptides by using electrospray ionization tandem mass spectrometry (ESI-MS(n) ) is desirable because of their fundamental importance from the view point of peptide mass spectrometry and also of their increasing importance in the area of research towards biomedical and material applications; hence, the present study is undertaken. Electrospray ionization ion-trap tandem mass spectrometry (ESI-MS(n)) was used to characterize and differentiate three pairs of positional isomers of Boc-N-protected hybrid peptides containing repeats of D-Ala-APyC and APyC-D-Ala (D-Ala = D-alanine and APyC = trans-3-aminopyran-2-carboxylic acid). ESI-MS(n) spectra of protonated and alkali-cationized positional isomeric peptides display characteristic fragmentation involving the peptide backbone, the Boc group, and the side chain. It is observed that abundant rearrangement ions [b(n-1) + OCH(3) + Na](+) or [b(n-1) + OH + Na](+) are formed when D-Ala is present at C-terminus and the presence of APyC at the C-terminus inhibits the formation of rearrangement ions. In addition, abundant b(n-1)(+) ions are formed, presumably with stable oxazolone structures, when the C-terminus of b(n-1) (+) ions possessed D-Ala. The present study demonstrates that ESI tandem mass spectrometry is very useful for differentiating positional isomers of hybrid peptides containing D-Ala and APyC amino acids. While the protonated peptides give rise to characteristic sequencing ions, the cationized peptides produce additional rearrangement ions ([b(n-1) + OCH(3) + Na](+) and [b(n-1) + OH + Na](+)) which helps distinguish between the presence of D-Ala and APyC amino acids at the C-terminus.

Determination of esculentoside A in dog plasma by LC–MS/MS method: Application to pre-clinical pharmacokinetics

A simple and rapid high performance liquid chromatography electrospray ionization ion-trap tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantitative determination of esculentoside A (EsA) in dog plasma using ginsenoside Rg1 as the internal standard (IS). After liquid–liquid extraction (LLE) with n-butanol, the analyte and IS were separated on a Diamonsil C18 (2.1mm×50mm, 3μm) column with the mobile phase of methanol–water containing 0.1% acetic acid (70:30, v/v) at a flow rate of 0.2ml/min. An ion trap mass spectrometer equipped with an electrospray ionization source performed in selected reaction monitoring (SRM) mode was used as the detector. The precursor-product ion transitions were m/z 849.3 [M+Na]+→m/z 805.3 for EsA and m/z 823.3 [M+Na]+→m/z 643.3 for IS. The total chromatographic run time was 5min. The method was sensitive enough with a lower limit of quantitation (LLOQ) of 5ng/ml and had a good linearity (r2>0.997) over the linear range of 5–500ng/ml. The mean extraction recovery of EsA from spiked plasma samples was over 75%. The intra- and inter-precisions were no more than 8.8% and accuracies were within the range of −4.6 to 8.7%. All the validated data were within the accepted criteria as stated in the FDA bioanalytical method validation guideline. The developed method was suitable for the quantification of EsA and successfully applied to the pharmacokinetic study of EsA after an oral administration to beagle dogs.

Use of electrospray ionization ion‐trap tandem mass spectrometry and principal component analysis to directly distinguish monosaccharides

Carbohydrates are good source of drugs and play important roles in metabolism processes and cellular interactions in organisms. Distinguishing monosaccharide isomers in saccharide derivates is an important and elementary work in investigating saccharides. It is important to develop a fast, simple and direct method for this purpose, which is described in this study. Stock solutions of monosaccharide with a concentration of 400 μM and sodium chloride at a concentration of 10 μM were made in water/methanol (50:50, v/v). The samples were subjected to electrospray ionization ion-trap tandem mass spectrometry (ESI-MS) and the detected [2M + Na - H(2)O](+) ions were further investigated by tandem mass spectrometry (MS/MS), followed by applying principal component analysis (PCA) on the obtained MS/MS data sets. The MS/MS spectra of the [2M + Na - H(2)O](+) ions at m/z 365 for hexoses and m/z 305 for pentoses yielded unambiguous fragment patterns, while rhamnose can be directly identified by its ESI-MS [M + Na](+) ion at m/z 187. PCA showed clustering of MS/MS data of identical monosaccharide samples obtained from different experiments. By using this method, the monosaccharide in daucosterol hydrolysate was successfully identified. A new strategy was developed for differentiation of the monosaccharides using ESI-MS/MS and PCA. In MS/MS spectra, the [2M + Na - H(2)O](+) ions yielded unambiguous distinction. PCA of the archived MS/MS data sets was applied to demonstrate the spatial resolution of the studied samples. This method presented a simple and reliable way for distinguishing monosaccharides by ESI-MS/MS.

