Increased Ionization Efficiency Research Articles

Strong field non-Franck–Condon ionization of H$$_2$$: a semi-classical analysis

Single ionization of H $$_2$$ molecules exposed to strong and short laser pulses is investigated by a semi-classical method. Three laser characteristics are considered: (i) The carrier-wave frequency corresponds to wavelengths covering and bridging the two ionization regimes: From tunnel ionization (TI) at 800 nm to multiphoton ionization (MPI) at 266 nm. (ii) Values of the peak intensity are chosen within a window to eliminate competing double ionization processes. (iii) Particular attention is paid to the polarization of the laser field, which can be linearly or circularly polarized. The results and their interpretation concern two observables, namely the end-of-pulse total ionization probability and vibrational distribution generated in the cation H $$_2^+$$ . The most prominent findings are an increased ionization efficiency in circular polarization and a vibrational distribution of the cation that favors lower-lying levels than those that would be populated in a vertical (Franck–Condon) ionization, leading to non-Franck–Condon distributions, both in linear and circular polarizations.

Quasi-continuous infrared matrix-assisted laser desorption electrospray ionization source coupled to a quadrupole time-of-flight mass spectrometer for direct analysis from well plates.

High-throughput screening (HTS) is a technique mostly used by pharmaceutical companies to rapidly screen multiple libraries of compounds to find drug hits with biological or pharmaceutical activity. Mass spectrometry (MS) has become a popular option for HTS given that it can simultaneously resolve hundreds to thousands of compounds without additional chemical derivatization. For this application, it is convenient to do direct analysis from well plates. Herein, we present the development of an infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled directly to an Agilent 6545 for direct analysis from well plates. The source is coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer to take advantage of the high acquisition rates without sacrificing resolving power as required with Orbitrap or Fourier-transform ion cyclotron resonance (FTICR) instruments. The laser used for this source operates at 100 Hz, firing 1 pulse-per-burst, and delivers around 0.7 mJ per pulse. Continuously firing this laser for an extended duration makes it a quasi-continuous ionization source. Additionally, a metal capillary was constructed to extend the inlet of the mass spectrometer, increase desolvation of electrospray charged droplets, improve ion transmission, and increase sensitivity. Its efficiency was compared with the conventional dielectric glass capillary by measured signal and demonstrated that the metal capillary increased ionization efficiency due to its more uniformly distributed temperature gradient. Finally, we present the functionality of the source by analyzing tune mix directly from well plates. This source is a proof of concept for HTS applications using IR-MALDESI coupled to a different MS platform.

Comparison of electrospray and UniSpray™ ion sources with post-column hydrochloric acid infusion for 119 abuse relevant substances

