Nano-electrospray Ionization Tandem Mass Spectrometry Research Articles

Proteomics-Based Identification of Retinal Protein Networks Impacted by Elevated Intraocular Pressure in the Hypertonic Saline Injection Model of Experimental Glaucoma.

Elevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time.

Malondialdehyde-Induced Post-translational Modifications in Hemoglobin of Smokers by NanoLC-NSI/MS/MS Analysis.

Malondialdehyde (MDA) is the most abundant α,β-unsaturated aldehyde generated from endogenous peroxidation of polyunsaturated fatty acids and is present in cigarette smoke. Post-translational modifications of blood hemoglobin can serve as biomarkers for exposure to chemicals. In this study, two types of MDA-induced modifications, the N-propenal and the dihydropyridine (DHP), were identified at multiple sites in human hemoglobin digest by the high-resolution mass spectrometry. The N-propenal and the DHP types of modification led to the increase of 54.0106 and 134.0368 amu, respectively, at the N-terminal and lysine residues. Among the 21 MDA-modified peptides, 14 with dose-response to MDA concentrations were simultaneously quantified in study subjects by the nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry under selected reaction monitoring (nanoLC-NSI-MS/MS-SRM) without prior enrichment. The results showed that the modifications of the N-propenal-type at α-Lys-11, α-Lys-16, α-Lys-61, β-Lys-8, and β-Lys-17, as well as the DHP-type at the α-N-terminal valine, are significantly higher in hemoglobin isolated from the blood of smokers than in nonsmoking individuals. This is the first report to identify and quantify multiple sites of MDA-induced modifications in human hemoglobin from peripheral blood. Our results suggest that the MDA-derived modifications on hemoglobin might represent valuable biomarkers for MDA-induced protein damage.

Characterization and Quantification of Acrolein-Induced Modifications in Hemoglobin by Mass Spectrometry─Effect of Cigarette Smoking.

Exposure to acrolein, the smallest α, β-unsaturated aldehyde, in humans originates from cigarette smoking and other environmental sources, food cooking, and endogenous lipid peroxidation and metabolism. The protein modification caused by acrolein is associated with various diseases, including cancer, cardiovascular, and neurodegenerative diseases. In this study, acrolein-modified human hemoglobin was reduced by sodium borohydride. Thus, three types of modifications, that is, Schiff base, Michael addition, and formyl-dehydropiperidion adducts, were converted to the corresponding stable adducts, leading to mass increases of 40.0313, 58.0419, and 96.0575 Da, respectively. These stable acrolein-modified hemoglobin peptides were identified by nanoflow liquid chromatography coupled to a high-resolution nanoelectrospray ionization tandem mass spectrometry. Among the 26 different types and sites of modifications, 15 of them showed a dose-dependent increase with increasing concentrations of acrolein. To investigate the role of acrolein-induced modifications in smoking and oral cancer, the 15 dose-responsive acrolein-modified peptides, together with three ethylated peptides previously identified, were quantified in oral cancer patients, healthy smokers, and healthy nonsmokers. The results reveal that the relative extents of the Michael-type adduct at α-Lys-16, α-His-50, and β-Lys-59 are significantly higher in smokers (oral cancer and healthy) than in nonsmokers. Areas under the receiver operating characteristic curve of these peptides range from 0.7500 to 0.9375, indicating the ability to discriminate smokers from nonsmokers. Additionally, these acrolein-modified peptides correlate with three ethylated peptides at the N-termini of α- and β-globin, as well as β-His-77, and with the number of cigarettes smoked per day. Therefore, measuring the reduced Michael adducts at α-Lys-16, α-His-50, and β-Lys-59 of hemoglobin from one drop of blood by this sensitive and specific method may reflect the increase of acrolein exposure due to cigarette smoking.

Rapid identification of bear bile powder from other bile sources using chip-based nano-electrospray ionization tandem mass spectrometry.

