Mass Spectrometric Performance Research Articles

Measurement report: The Fifth International Workshop on Ice Nucleation phase 1 (FIN-01): intercomparison of single-particle mass spectrometers

Abstract. Knowledge of the chemical composition and mixing state of aerosols at a single-particle level is critical for gaining insights into atmospheric processes. One common tool to make these measurements is single-particle mass spectrometry. There remains a need to compare the performance of different single-particle mass spectrometers (SPMSs). An intercomparison of SPMSs was conducted at the Aerosol Interaction and Dynamics in the Atmosphere (AIDA) chamber at the Karlsruhe Institute of Technology (KIT) in November 2014, as part of the first phase of the Fifth International Workshop on Ice Nucleation (FIN-01). In this paper we compare size distributions and mass spectra of atmospherically relevant particle types measured by five SPMSs. These include different minerals, desert and soil dusts, soot, bioaerosol (Snomax; protein granule), secondary organic aerosol (SOA), and SOA-coated mineral particles. Most SPMSs reported similar vacuum aerodynamic diameter (dva) within typical instrumental ranges from ∼100–200 nm (lower limit) to ∼2–3 µm (upper limit). In general, all SPMSs exhibited a wide dynamic range (up to ∼103) and high signal-to-noise ratio (up to ∼104) in mass spectra. Common spectral features with small diversities in mass spectra were found with high average Pearson's correlation coefficients, i.e., for average positive spectra ravg-pos=0.74 ± 0.12 and average negative spectra ravg-neg=0.67 ± 0.22. We found that instrument-specific detection efficiency (DE) was more dependent on particle size than particle type, and particle identification favored the use of bipolar, rather than monopolar, instruments. Particle classification from “blind experiments” showed that all instruments differentiated SOA, soot, and soil dust and detected subtle changes in the particle internal mixing but had difficulties differentiating among specific mineral types and dusts. This study helps to further understand the capabilities and limitations of the single-particle mass spectrometry technique in general and the specific performance of the instrument in characterizing atmospheric aerosol particles.

Offline analysis of secondary formation markers in ambient organic aerosols by liquid chromatography coupled with time-of-flight mass spectrometry

The present study provides a comprehensive assessment of the quantitative analysis by high-performance liquid chromatography coupled with dual orthogonal electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) of pinene markers, biomass-burning related phenols, and other relevant carboxylic acids in atmospheric aerosol samples. Significant insights into the quantitative determination are offered on the basis of systematic experiments targeting the optimization of chromatographic separation, ionization source, and mass spectrometer performance. After testing three analytical columns, the best separation of the compounds of interest was achieved on a Poroshell 120 ECC18 column (4.6 × 50 mm, 2.7 µm) thermostated at 35 °C, operating in gradient elution mode with 0.1% acetic acid in water and acetonitrile at a 0.8 mL min−1 flow rate. Optimal operational conditions for the ESI-TOF-MS instrument were identified as a 350 °C drying gas temperature, 13 L min−1 drying gas flow rate, 60 psig nebulizer pressure, 3000 V for the ion transfer capillary, 60 V for the skimmer, and 150 V for the fragmentor. Additionally, the matrix effect on the ESI efficiency and the spike recovery factors of the compounds were tested. Method quantification limits can go as low as in the 0.88–48.0 μg L − 1 (3.67–200 pg m − 3, at 120 m3 of sampled air) range. The developed method was shown to be reliable for the quantification of the targeted compounds in real atmospheric aerosol samples. The accuracy in the molecular mass determination of less than 5 ppm and the acquisition in the full scan mode were shown to bring additional insights into the organic constituents in atmospheric aerosols.

A comprehensive and high-throughput screening method for multiple prohibited substances by UPLC-QE Plus-HRMS and HPLC-QQQ-MS in human urine for doping control.