Comparing the sensitivity of the Waters UniSpray™ ion source (USI) with the Waters electrospray ion source (ESI) on a Waters Xevo TQ-XS mass spectrometer with and without additional post-column infusion of low concentrated hydrochloric acid (HCl). To reduce sample volumes or increase analytical sensitivity for drugs of abuse detection in human body fluids, technological advances in mass spectrometry often target an increase in ionization efficiency. One example for this development is the USI, which claims higher ion yields and therefore higher sensitivity due to a unique geometry. Another possibility to increase sensitivity through increased ion yield is the post-column infusion of low concentrated HCl. For sensitivity comparison, we choose the, in our opinion, 119 most relevant medications and drugs of abuse. These were prepared in eleven acetonitrile solutions containing 10 analytes each and one with 9 analytes at a concentration of 1 μg/mL for each substance. For analysis, every solution was diluted 1:1000 in mobile phase A/ethylene glycol (70:30 v/v) to a concentration of 1.0 ng/mL. Solutions were analysed in triplicate on the same day interrupted by the ion source change, to decrease inter-day variations in sensitivity. Chromatographic separation was performed on a Waters 2.1 × 150 mm, 1.8 μm BEH Phenyl column kept at 60 °C on an Acquity UPLC connected to a Xevo TQ-XS detector (Waters). The standard ESI ion source or the USI ion source was mounted on the detector. Mobile phase A consisted of 20 mM ammonium formate and 0.1% formic acid (pH 3) and mobile phase B was 0.1% formic acid in methanol. Gradient elution was conducted within 6.0 min at a flow rate of 0.5 mL/min with 85% A and 15% B at the beginning and 100% B at the end. Data were acquired with the ion sources operating in the positive ionization MRM mode. Two transitions for each analyte were monitored at a constant dwell time of 10 ms over the full chromatographic run time. In an additional experiment, both ion sources were compared with additional post-column infusion of 0.0037% HCl at a flow rate of 5.0 μL/min. Injection volume was 2.0 μL in all experiments. USI provided a general gain in signal intensity (peak area): 12% ( n = 14) of the analytes showed a peak area increase up to 2-fold, 85% ( n = 101) increased between 2- and 4-fold and 3% ( n = 4) increased above 4-fold in peak area. Post-column HCl infusion with the ESI source mounted, resulted in an analyte dependent additional gain in signal intensity for 15% ( n = 18) of the analytes ranging from 2- to 21-fold. 78% ( n = 93) ranged from 0.8-fold to 2-fold. In contrast 7% ( n = 8) showed a signal decrease between 0.4-fold and 0.8-fold. The combination of USI and post-column HCl infusion resulted in an increase in peak area from 2-fold up to 62-fold compared to ESI without infusion for 91% (n = 108) of the analytes. 8% ( n = 9) where between 0.8-fold and 2-fold and 1% ( n = 2) ranged from 0.4-fold to 0.8-fold. With USI, a general improvement in analytical sensitivity under identical conditions could be achieved for all of our 119 “favorite analytes” separated in one chromatographic run. Additional post-column infusion of HCl increased sensitivity for many substances. This approach can be used very selectively when applied to certain retention time windows only. Further experiments are needed to understand how different adjustments of ESI and USI and different matrices will affect ionization. USI, especially in combination with post-column HCl infusion, is a promising tool to increase sensitivity in toxicological multi-target LC-MS/MS analysis.

A miniatured built-in surface ionization oven for the compact microwave ion source

At Peking University (PKU), a compact 2.45 GHz microwave ion source was developed to obtain high-intensity Li+ beam. To improve the generation efficiency of Li+ ions, we developed a built-in lithium oven named surface ionization oven (SIO), which consists of a heating oven for lithium vapor production and a surface ionizer for the ionization of lithium neutrals. Consequently, the surface ionization and electron cyclotron resonance heating (ECRH) principles are combined within the compact microwave ion source. In this work, the structure and materials of the SIO are investigated systemically to lower thermal risk and increase ionization efficiency. The oven is heated by resistance heating. To minimize the heat loss, polished stainless steel shields are used to surround the oven. The surface ionizer is installed at the exit of the nozzle of the heating oven. It consists of six rhenium wires with the diameter of 0.25 mm inside the ionizer tube. Limited space near the microwave window, the dimension of SIO is ⊘ 13.5 mm × 40 mm. This source has been tested with only heating oven and SIO. With only heating oven, which means the absence of surface ionization, and a 200 eμA pulsed Li+ ion beam was stably extracted from this ion source. When the surface ionizer and the heating oven work together, a pulsed Li+ ion beam of 800 eμA is obtained, which increased by about 300%. The experiment proved that surface ionization could dramatically increase the discharge efficiency of lithium plasma in ECRIS.

Supercharging reagents in LC-MS/MS hormone analyses: Enhancing ionization, not limit of quantification

One of the critical steps during LC-MS/MS hormone analyses that affects the sensitivity of the assay is the ionization process. Enhancing ionization efficiencies by the addition of supercharging reagents might be one way to improve sensitivity and reduce the limit of quantification (LOQ). Therefore, we investigated whether the addition of the supercharging reagents m-nitrobenzyl alcohol (m-NBA), sulfolane, propylene carbonate, and o-nitroanisole (o-NA) increased ionization efficiency and improved assay LOQ of insulin, oxytocin, sex steroids, and corticosteroids in test solutions. Additionally, the influence of the supercharging reagents was tested in serum samples after sample pretreatment to determine whether ionization would be enhanced similarly in routine analyses and, subsequently, lead to improved sensitivity. The screening experiments showed that the impact of the supercharging reagents varied for each hormone; although the addition of m-NBA increased the signal of all hormones, the other reagents only enhanced ionization efficiencies for some hormones. While the addition of 0.05 v/v% m-NBA and 0.05 v/v% o-NA did result in an increase in peak area in both test solutions and serum samples, it did not significantly improve the signal-to-noise ratio, as a simultaneous increase in noise was observed. In conclusion, even though supercharging reagents can enhance ionization efficiencies of hormones significantly, the addition of these reagents does not result in an improved LOQ for hormone measurements with LC-MS/MS.