Bear bile powder (BBP) is a widely used traditional Chinese medicine (TCM), and bile acids (BAs) are the main active components in BBP. Due to the scarcity of BBP resources, adulterations often occur in the market. Conventional methods to distinguish them are usually complicated and time-consuming. To enhance effectiveness and accuracy, a rapid and rough analytical method is desperately needed. In this study, a rapid strategy using chip-based nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was established to distinguish BBP from other sources of bile powder (BP). In addition, the results were further verified by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry (UPLC/MS). The precision of the chip-based nano-ESI-MS/MS method was validated to be acceptable with relative standard deviation (RSD) <15%. The distinction between BBP and other sources of BP, including common adulterants of pig bile powder (PBP), cattle bile powder (CBP), sheep bile powder (SBP), and chicken bile powder (CkBP), can be observed in the spectra. By using orthogonal partial least-squares discriminant analysis (OPLS-DA), more potential m/z markers were investigated. A BAs-related m/z marker of 498.3 was discovered as a typical differential molecular ion peak and was identified as tauroursodeoxycholic acid (TUDCA) and taurochenodeoxycholic acid (TCDCA) in BBP. The proposed strategy has simple sample pretreatment steps and significantly shortened analysis time. As an emerging technology, chip-based nano-ESI-MS not only provides a reference for the rapid distinction of adulterated Chinese medicines, but also provides some insights into the identification of other chemicals and foods.

Multiple oxidative and advanced oxidative modifications of hemoglobin in gastric cancer patients measured by nanoflow LC-MS/MS

Overproduction of the reactive oxygen, nitrogen, and chlorine species by the immune systems during chronic infection and inflammation can cause structural and functional changes of cellular proteins. The high abundance of hemoglobin in blood makes hemoglobin adducts suitable biomarkers for assessing the damage of these reactive species in the body. In this study, a total of 23 types and sites of modification in human hemoglobin were simultaneously analyzed, including monooxygenation of histidine, tyrosine, methionine, and aspartate, conversion of histidine to aspartate and hydroxyaspartate, as well as chlorination and nitration of tyrosine residues. Hemoglobin was isolated from the blood of the study subjects, digested into peptides, and the extents of these modifications were quantified relative to their parent peptides using nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry under selected reaction monitoring (nanoLC − NSI-MS/MS-SRM). The extents of monooxygenation at β-His-77 and β-Tyr-130, chlorination at α-Tyr-24 and β-Tyr-130, and nitrosylation at α-Tyr-24 were elevated in gastric cancer patients. Conversely, conversion of histidine to aspartate at α-His-20, α-His-50, β-His-2, β-His-143, and monooxygenation at β-His-143 were decreased in gastric cancer patients. The areas under the receiver operating characteristics (AUC of ROC) curve of these ten types and sites of hemoglobin modifications were between 0.7644 and 0.9644. The ratio of conversion of histidine to hydroxyaspartate versus conversion of histidine to aspartate was significantly higher in gastric cancer patients at α-His-20, α-His-50, and β-His-143 (p < 0.05) with AUC of ROC ranging between 0.7689 and 0.9178. To our knowledge, this is the first report of simultaneous measurement of multiple types of oxidative and advanced oxidative hemoglobin modifications in gastric cancer patients. The results revealed elevated levels of oxidative stress-induced protein damage in gastric cancer patients and the potential of using these modifications of hemoglobin as biomarkers for evaluation of oxidative stress in one drop of blood.

Mass Spectrometric Evaluation of β-Cyclodextrins as Potential Hosts for Titanocene Dichloride.