Since the World Anti-Doping Agency's (WADA) Prohibited List is updated on an annual basis, screening methods must be continually adapted to align with these changes. In accordance with Technical Document-MRPL 2022, a newly combined, comprehensive, rapid and high-throughput doping control screening method has been developed for the analysis of 350 substances with different polarities in human urine using ultra-high performance liquid chromatography coupled with Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (UPLC-QE Plus-HRMS) and ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UPLC-QQQ-MS). The limits of detection were in the range of 0.12-50 ng mL-1 for beta-2 agonists, hormone and metabolic modulators, narcotics, cannabinoids and glucocorticoids, 0.1-14 ng mL-1 for the manipulation of blood and blood components, beta blockers, anabolic agents and hypoxia-inducible factor (HIF) activating agents, and 2.5-100 000 ng mL-1 for substances of Appendix A, diuretics & masking agents and stimulants. The sample preparation consisted of two parts: one is the dilute & shoot part analyzed in UPLC-QQQ-MS, another is a mixture of the dilute & shoot part and a liquid-liquid extraction part of hydrolyzed human urine analyzed in UPLC-QE Plus-HRMS in full scan mode with polarity switching and parallel reaction monitoring (PRM) mode. The method has been fully validated for doping control purposes. All the substances were compliant with WADA's required 1/2 minimum requirement performance level (MRPL) or minimum reporting level (MRL), and this method was successfully used in the 2022 Beijing Winter Olympic Games and Winter Paralympic Games for anti-doping purpose.

Distribution of Heavy Metals and Organic Compounds: Contamination and Associated Risk Assessment in the Han River Watershed, South Korea

Given water pollution increases in aquatic ecosystems resulting from industrialization and rapid urbanization, appropriate treatment strategies to alleviate water pollution are crucial. The spatiotemporal distribution, sources, and potential risk of heavy metals and organic compounds were determined in surface water from the Han River watershed (n = 100) in wet and dry seasons. The inductively coupled plasma mass spectrometer (Cr and As), mercury analyzer (Hg), and ultra-high performance liquid chromatography tandem mass spectrometer (organic compounds) were used to analyze the target compounds. Total concentration and detection frequency were in the order: Cr (2.375 µg/L, 100%) > As (1.339 µg/L, 100%) > Hg (0.007 µg/L, 100%) for heavy metals, and carbofuran (0.051 µg/L, 75%) > bisphenol A (0.040 µg/L, 47%) > quinoline (0.020 µg/L, 32%) for organic compounds. The target compounds showed the highest concentration in the area near industrial facilities. High concentrations and risk levels of all target compounds, except quinoline, were observed during the wet season. Principal component analysis indicated anthropogenic activities were the primary source of pollution. Cr showed the most prominent environmental impact in the wet season, suggesting its ecological risk. Additional monitoring is required for clear risk pollutant assessments in aquatic ecosystems to aid policy implementation.

Real-Time Investigation of Primary Ship Engine Emissions by Vacuum Resonance-Enhanced Multiphoton Ionization High-Resolution Orbitrap Mass Spectrometry.

The comprehensive chemical description of air pollution is a prerequisite for understanding atmospheric transformation processes and effects on climate and environmental health. In this study, a prototype vacuum photoionization Orbitrap mass spectrometer was evaluated for field-suitability by an online on-site investigation of emissions from a ship diesel engine. Despite remote measurements in a challenging environment, the mass spectrometric performance could fully be exploited. Due to the high resolution and mass accuracy in combination with resonance-enhanced multiphoton ionization, the aromatic hydrocarbon profile could selectively and sensitively be analyzed. Limitations from commonly deployed time-of-flight platforms could be overcome, allowing to unraveling the oxygen- and sulfur-containing compounds. Scan-by-scan evaluation of the online data revealed no shift in exact m/z, assignment statistics with root mean square error (RMSE) below 0.2 ppm, continuous high-resolution capabilities, and good isotopic profile matches. Emissions from three different feed fuels were investigated, namely, diesel, heavy fuel oil (HFO), and very low sulfur fuel oil (VLSFO). Regulations mainly concern the fuel sulfur content, and thus, exhaust gas treatment or new emerging fuels, such as the cycle-oil-based VLSFO, can legally be applied. Unfortunately, despite lower CHS-class emissions, a substantial amount of PAHs is emitted by the VLSFO with higher aromaticity compared to the HFO. Hence, legislative measures might need to take further chemical criteria into account.