Effects of racetrack magnetic field strength on structure and properties of amorphous carbon coatings deposited by HiPIMS using deep oscillation pulses

In this study, non‑hydrogenated diamond like carbon (DLC) coatings were deposited by sputtering a graphite target in argon (Ar) by high power impulse magnetron sputtering (HiPIMS) using deep oscillation pulses. The effects of the magnetic field strength (B) tangential to surface of the target racetrack on the deposition parameters were studied. The structure, adhesion, and mechanical properties of DLC coatings deposited at different B were investigated by scanning electron microscopy (SEM), Raman spectroscopy, atomic force microscopy (AFM), scratch test, and nanoindentation. The tribological properties of the DLC coatings were evaluated using ball on disk wear test under dry and lubricated test conditions. It was found that the peak target current and substrate current density increased as B increased due to the enhanced electron confinement which results in an increased ionization efficiency. The enhanced ion bombardment was beneficial for improving the structure and properties of DLC coatings. As B increased, the DLC coatings exhibited more compact structure, smaller carbon mounds, and higher sp3 bond fractions. The improvement of the microstructure of the DLC coatings contributed to improved hardness and wear resistance of the coatings in both dry and lubricated test conditions. However, the deposition rate decreased at higher B owing to the high probability of back-attraction of carbon ions. The results suggested that controlling B in the range of 600 G to 700 G is important for achieving fully dense structure, high sp3 bond fractions, and high hardness for the DLC coatings deposited using HiPIMS. A further increase in B to 850 G showed no significant changes in the magnetic field induced effects on the structure and properties of the coatings.

Developing Accelerator Mass Spectrometry Capabilities for Anthropogenic Radionuclide Analysis to Extend the Set of Oceanographic Tracers

Recent major advances in Accelerator Mass Spectrometry (AMS) at the Vienna Environmental Research Accelerator (VERA) regarding detection efficiency and isobar suppression have opened possibilities for the analysis of additional long-lived radionuclides at ultra-low environmental concentrations. These radionuclides, including 233U, 135Cs, 99Tc, and 90Sr, will become important for oceanographic tracer application due to their generally conservative behavior in ocean water. In particular, the isotope ratios 233U/236U and 137Cs/135Cs have proven to be powerful fingerprints for emission source identification as they are not affected by elemental fractionation. Improved detection efficiencies allowed us to analyze all major long-lived actinides, i.e., 236U, 237Np, 239,240Pu, 241Am as well as the very rare 233U, in the same 10 L water samples of a depth profile from the northwest Pacific Ocean. For this purpose, a simplified and very flexible chemical purification procedure based on extraction chromatography (a single UTEVA® column) was implemented which can be extended by a DGA® column for Am purification. The procedure was validated with the reference materials IAEA-381/385. With the additional increase in ionization efficiency expected for the extraction of actinides as fluoride molecules from the AMS ion source, a further reduction of chemical processing may become possible. This method was successfully applied to an exemplary set of air filter samples. In order to determine the quantitative 237Np concentration reliably, a 236Np spike material is being developed in collaboration with the University of Tsukuba, Japan. Ion-Laser Interaction Mass Spectrometry (ILIAMS), a novel technique for the efficient suppression of stable isobaric background, has been developed at VERA and provides unprecedented detection sensitivity for the fission fragments 135Cs, 99Tc, and 90Sr. The corresponding setup is fully operational now and the isobar suppression factors of >105 achieved, in principle, allow for the detection of the mentioned radionuclides in the environment. Especially for 90Sr analysis, this new approach has already been validated for selected reference materials (e.g., IAEA-A-12) and is ready for application in oceanographic studies. We estimate that a sample volume of only (1–3) L ocean water is sufficient for 90Sr as well as for 135Cs analysis, respectively.