Bent metallocene dichlorides (Cp2MCl2, M = Ti, Mo, Nb, …) have found interest as anti-cancer drugs in order to overcome the drawbacks associated with platinum-based therapeutics. However, they suffer from poor hydrolytic stability at physiological pH. A promising approach to improve their hydrolytic stability is the formation of host-guest complexes with macrocyclic structures, such as cyclodextrins. In this work, we utilized nanoelectrospray ionization tandem mass spectrometry to probe the interaction of titanocene dichloride with β-cyclodextrin. Unlike the non-covalent binding of phenylalanine and oxaliplatin to β-cyclodextrin, the mixture of titanocene and β-cyclodextrin led to signals assigned as [βCD + Cp2Ti–H]+, indicating a covalent character of the interaction. This finding is supported by titanated cyclodextrin fragment ions occurring from collisional activation. Employing di- and trimethylated β-cyclodextrins as hosts enabled the elucidation of the influence of the cyclodextrin hydroxy groups on the interaction with guest structures. Masking of the hydroxy groups was found to impair the covalent interaction and enabling the encapsulation of the guest structure within the hydrophobic cavity of the cyclodextrin. Findings are further supported by breakdown curves obtained by gas-phase dissociation of the various complexes.

Sphingolipid Δ4-desaturation is an important metabolic step for glycosylceramide formation in Physcomitrium patens.

Glycosylceramides are abundant membrane components in vascular plants and are associated with cell differentiation, organogenesis, and protein secretion. Long-chain base (LCB) Δ4-desaturation is an important structural feature for metabolic channeling of sphingolipids into glycosylceramide formation in plants and fungi. In Arabidopsis thaliana, LCB Δ4-unsaturated glycosylceramides are restricted to pollen and floral tissue, indicating that LCB Δ4-desaturation has a less important overall physiological role in A. thaliana. In the bryophyte Physcomitrium patens, LCB Δ4-desaturation is a feature of the most abundant glycosylceramides of the gametophyte generation. Metabolic changes in the P. patens null mutants for the sphingolipid Δ4-desaturase (PpSD4D) and the glycosylceramide synthase (PpGCS), sd4d-1 and gcs-1, were determined by ultra-performance liquid chromatography coupled with nanoelectrospray ionization and triple quadrupole tandem mass spectrometry analysis. sd4d-1 plants lacked unsaturated LCBs and the most abundant glycosylceramides. gcs-1 plants lacked all glycosylceramides and accumulated hydroxyceramides. While sd4d-1 plants mostly resembled wild-type plants, gcs-1 mutants were impaired in growth and development. These results indicate that LCB Δ4-desaturation is a prerequisite for the formation of the most abundant glycosylceramides in P. patens. However, loss of unsaturated LCBs does not affect plant viability, while blockage of glycosylceramide synthesis in gcs-1 plants causes severe plant growth and development defects.

Quantitation of Nitration, Chlorination, and Oxidation in Hemoglobin of Breast Cancer Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry.

Cells are continually exposed to endogenous reactive oxygen, nitrogen, and halogen species, causing damage to biomolecules. Among them, peroxynitrite and hypochlorous acid are not only oxidants but also biological nitrating and chlorinating agents, leading to the formation of 3-nitrotyrosine and 3-chlorotyrosine, respectively, in proteins. 3-Nitrotyrosine has been detected in vivo under several pathophysiological conditions, including breast cancer. Studies show that the concentrations of 3-nitrotyrosine in plasma proteins and platelets were significantly elevated in breast cancer patients. Compared to blood serum albumin, hemoglobin adducts represent biomonitoring of exposure with a longer lifetime. In this study, human hemoglobin was freshly isolated from blood and digested into peptides with trypsin, and the levels of protein adducts, including nitration, nitrosylation, and chlorination of tyrosine as well as oxidation of methionine residues, were simultaneously quantified by nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) with selected reaction monitoring. The results demonstrated that the relative extents of nitration at α-Tyr-42 and β-Tyr-130, nitrosylation at α-Tyr-24, and chlorination at α-Tyr-24 and β-Tyr-130 are significantly higher in globin of 25 breast cancer patients compared to those in 25 healthy subjects (p < 0.05). In particular, nitration at α-Tyr-42 and chlorination at α-Tyr-24 showed the area under the receiver operating characteristic curve of >0.8. While the age of the subjects is correlated with the extents of some of these adducts, the body mass index does not have an effect on any of them. Starting with 1 drop of blood, our results indicated that this highly sensitive and specific nanoLC-NSI/MS/MS is useful in investigating the role of reactive nitrogen oxide species and reactive chlorine species in the etiology of breast cancer.