Enhancement of Portable Mass Spectrometer Sensitivity and Selectivity by a Qualitative Pre-Scan Waveform (QPSW)

While portability is a key aspect of small mass spectrometers, they also need to be able to effectively screen the analytes from complex matrices. The accuracy and performance of mass spectrometers depend not only on the identification of parent ion signals in targets but also fragment ion signals. This paper reports on the construction of a device that uses a new time-domain excitation waveform called qualitative pre-scan waveform (QPSW) for its quadrupole ion trap mass spectrometry analyzer. This approach uses scanning to eliminate interfering ions, thus improving the sensitivity for the analyte and removing the cumbersome steps required to convert conventional stored waveform inverse Fourier transform (SWIFT) waveforms from the frequency domain to the time domain. Tests show that the target ions can be fragmented just by introducing air at the moment when the pinch valve is opened without any need for a carrier gas. This simplifies the equipment required, making the mass spectrometer more portable. Tests were undertaken to compare the performance with the use of conventional SWIFT waveforms and to optimize its experimental parameters so that the target ions were effectively accumulated in the ion trap. An approximate 5-fold improvement in sensitivity was achieved by QPSW compared to the SWIFT for 2 μg/mL ketamine. A 5-fold reduction in the limit of detection (LOD) to 20 ng/mL was also obtained for enrofloxacin. In addition, the stability was improved by QPSW with a relative standard deviation less than 13% compared to 16% for SWIFT.

Comparison of High-Resolution Fourier Transform Mass Spectrometry Platforms for Putative Metabolite Annotation.

Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap mass spectrometry (MS) are among the highest-performing analytical platforms used in metabolomics. Non-targeted metabolomics experiments, however, yield extremely complex datasets that make metabolite annotation very challenging and sometimes impossible. The high-resolution accurate mass measurements of the leading MS platforms greatly facilitate this process by reducing mass errors and spectral overlaps. When high resolution is combined with relative isotopic abundance (RIA) measurements, heuristic rules, and constraints during searches, the number of candidate elemental formula(s) can be significantly reduced. Here, we evaluate the performance of Orbitrap ID-X and 12T solariX FT-ICR mass spectrometers in terms of mass accuracy and RIA measurements and how these factors affect the assignment of the correct elemental formulas in the metabolite annotation pipeline. Quality of the mass measurements was evaluated under various experimental conditions (resolution: 120, 240, 500 K; automatic gain control: 5 × 104, 1 × 105, 5 × 105) for the Orbitrap MS platform. High average mass accuracy (<1 ppm for UPLC-Orbitrap MS and <0.2 ppm for direct infusion FT-ICR MS) was achieved and allowed the assignment of correct elemental formulas for over 90% (m/z 75-466) of the 104 investigated metabolites. 13C1 and 18O1 RIA measurements further improved annotation certainty by reducing the number of candidates. Overall, our study provides a systematic evaluation for two leading Fourier transform (FT)-based MS platforms utilized in metabolite annotation and provides the basis for applying these, individually or in combination, to metabolomics studies of biological systems.