α-Active Pyrylium Salt 2,4,5-Triphenylpyrylium for Improved Mass Spectrometry-Based Detection of Peptides

Pyrylium salts are considered efficient chemical tags for amino groups. However, the apparent steric selectivity of pyrylium salts limits their application in the field of chemical labeling, especially during the labeling of sterically hindered compounds like amino acids, peptides, and proteins. Herein, we have investigated the effects of the α-substitution of pyrylium salts on their reactivity. We have also investigated the mechanism of nucleophilic reactions with pyrylium salts and further proposed that the reactivity of pyrylium salts mainly depends on the position and type of their substituents. A series of pyrylium salts were synthesized, and a highly active α-monosubstituted pyrylium salt, 2,4,5-triphenylpyrylium, was developed for efficient chemical labeling. All of the 15 amino acids studied were efficiently labeled under optimized reaction conditions. The 2,4,5-triphenylpyrylium salt was highly efficient in comparison to the previously reported 2,4,6-triphenylpyrylium salt developed for lysine-specific modifications. Furthermore, we successfully used 2,4,5-triphenylpyrylium salt for the hydrophobic labeling of peptides and protein hydrolysates. The most striking observation was that the ionization efficiency of short-chain multilabeled peptides in mixed samples, after derivatization, increased by up to 60 times. The increase in ionization efficiency gradually decreased with increasing peptide chain length. During the "soft" collision-induced dissociation (CID) process, the peptide was tagged at the N-terminus with 2,4,5-triphenylpyrylium, producing abundant a-type ions and b-type ions (Δ = 28), which eases the peptide resequencing process and assists in cracking the peptide codes. Moreover, 2,4,5-triphenylpyrylium has been utilized for the proteomic analysis of HeLa cell digests. In addition, 215 additional proteins were identified in the labeled products and the coverage of most proteins was improved.

Spatial distribution of isobaric androgens in target tissues using chemical derivatization and MALDI-2 on a trapped ion mobility quadrupole time-of-flight instrument.

Prostate cancer is initially treated via androgen deprivation therapy (ADT), a highly successful treatment in the initial pursuit of tumour regression, but commonly restricted by the eventual emergence of a more lethal ‘castrate resistant’ (CRPC) form of the disease. Intracrine pathways that utilize dehydroepiandrosterone (DHEA) or other circulatory precursor steroids are thought to generate relevant levels of growth-stimulating androgens such as testosterone (T) and dihydrotestosterone (DHT). Decoding this tissue-specific metabolic pathway is key for the development of novel therapeutic treatments. Mass spectrometry imaging (MSI) is an analytical technique that allows the visualization of the distribution of numerous classes of biomolecules within tissue sections. The analysis of androgens by liquid chromatography mass spectrometry (LC/MS)-based methods however presents a challenge due to their generally poor ionization efficiency and low physiological endogenous levels. In MSI, on-tissue chemical derivatization (OTCD) has enabled the limits of steroids to be imaged within tissues to be pushed by overcoming poor ionization performance. However, isobaric interference of key androgen derivatives such as T and DHEA can severely hamper studying the intracrinology in several diseases. Here, we have evaluated the use of laser induced post-ionization (MALDI-2) combined with trapped ion mobility separation (TIMS) and orthogonal time-of-flight (QTOF) MS for the visualization of isobaric derivatized androgens in murine tumour xenograft at about 50 μm spatial resolution. With this combination, isobaric T and DHEA were separated in tissue sections and the signals of derivatized steroids enhanced by about 20 times. The combination of TIMS and MALDI-2 thus shows unique potential to study tissue intracrinology within target tissues. This could offer the opportunity for many novel insights into tissue-specific androgen biology.