Structure revision of hupehensis saponin F and G and characterization of new trace triterpenoid saponins from Anemone hupehensis by tandem electrospray ionization mass spectrometry

Electrospray ionization ion-trap tandem mass spectrometry (ESI-MSn) was first employed for reinvestigating the structures of hupehensis saponin F and G previously isolated from Anemone hupehensis in our lab. Hupehensis saponin G was determined to contain one more trisaccharide unit (Rha-(1→4)-Glc-(1→6)-Glc-), not a glucose residue, than saponin F based on their molecular weights deduced from their [M+Na]+ ions in ESI-MS spectra. The 2,4A4α-ion at m/z 551.3 formed by retro-Diels–Alder (RDA) rearrangement in positive mode illustrated that the C-28 sugar chains of the two saponins were composed of trisaccharide repeating moieties with (1→4) linkages rather than (1→3) linkages. The interpretation of 2D-NMR spectra of the two compounds also confirmed the results obtained by ESI-MSn. Moreover, from the water soluble part of A. hupehensis, two novel triterpene saponins were tentatively characterized to contain 4 and 5 (1→4)-linked above trisaccharide repeating moieties at C-28 position according to their ESI-MSn behaviors, respectively.

Characterization of Phospholipid Molecular Species in the Edible Parts of Bony Fish and Shellfish

The phospholipid molecular species of freshwater (pangasius, Nile perch, trout), marine fish fillets (horse mackerel, European hake, common sole, European anchovy, European pilchard, Atlantic mackerel) and the edible muscle foot of bivalves (clam, mussel, oyster) commonly available in the Italian market during spring and summer were characterized by means of normal-phase high performance liquid chromatography coupled online with positive electrospray ionization ion-trap tandem mass spectrometry. From principal component analysis (PCA), it was observed that the total fatty acid profile was not suitable to differentiate among the shellfish genera. The fatty acid molecular combinations of phosphatidylcholine, the main phospholipid class, as well as phosphatidylinositol and phosphatidylethanolamine allowed for the differentiation of shellfish from the bony fishes. Phosphatidylserine and phosphatidylethanolamine plasmalogen profile allowed for the discrimination of each bony fish or shellfish genus since PS and pPE classes included a large number of fatty acid combinations that were specific for a fish genus or group.

Characterization of Gallotannins from Astronium Species by Flow Injection Analysis-Electrospray Ionization-Ion Traptandem Mass Spectrometry and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The species Astronium urundeuva (Allemao) Engl. and Astronium graveolens Jacq., which are used in Brazilian folk medicine to treat allergies, inflammation, diarrhea and ulcers, were investigated for their composition. The aim of this study was to define a rapid and reliable analytical approach, based on the flow-injection analysis-electrospray ionization-ion trap-tandem mass spectrometry (FIA-ESI-IT-MS-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF-MS), to investigate the full range of hydrolyzable tannins present in the extracts of these Astronium species. The MALDI-ToF-MS analysis allowed us to ascertain the presence of hydrolysable tannins in both Astronium species as a series of gallotannins with degrees of polymerization of 7 to 13 galloyl units. Moreover, the analysis by FIA-ESI-IT-MS-MS, as well as confirming this result and chemically defining gallotannins as galloylglucose compounds, highlighted the presence of further classes of hydrolysable tannins, such as hexahydrodiphenoyl esters of glucose and some gallic acid derivatives, providing information about their structure by a careful study of their fragmentation patterns. Finally, the evaluation of the number of positional isomers of gallotannins occurring in both Astronium species was obtained by high-performance liquid chromatography-electrospray ionization-ion trap mass spectrometry (HPLC/ESI-IT-MS). This is the first mass spectrometric evidence relating to the existence of gallotannins in Astronium genus.

Characterization of Nα‐Fmoc‐protected dipeptide isomers by electrospray ionization tandem mass spectrometry (ESI‐MSn): effect of protecting group on fragmentation of dipeptides