Dilute-and-shoot-based direct nano-electrospray ionization tandem mass spectrometry as screening methodology for multivitamins in dietary supplement and human urine

IntroductionIn recent years, analytical screening methods for simultaneous detection of multivitamins have gained substantial attention to ensure quality and public confidence in dietary supplements. Even so, few analytical methods have been proposed for simultaneous analysis of multivitamin constituents due to the large divergence in chemical characteristics. ObjectivesIn the present study, the objective was to develop a simple and rapid direct nano-electrospray ionization-tandem mass spectrometry (DI-nano-ESI-MS/MS) method for targeted detection of water soluble vitamins, fat soluble vitamins, amino acids, royal jelly, ginkgo biloba, and ginseng in a dietary supplement. The applicability of dilute-and-shoot-based DI-nano-ESI-MS/MS to analyze the same tested compounds and their related metabolites in clinical samples was also examined. MethodsIntact urine mixed with the ionization solvent was loaded (4-μL aliquot) into a nanospray (NS) capillary of 1-μm tip diameter. The NS capillary was then fitted into an off-line ion source at a distance of 5 mm from MS aperture. The sample was directly injected by applying a voltage of 1.1 kV, producing a numerous of m/z peaks for analysis in mere minutes. ResultsThe DI-nano-ESI-MS/MS method successfully identified almost all dietary supplement components, as well as a plethora of component-related metabolites in clinical samples. In addition, a new merit of the proposed method for the detection of index marker and chemical contaminants as well as subspecies identification was investigated for further quality evaluation of the dietary supplement. ConclusionsThe previous findings illustrated that DI-nano-ESI-MS/MS approach can emerge as a powerful, high throughput, and promising analytical tool for screening and accurate detection of various pharmaceuticals and ingredient in dietary supplements as well as biological fluids.

Direct nano-electrospray ionization tandem mass spectrometry for the quantification and identification of metronidazole in its dosage form and human urine.

A rapid, sensitive and direct nano-electrospray ionization-tandem mass spectrometry (NS-ESI-MS/MS) method, using an offline nanospray (NS) capillary, has been developed and validated for the analysis of metronidazole (MTZ). A mixture of 2 µl MTZ sample solution prepared in an ionization solvent consisting of methanol : water : formic acid in a ratio of 80 : 20 : 0.3, together with 2 µl of an internal standard (IS), 1,3,6-polytyrosine, is loaded into the back of the NS capillary. The NS capillary was fitted into the ion source at a distance of 3 mm between the NS tip and MS orifice. The sample is then analysed and acquired a sustainable signal that allowed for data compilation across various data points for MTZ identification and quantification. The quantification relied on the ratio of the [M + H]+ peaks of MTZ and IS with m/z values of 172.0717 and 182.0812, respectively, while the identification relied on the MS/MS of the precursor ions [M + H]+ of both compounds and their fragments at 128.05 for MTZ and 165.1 and 136.07 for the IS. The NS-ESI-MS/MS method was accurate and precise for the quantification of MTZ over the concentration range from 2.5 to 25 000 ng ml−1. The applicability of the method was confirmed by MTZ analysis in its pharmaceutical dosage form and detection of the analyte in clinical human urine samples without any sample treatment procedure.