Combined secondary electrospray and corona discharge ionization (SECDI) for improved detection of explosive vapors using drift tube ion mobility spectrometry

Secondary electrospray corona discharge ionization (SECDI) combines the principles of secondary electrospray ionization (SESI) and corona discharge (CD) to achieve higher sensitivity, which is demonstrated through the detection of 2,4,6-trinitrotoluene (TNT) and 2,6-dinitrotoluene (2,6-DNT) vapors using ion mobility spectrometry (IMS). Using SECDI, enhancements in the IMS signal for TNT and 2,6-DNT vapors at trace concentrations are as much as 2–26 times that observed with CD or SESI alone. The enhancement in sensitivity is hypothesized to result from an increase in ionization efficiency driven by a higher number of reactant ions associated with SECDI compared to either technique individually. The ability of SECDI to achieve higher sensitivity without the aid of dopant molecules demonstrates its merit as an alternative ionization technique.

Very complex internal standard response variation in LC-MS/MS bioanalysis: root cause analysis and impact assessment.

Internal standards (ISs) are essential for the development and use of reliable quantitative bioanalytical LC-MS/MS methods, because they correct for fluctuations in the analytical response that are caused by variations in experimental conditions. Sample-to-sample differences in the IS response are thus to be expected, but a large variability often is an indication of nonoptimal sample handling or analysis settings. This paper discusses a number of cases of very complex variation of IS responses that could be attributed to analytical problems such as injection errors and sample inhomogeneity, and matrix-related issues such as degradation and increased ionization efficiency. A decision tree is proposed to help find the underlying root cause for extreme IS variability.

Supercharging and multiple reaction monitoring of high-molecular-weight intact proteins using triple quadrupole mass spectrometry.

Different supercharging agents were tested to study their effect on the intensity and charge state distributions of high-molecular-weight intact proteins. The goal of this work was to increase chargeability and ionization efficiency for proteins ranging from 66 to 150 kDa, to enable subsequent optimization of multiple reaction monitoring (MRM) mode transitions with a triple quadrupole mass spectrometer for potential top-down quantitative analysis. Supercharging agents, such as meta-nitrobenzyl alcohol (m-NBA), dimethylsulfoxide, trifluoroethanol (TFE), and sulfolane were tested in different concentrations in 50/50 acetonitrile/water with 0.5% formic acid to examine the electrospray ionization response for three model proteins: bovine serum albumin (66 kDa), holo-transferrin (78 kDa), and immunoglobulin G (150 kDa). The settings of ionization source temperature and mobile phase flow rate were also examined. MRM transitions were developed for a wide range of precursor ions for each protein, and limits of detection were determined for the proteins in the presence of favorable additive combinations. For most of the proteins, m-NBA (1%) and TFE (5%) worked most effectively, both to shift the charge state and increase intensity. This is the first report of the use of TFE as an effective agent for both increasing protein chargeability and ionization response. TFE increased ionization efficiency between 3- and 14-fold for the model proteins studied. Increases in both source temperature and flow rate reduced the magnitude of the average charge state observed. The MRM transitions of six to eight different precursor ions of the proteins were optimized and limits of detection in the nanogram quantity on column were determined. The feasibility for top-down quantitative analysis of high-molecular-weight proteins with a triple quadrupole mass spectrometer was demonstrated. Further, additives such as TFE can be highly beneficial for increased chargeability and response of the proteins.

A pyrene linked peptide probe for quantitative analysis of protease activity via MALDI-TOF-MS

We report herein a rationally designed pyrene linked substrate for quantitative protease activity assay via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this proof-of-concept study, a trypsin-specific peptide with the sequence of GGGGRG was selected to conjugate with pyrene forming a pyrene linked peptide probe, Py-GGGGRG. In the presence of trypsin, the Py-GGGGRG probe can be specifically hydrolyzed into Py-GGGGR. The introduction of pyrene greatly increased ionization efficiency of Py-peptides, and Py-peptides could be selectively captured from complex mixtures by a facially fabricated polystyrene coated MALDI plate through hydrophobic and π-π stacking interactions. As a result, trypsin activity can be directly quantified by relative intensity ratio of product and substrate via MALDI-TOF-MS without the use of external internal standard. A linear range of 0.1–10 μg/mL and a relatively low detection limit of 29 ng/mL were obtained. This method has also been successfully used for quantification of trypsin activity in urine and screening the inhibitors of trypsin. Besides, the proposed strategy was also validated for another protease, chymotrypsin, by using the probe Py-GGGGGGYG. Therefore, owing to simplicity, high-throughput capacity and quantificational accuracy, the proposed method shows great potential for activity assay of various proteases and screening their inhibitors via application of specific peptide sequences.