A series of positional isomeric pairs of Fmoc-protected dipeptides, Fmoc-Gly-Xxx-OY/Fmoc-Xxx-Gly-OY (Xxx=Ala, Val, Leu, Phe) and Fmoc-Ala-Xxx-OY/Fmoc-Xxx-Ala-OY (Xxx=Leu, Phe) (Fmoc=[(9-fluorenylmethyl)oxy]carbonyl) and Y=CH(3)/H), have been characterized and differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MS(n)). In contrast to the behavior of reported unprotected dipeptide isomers which mainly produce y(1)(+) and/or a(1)(+) ions, the protonated Fmoc-Xxx-Gly-OY, Fmoc-Ala-Xxx-OY and Fmoc-Xxx-Ala-OY yield significant b(1)(+) ions. These ions are formed, presumably with stable protonated aziridinone structures. However, the peptides with Gly- at the N-terminus do not form b(1)(+) ions. The [M+H](+) ions of all the peptides undergo a McLafferty-type rearrangement followed by loss of CO(2) to form [M+H-Fmoc+H](+). The MS(3) collision-induced dissociation (CID) of these ions helps distinguish the pairs of isomeric dipeptides studied in this work. Further, negative ion MS(3) CID has also been found to be useful for differentiating these isomeric peptide acids. The MS(3) of [M-H-Fmoc+H](-) of isomeric peptide acids produce c(1)(-), z(1)(-) and y(1)(-) ions. Thus the present study of Fmoc-protected peptides provides additional information on mass spectral characterization of the dipeptides and distinguishes the positional isomers.

Differentiation of Positional Isomers of Hybrid Peptides Containing Repeats of β-Nucleoside Derived Amino Acid (β-Nda-) and L-Amino Acids by Positive and Negative Ion Electrospray Ionization Tandem Mass Spectrometry (ESI-MSn)

A new class of positional isomeric pairs of -Boc protected oligopeptides comprised of alternating nucleoside derived β-amino acid (β-Nda-) and L-amino acid residues (alanine, valine, and phenylalanine) have been differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-MS(n)). The protonated dipeptide positional isomers with β-Nda- at the N-terminus lose CH(3)OH, NH(3), and C(2)H(4)O(2), whereas these processes are absent for the peptides with L-amino acids at the N-terminus. Instead, the presence of L-amino acids at the N-terminus results in characteristic retro-Mannich reaction involving elimination of imine. A good correlation has been observed between the conformational structure of the peptides and the abundance of y(n)(+) and b(n)(+) ions in MS(n) spectra. In the case of tetrapeptide isomers that are reported to form helical structures in solution phase, no y(n)(+) and b(n)(+) ions are observed when the corresponding amide -NH- participates in the helical structures. In contrast, significant y(n)(+) and b(n)(+) ions are formed when the amide -NH- is not involved in the H-bonding. In the case of tetra- and hexapeptides, it is observed that abundant b(n)(+) ions are formed, presumably with stable oxazolone structures when the C-terminus of the b(n)(+) ions possessed L-amino acid and the β-Nda- at the C-terminus appears to prevent the cyclization process leading to the absence of corresponding b(n)(+) ions.

High performance liquid chromatography-tandem mass spectrometry of phospholipid molecular species in eggs from hens fed diets enriched in seal blubber oil

The total lipid fraction of eggs from hens fed diets enriched in seal blubber oil (1.25–5.0% SBO) was directly analysed with normal-phase high performance liquid chromatography coupled on-line with electrospray ionization ion-trap tandem mass spectrometry (HPLC-ESI-MS–MS) for the identification of the molecular species of phospholipids (PLs). The species of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were all detected as the [M − H] − ions. The phosphatidylcholine (PC), sphingomyelin (Sph) and lysophosphatidylcholine (LPC) classes, were detected as formate adducts [M + HCOO] −. Tandem MS of PE and PI showed the loss of the carboxylate anions, and, for PI, also the loss of water and inositol. Product ion spectrum of PC, LPC and Sph contained only the [M − CH 3] − ion fragment. Feeding different levels of SBO for 5 weeks resulted in a significant increase of PE, PC and PI molecular species carrying eicosapentaenoic acid (C 20:5 ω3 , EPA), docosapentaenoic acid (C 22:5 ω3 , DPA) and docosahexaenoic acid (C 22:6 ω3 , DHA), but not Sph nor LPC. The highest increase of the ω3/ω6 ratio occurred for PE and PC. On the contrary, PI was less affected by the increase of SBO in the diet.

ESI-MS quantitation of increased sphingomyelin in Niemann-Pick disease type B HDL

HDLs have been proposed to have antiatherogenic properties because of their role in reverse cholesterol transport as lipid acceptors. To elucidate the phospholipid profile of these particles, we used electrospray ionization mass spectrometry to examine the phosphatidylcholine (PC) and sphingomyelin (SM) composition of HDLs purified from plasma and nascently generated in vitro from fibroblasts. We also quantitatively compared the phospholipids present in these lipoproteins between normal and Niemann-Pick disease type B (NPD-B) subjects characterized by sphingomyelinase (SMase) deficiency. We demonstrated that plasma HDLs from NPD-B were significantly enriched in SM by an average of 28%, particularly the palmitoyl SM (with an increase of 95%), which accounted for approximately 25-44% of total SM molecular species. Similarly, we observed an increase of approximately 63% in total SM levels in nascent HDLs prepared from NPD-B fibroblasts. Although PC levels in nascent HDLs were comparable between control and NPD-B cells, there was a 95% increase in total PC levels similar to that of SM in plasma HDLs extracted from NPD-B subjects. These data provide insight into the structure of HDLs and identify potential new roles for SMase in lipoprotein metabolism.