Characterization of N-glycosylations in Entamoeba histolytica ubiquitin

Entamoeba histolytica harbors an extensive intracellular distribution of ubiquitin-proteasome systems important for numerous cellular processes. However, glycosylation studies of ubiquitin-proteasome components have not yet been elucidated. Here we report the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE), Nanoelectrospray Ionization-Tandem Mass Spectrometry (NSI-MS), Matrix-Assisted Laser-Desorption time-of-flight Mass Spectrometry (MALDI-TOF MS) and Gas Chromatography–Mass Spectrometry (GC–MS) analysis. To our knowledge, the data presented in this report represents the first structural glycomics analysis of E. histolytica ubiquitin, while most of the reports are performed on whole parasitic glycan profiles. The glycan profile of E. histolytica ubiquitin has high mannose N-glycan structures. The N-linked glycan profile showed fragments from Hex3HexNAc2 to Hex9HexNAc2. Based in our findings and ubiquitin function, we hypothesize that the same ubiquitin Asn-Asp-Ser sequon carries heterogenic glycosylations, at different metabolic pathway stages according to ubiquitin functional requirements. Finally, we propose a set of possible high mannose N-glycan structures that will help to elucidate the ubiquitin biochemical composition and may well represent good targets for anti-amoebic drugs.

A Protein Decomplexation Strategy in Snake Venom Proteomics.

Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).

Trisaminohexyl isocyanurate, a urinary biomarker of HDI isocyanurate exposure

Biological monitoring of occupational exposure to 1,6-hexamethylene diisocyanate (HDI)-containing spray-paints is limited to analysis of metabolites of HDI monomer although polymeric HDI isocyanurate constitutes the predominant inhalation and skin exposure for workers in the automotive paint industry. A novel method using nanoflow ultra-performance liquid chromatography coupled to nano-electrospray ionization tandem mass spectrometry (nano-UPLC-ESI-MS/MS) was developed to quantify trisaminohexyl isocyanurate (TAHI), a hydrolysis product of HDI isocyanurate, in the urine of spray-painters. Analytical and internal standards were synthesized in-house and weighted linear regression calibration curves were generated using spiked control urine from non-exposed persons (0.06–7.98 μg/L; N = 13; w = x−2; r = 0.998). Urine samples collected from 15 exposed workers (N = 111) were subjected to acid hydrolysis and extracted with dichloromethane, then derivatized with acetic anhydride. The derivatized product, trisacetamidohexyl isocyanurate (TAAHI), was analyzed using nano-UPLC-ESI-MS/MS. The protocol was sensitive and specific for analysis of TAHI in the urine of exposed workers with a method detection limit at 0.03 μg/L. TAHI was detected in 33 of 111 urine samples and in 11 of 15 workers. This biomarker for HDI isocyanurate is critical to determine the relative potency and dose-relationships between the monomer and oligomer exposure on the development of diisocyanate induced health effects in future studies.

Screening Serum Differential Proteins for Childhood Asthma at Different Control Levels by Isobaric Tags for Relative and Absolute Quantification-based Proteomic Technology.

Objective To screen serum differential proteins for childhood asthma at different control levels,which provided the basis for the prevention and treatment of childhood asthma. Methods Isobaric tags for relative and absolute quantification,two-dimensional liquid chromatography,nanoelectrospray ionization,and high-resolution tandem mass spectrometry using the hybrid quadrupole time-of-flight platform was used to screen the differential proteins in serum samples from pediatric patients with controlled,partly controlled,or uncontrolled childhood asthma. Differential proteins were validated using enzyme-linked immunosorbent assay (ELISA). Results A total of 260 expressed proteins were identified. Among them 57 differentially expressed proteins were found among the different control levels of childhood asthma (fold<0.8 or fold>1.2). The differentially expressed proteins were involved mainly in 21 biological processes and 8 molecular functions and were located in 17 cellular components. ELISA showed that the serum vitronectin level was significantly higher in controlled group [(573.92±412.43) μg/ml] than in uncontrolled group[(382.27±238.64)]μg/ml (P=0.0399). Conclusion We identified 57 differential proteins for childhood asthma at different control levels,which may be used as potential biological targets for the control of childhood asthma.