Liquid chromatography – high resolution mass spectrometry method for monitoring of 17 mycotoxins in human plasma for exposure studies

Mycotoxins are secondary metabolites produced by filamentous fungi. Primary route of human exposure to mycotoxins is the intake of the contaminated food. Minimizing mycotoxin exposure is important for population health, as their chronic toxic effects have been associated with kidney and liver diseases, some types of cancer and immunosuppression. The objective of this work was to develop and validate a multi-class mycotoxin method suitable for exposure monitoring of mycotoxins in human plasma. A sensitive liquid chromatography – mass spectrometry method was developed for 17 mycotoxins: nivalenol (NIV), deoxynivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 toxin, HT-2 toxin, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, zearalenone, α-zearalenol (α-ZOL), β-zearalenol, zearalanone, α-zeranol and, and β-zeranol. The method relies on three-step liquid–liquid extraction with ethyl acetate to eliminate the need for immunoaffinity extraction and minimize ionization matrix effects. Chromatographic separation of mycotoxins, including all isomers, was achieved with pentafluorophenyl column and water/methanol mobile phase. Mycotoxin detection and quantitation were performed using high-resolution mass spectrometry on LTQ Velos Orbitrap, in both positive and negative electrospray ionization (ESI(+) and (ESI(−)). The use of 0.02% acetic acid as mobile phase additive for ESI(−) resulted in significant increase in ionization efficiency ranging from 1.7 to 26 times for mycotoxins that ionize better in ESI(−). The optimized method was validated according to FDA guidance procedures. LOQs of all mycotoxins ranged from 0.1 to 0.5 ng/ml, except NIV which resulted in LOQ of 3 ng/ml because of low extraction recovery of this highly polar mycotoxin. Mean intra-day accuracy ranged from 85.8% to 116.4%, and intra-day precision (n = 6) ranged from 1.6% to 12.5% RSD for all mycotoxins except α-ZOL where mean accuracy ranged from 72.9% to 97.2%. Inter-day accuracy and precision were 85.6%–111.5% and 2.7–15.6% RSD respectively, showing good analytical performance of the method for biomonitoring.

Mechanistic Understanding Leads to Increased Ionization Efficiency and Selectivity in Dielectric Barrier Discharge Ionization Mass Spectrometry: A Case Study with Perfluorinated Compounds.

Perfluorinated compounds have unique properties and many practical applications, but are difficult to ionize efficiently with soft ionization methods. An active capillary plasma ionization source based on dielectric barrier discharge ionization (DBDI) coupled with mass spectrometry was used to study the ionization pathway of perfluorinated compounds (PFCs), with the aim of both increasing the ionization efficiency and influencing the selectivity for generating product ions in negative ion mode. Cyclic and linear perfluorinated alkanes were found to mainly form [M - F]- and [M - F + O]- ions, respectively; the [M]-• ion was only obtained at low discharge voltage. Additionally, fluorine attachment [M + F]- was observed mostly for perfluorinated alkenes. An isotope labeling experiment with 18O2 showed that the primary source of oxygen in the substitution reaction is molecular oxygen, reacting with the analyte in the form of O-• ions. The abundance of [M - F + O]- ions can thus be enhanced by increasing the plasma voltage to produce a higher O-• ion density. The loss of the fluorine (without substitution by oxygen) was mainly observed at high frequency, a fact which can be exploited for tuning the ionization toward specific product ions. Overall, the mechanistic understanding of the ionization of PFCs allowed to increase the selectivity of the product ions, resulting in increased ionization efficiency.

Demonstration of hydrazide tagging for O-glycans and a central composite design of experiments optimization using the INLIGHT™ reagent.