Stereoselective Discrimination and Quantification of Arginine and N-Blocked Arginine Enantiomers by Formation and Dissociation of Calcium-Mediated Diastereomeric Trimer Complexes with a Chiral Reference Compound Using Electrospray Ionization-Ion Trap Tandem Mass Spectrometry

Chiral resolution of arginine (Arg) and Arg derivatives is demonstrated using electrospray ionization-tandem mass spectrometry (ESI-MS). Calcium ion (Ca(II))-mediated trimeric clusters are generated, which incorporate the analyte of interest and an enantiomerically pure reference molecule of similar metal ion affinity. Two methods, one based on the measurement of a competitive-dissociation-based branching ratio (R(chir)) by the kinetic method (KM) and one based on the measurement of a chiral recognition ratio (CR) by a similar method, are compared. Incorporating N-blocked Arg derivatives (Z-Arg and Boc-Arg) as chiral references provides chiral resolution greater than that previously reported for Arg enantiomers. In a reciprocal manner, pure Arg enantiomers can be used as references for discriminating enantiomers of these N-blocked Arg derivatives. Condensed-phase and gas-phase Ca(II) ion affinity relative to Arg is also addressed qualitatively for other acidic, basic, and neutral amino acids. In some cases, when only one offspring ion is observed (insufficient for KM analysis), the CR method can be applied as an alternative to obtain a measurable stereoselectivity value for the system. The results of these experiments demonstrate the applicability of, and the difference between, the KM and the CR method for improved quantitative analysis of enantiomeric excess for Arg.

Fractionation of peptides and identification of proteins from Saccharomyces cerevisiae in proteomics with the use of reversed-phase capillary liquid chromatography and pI-based approach

The aim of the work was to explore the identification of proteins from Saccharomyces cerevisiae using combined capillary reversed-phase liquid chromatography (RPLC) and in-solution isoelectric focusing (sIEF) for fractionation of peptides prior to mass spectrometry analysis. That method was proved to be the alternative separation method for complex mixtures of protein tryptic digests in proteomics. Analysis of the identification of peptides was performed with the use of electrospray ionization-ion trap tandem mass spectrometry (ESI-IT-MS/MS). First, the sIEF fractionation was carried out prior to separation and mass spectrometry identification by nano-LC/ESI-MS/MS instrument. The proposed approach based on sIEF and nano-LC/ESI-MS/MS analysis was proved to be an efficient and accurate alternative fractionation method of complex protein digests and can be considered as the useful tool for identification of proteins. Moreover, analytical information from that approach can be considered as the additional source of database matching constraint and can be valuable tool for analytical and bioinformatics studies of peptides fractionation in proteomics. Based on the MS/MS results obtained with ESI-IT-MS/MS instrument, 851 proteins from S. cerevisiae were identified. However, after careful analysis of the data reduction in number of proteins to 126 was obtained. Those results are discussed and interpreted in the view of the evaluation method used.

Determination of γ-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 m M nicotinic acid (compound for indirect detection), 3 m M spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ, respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→ m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ.

Direct analysis of microcystins by microbore liquid chromatography electrospray ionization ion-trap tandem mass spectrometry

Microcystins are a group of structurally similar cyclic heptapeptide hepatotoxins and tumor promoters, produced by cyanobacteria. A microbore liquid chromatography electrospray ionization ion-trap mass spectrometry (LC-ESI-ITMS) method has been developed which is capable of separating and detecting trace amounts of microcystin variants in environmental samples. Extracted water sample was loaded onto a LC trapping column and, using a column switching technique, the compounds of interest were back-flushed onto a 1-mm LC column. Structural elucidation was achieved using ion-trap with tandem mass spectrometry in the data dependent scan mode. Collision-induced dissociation to MS 3 allowed tentative identification of these cyclic peptides. Full-scan LC-ESI-MS mass spectrum was obtained when 250 pg of the authentic compound was injected onto the HPLC column, which represents the detection limit for microcystin-LR. This study demonstrated that LC-ESI-ITMS is a reliable and sensitive technique for analysing trace levels of microcystins.