Nanocoating cellulose paper based microextraction combined with nanospray mass spectrometry for rapid and facile quantitation of ribonucleosides in human urine

A rapid and facile analytical method for quantification of ribonucleosides in human urine was developed by the combination of nanocoating cellulose paper based microextraction and nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). Cellulose paper used for microextraction was modified by nano-precision deposition of uniform ultrathin zirconia gel film using a sol-gel process. Due to the large surface area of the cellulose paper and the strong affinity between zirconia and the cis-diol compounds, the target analytes were selectively extracted from the complex matrix. Thus, the detection sensitivity was greatly improved. Typically, the nanocoating cellulose paper was immersed into the diluted urine for selective extraction of target analytes, then the extracted analytes were subjected to nESI-MS/MS detection. The whole analytical procedure could be completed within 10min. The method was evaluated by the determination of ribonucleosides (adenosine, cytidine, uridine, guanosine) in urine sample. The signal intensities of the ribonuclesides extracted by the nanocoating cellulose paper were greatly enhanced by 136–459-folds compared with the one of the unmodified cellulose paper based microextraction. The limits of detection (LODs) and the limits of quantification (LOQs) of the four ribonucleosides were in the range of 0.0136–1.258μgL−1 and 0.0454–4.194μgL−1, respectively. The recoveries of the target nucleosides from spiked human urine were in the range of 75.64–103.49% with the relative standard deviations (RSDs) less than 9.36%. The results demonstrate the potential of the proposed method for rapid and facile determination of endogenous ribonucleosides in urine sample.

Analysis of N'-nitrosonornicotine enantiomers in human urine by chiral stationary phase liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry.

We have developed a chiral stationary phase liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method to investigate the enantiomeric composition of low parts per trillion amounts of the carcinogen N'-nitrosonornicotine (NNN) in the urine of cigarette smokers and smokeless tobacco users. (S)-NNN is the major enantiomer in tobacco and is more carcinogenic than (R)-NNN in rats, but no data are available on the enantiomeric composition of NNN in humans. The method used [13C6]NNN as an internal standard and [pyridine-D4]nornicotine to monitor possible artifactual formation of NNN, which was found to be less than 2% of the quantified NNN. The enantiomeric composition of NNN (20.5±27.1fmol/mL urine) in 20 cigarette smokers was 67±5% (S)-NNN while that in 10 smokeless tobacco users (67.1±56.7 fmol/mL urine) was 56±3% (S)-NNN. These results demonstrate that the highly carcinogenic (S)-NNN is the major enantiomer in human urine, and that the enantiomeric composition of NNN in human urine is remarkably similar to that in cigarette smoke and smokeless tobacco. This is the first study to combine chiral stationary phase separations with nanoelectrospray ionization and high resolution tandem mass spectrometry to quantify trace levels of enantiomeric metabolites in human urine.

Ethylene glycol improves electrospray ionization efficiency in bottom-up proteomics.

Liquid chromatography coupled online to nano-electrospray ionization (nESI) tandem mass spectrometry is the analytical workhorse in the field of proteome research. Dimethyl sulfoxide (DMSO) was recently shown to improve nESI efficiency by a factor of three to ten thus improving the sensitivity and coverage of proteomic experiments. However, relatively few investigations into which solvent additives promote nESI response have been performed at a proteomic scale. Here, we systematically evaluated the concept by screening about 30 compounds with various physico-chemical properties. Detailed further analysis showed that ethylene glycol performed similarly to DMSO and the results indicate that enhancing the nESI response of peptides by simple solvent additives is a valid and promising approach. Ethylene glycol may serve as a viable alternative to DMSO in applications where DMSO has disadvantages. In keeping with nESI theory, the key properties of an effective solvent additive for proteomic applications are a boiling point higher than water, low surface tension, and preferably high polarity for reversed phase LC-MS/MS applications. Graphical Abstract Ethylene glycol substantially improves peptide ionization.