The INLIGHT™ strategy for N-linked glycan derivatization has been shown to overcome many of the challenges associated with glycan analysis. The hydrazide tag reacts efficiently with the glycans, increasing their non-polar surface area, allowing for reversed-phase separations and increased ionization efficiency. We have taken the INLIGHT™ strategy and adopted it for use with O-linked glycans. A central composite design was utilized to find optimized tagging conditions (45% acetic acid, 0.1μg/μL tag concentration, 37 C, 1.75h). Derivatization at optimized conditions was much quicker than any hydrazide derivatization strategy used previously. Human immunoglobulin A (IgA) and bovine submaxillary mucin (BSM) were then deglycosylated through hydrazinolysis and the removed glycans were tagged under optimum conditions. XIC of tagged glycans and MS2 data show successful hydrazide tagging of O-linked glycans for the first time. Graphical abstract The INLIGHT™ hydrazide tag was optimized using a central composite design for derivatization of O-linked glycans. Two glycoprotein standards were deglycosylated through hydrazinolysis and tagged at the optimized conditions. MS/MS data shows INLIGHT™ derivatization of glycans demonstrating successful hydrazide tagging of O-glycans for the first time.

Microfluidic Separation Coupled to Mass Spectrometry for Quantification of Peanut Allergens in a Complex Food Matrix.

Peanut is an important food allergen, but it cannot currently be reliably detected and quantified in processed foods at low levels. A level of 3 mg protein/kg is increasingly being used as a reference dose above which precautionary allergen labeling is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred with 0, 3, 10, or 50 mg/kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid-labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on the detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionization efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimized MRM method. Although other peptide targets were detected with three transitions at the 50 mg/kg peanut protein level in both matrices, only Arah2(Q6PSU2)147-155 could be quantified reliably and only in the chocolate dessert at 10 mg/kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30-50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using a simple extraction methodology.

Simultaneous quantification of estrogens, their precursors and conjugated metabolites in human breast cancer cells by LC–HRMS without derivatization

Liquid chromatography–mass spectrometry (LC–MS) is the state of the art technique for quantification of steroid hormones. Currently used methods are typically limited by the need of pre-column derivatization to increase ionization efficiency; however, this causes hydrolysis of conjugated metabolites. Our newly established LC–HRMS method is able to simultaneously quantify conjugated and unconjugated steroids without prior derivatization using deuterated internal standards and solid-phase extraction. This assay was validated according to ICH Q2(R1) guidelines for the analysis of the 10 main steroids of the estrogenic pathway, namely 4-androstene-3,17-dione, dehydroepiandrosterone (DHEA), DHEA-3-sulfate, estrone, 17β-estradiol, estriol (16α-OH-17β-estradiol), estrone-3-sulfate, 17β-estradiol-3-(β-d-glucuronide), 17β-estradiol-3-sulfate and testosterone. Assay performance characteristics were excellent with results for accuracy (98.8–101.2%), precision (mean: 2.05%, all ≤2.80%), stability over five freeze–thaw-cycles (95.7–100.4%) and SPE accuracy (96.9–102.0%), as well as suitable lower and upper limits of quantification for cell culture experiments (LLOQ 0.005–2ng/ml, ULOQ 3–2000ng/ml). Furthermore, we demonstrated the functionality of our method for the monitoring of steroid levels in the human breast cancer cell line MCF-7. This sensitive assay allows for the first time detailed investigations on estrogen metabolomics in breast cancer cells and may also apply to other estrogen-dependent tumor entities.

PH Effects on Electrospray Ionization Efficiency.

Electrospray ionization efficiency is known to be affected by mobile phase composition. In this paper, a detailed study of analyte ionization efficiency dependence on mobile phase pH is presented. The pH effect was studied on 28 compounds with different chemical properties. Neither pK a nor solution phase ionization degree by itself was observed to be sufficient at describing how aqueous phase pH affects the ionization efficiency of the analyte. Therefore, the analyte behavior was related to various physicochemical properties via linear discriminant analyses. Distinction between pH-dependent and pH-independent compounds was achieved using two parameters: number of potential charge centers and hydrogen bonding acceptor capacity (in the case of 80% acetonitrile) or polarity of neutral form of analyte and pK a (in the case of 20% acetonitrile). It was also observed that decreasing pH may increase ionization efficiency of a compound by more than two orders of magnitude. Graphical Abstract ᅟ.

Effects of peptide acetylation and dimethylation on electrospray ionization efficiency.

Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N-termini and the ε-amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC-ESI-MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two-fold improved protein identification sensitivity in comparison with acetylation.