Comparative analyses of secreted proteins from the phytopathogenic fungus Verticillium dahliae in response to nitrogen starvation

The soilborne fungus Verticillium dahliae is the major pathogen that causes the verticillium wilt disease of plants, which leads to huge economic loss worldwide. At the early stage of infection, growth of the pathogen is subject to the nutrition stress of limited nitrogen. To investigate the secreted pathogenic proteins that play indispensable roles during invasion at this stage, we compared the profiles of secreted proteins of V. dahliae under nitrogen starvation and normal conditions by using in-gel and in-solution digestion combined with liquid chromatography–nano-electrospray ionization tandem mass spectrometry (LC–nanoESI-MS). In total, we identified 212 proteins from the supernatant of liquid medium, including 109 putative secreted proteins. Comparative analysis indicated that the expression of 76 proteins was induced, whereas that of 9 proteins was suppressed under nitrogen starvation. Notably, 24 proteins are constitutively expressed. Further bioinformatic exploration enabled us to classify the stress-induced proteins into seven functional groups: cell wall degradation (10.5%), reactive oxygen species (ROS) scavenging and stress response (11.8%), lipid effectors (5.3%), protein metabolism (21.1%), carbohydrate metabolism (15.8%), electron–proton transport and energy metabolism (14.5%), and other (21.0%). In addition, most stress-suppressed proteins are involved in the cell-wall remodeling. Taken together, our analyses provide insights into the pathogenesis of V. dahliae and might give hints for the development of novel strategy against the verticillium wilt disease.

A Comparison of Patient Matched Meibum and Tear Lipidomes

To quantify the molecular lipid composition of patient-matched tear and meibum samples and compare tear and meibum lipid molecular profiles. Lipids were extracted from tears and meibum by bi-phasic methods using 10:3 tert-butyl methyl ether:methanol, washed with aqueous ammonium acetate, and analyzed by chip-based nanoelectrospray ionization tandem mass spectrometry. Targeted precursor ion and neutral loss scans identified individual molecular lipids and quantification was obtained by comparison to internal standards in each lipid class. Two hundred and thirty-six lipid species were identified and quantified from nine lipid classes comprised of cholesterol esters, wax esters, (O-acyl)-ω-hydroxy fatty acids, triacylglycerols, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine. With the exception of phospholipids, lipid molecular profiles were strikingly similar between tears and meibum. Comparisons between tears and meibum indicate that meibum is likely to supply the majority of lipids in the tear film lipid layer. However, the observed higher mole ratio of phospholipid in tears shows that analysis of meibum alone does not provide a complete understanding of the tear film lipid composition.

Wax ester profiling of seed oil by nano-electrospray ionization tandem mass spectrometry

BackgroundWax esters are highly hydrophobic neutral lipids that are major constituents of the cutin and suberin layer. Moreover they have favorable properties as a commodity for industrial applications. Through transgenic expression of wax ester biosynthetic genes in oilseed crops, it is possible to achieve high level accumulation of defined wax ester compositions within the seed oil to provide a sustainable source for such high value lipids. The fatty alcohol moiety of the wax esters is formed from plant-endogenous acyl-CoAs by the action of fatty acyl reductases (FAR). In a second step the fatty alcohol is condensed with acyl-CoA by a wax synthase (WS) to form a wax ester. In order to evaluate the specificity of wax ester biosynthesis, analytical methods are needed that provide detailed wax ester profiles from complex lipid extracts.ResultsWe present a direct infusion ESI-tandem MS method that allows the semi-quantitative determination of wax ester compositions from complex lipid mixtures covering 784 even chain molecular species. The definition of calibration prototype groups that combine wax esters according to their fragmentation behavior enables fast quantitative analysis by applying multiple reaction monitoring. This provides a tool to analyze wax layer composition or determine whether seeds accumulate a desired wax ester profile. Besides the profiling method, we provide general information on wax ester analysis by the systematic definition of wax ester prototypes according to their collision-induced dissociation spectra. We applied the developed method for wax ester profiling of the well characterized jojoba seed oil and compared the profile with wax ester-accumulating Arabidopsis thaliana expressing the wax ester biosynthetic genes MaFAR and ScWS.ConclusionsWe developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.