Effects of solvent composition on ionization and fragmentation within the solution-cathode glow discharge

Recently, solution-electrode glow discharges (SEGDs) have shown great utility as ionization sources for mass spectrometry (MS). The solution composition of SEGD electrodes is pivotal to their performance as it influences analyte-ion formation. The performance of electrospray ionization is heavily dictated by spray-solution composition, which can alter ionization efficiency and pathways. While SEGDs produce Taylor cones similar to electrospray ionization, the influence of solution-electrode composition on molecular-ion formation has not been studied. Here, we examine how additives to an atmospheric-pressure solution-cathode glow discharge (SCGD) influence molecular ionization and fragmentation. The impact of several additives to the acidic solution of an SCGD ionization source was evaluated based on mass-spectrometric performance. Addition of methanol increased molecular- and fragment-ion signals for peptide angiotensin II. This effect is likely due to improved desolvation and a greater interaction of analyte molecules with glow-discharge species. Several high-boiling-point reagents were tested to examine changes in the ion signal, the average charge state, and the degree of fragmentation. Overall, these additives inhibited fragmentation but significantly lowered intact molecular-ion signals. Interestingly, loss of fragment ions trended with the boiling point of the reagent used. We hypothesize that analyte molecules become trapped in droplets produced at the solution-cathode surface. These droplets do not fully desolvate before escaping the discharge region, sparing analyte molecules from fragmentation. For low volatility additives, droplets do not desolvate, even as they enter the heated MS, which yields a loss in molecular ions. The changing composition of the SCGD solution alters analytical performance, but also provides insight into analyte ionization and fragmentation processes.

Mass Spectral Analysis of Synthetic Peptides: Implications in Proteomics.

Sequence determination of peptides is a crucial step in mass spectrometry-based proteomics. Peptide sequences are determined either by database search or by de novo sequencing using tandem mass spectrometry. Determination of all the theoretical expected peptide fragments and eliminating false discoveries remains a challenge in proteomics. Developing standards for evaluating the performance of mass spectrometers and algorithms used for identification of proteins is important for proteomics studies. The current study is focused on these aspects by using synthetic peptides. A total of 599 peptides were designed from in silico tryptic digest with 1 or 2 missed cleavages from 199 human proteins, and synthetic peptides corresponding to these sequences were obtained. The peptides were mixed together, and analysis was carried out using liquid chromatography-electrospray ionization tandem mass spectrometry on a Q-Exactive HF mass spectrometer. The peptides and proteins were identified with SEQUEST program. The analysis was carried out using the proteomics workflows. A total of 573 peptides representing 196 proteins could be identified, and a spectral library was created for these peptides. Analysis parameters such as "no enzyme selection" gave the maximum number of detected peptides as compared with trypsin in the selection. False discoveries could be identified. This study highlights the limitations of peptide detection and the need for developing powerful algorithms along with tools to evaluate mass spectrometers and algorithms. It also shows the limitations of peptide detection even with high-end mass spectrometers. The mass spectral data are available in ProteomeXchange with accession no. PXD017992.

Applications and advancements of FT-ICR-MS for interactome studies.

The set of all intra- and intermolecular interactions, collectively known as the interactome, is currently an unmet challenge for any analytical method, but if measured, could provide unparalleled insight on molecular function in living systems. Developments and applications of chemical cross-linking and high-performance mass spectrometry technologies are beginning to reveal details on how proteins interact in cells and how protein conformations and interactions inside cells change with phenotype or during drug treatment or other perturbations. A major contributor to these advances is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) technology and its implementation with accurate mass measurements on cross-linked peptide-pair precursor and fragment ions to enable improved identification methods. However, these applications place increased demands on mass spectrometer performance in terms of high-resolution spectral acquisition rates for on-line MSn experiments. Moreover, FT-ICR-MS also offers unique opportunities to develop and implement parallel ICR cells for multiplexed signal acquisition and the potential to greatly advance accurate mass acquisition rates for interactome studies. This review highlights our efforts to exploit accurate mass FT-ICR-MS technologies with chemical cross-linking and developments being pursued to realize parallel MS array capabilities that will further advance visualization of the interactome.

Mass Spectral Analysis of Synthetic Peptides: Implications in Proteomics.

Sequence determination of peptides is a crucial step in mass spectrometry-based proteomics. Peptide sequences are determined either by database search or by de novo sequencing using tandem mass spectrometry. Determination of all the theoretical expected peptide fragments and eliminating false discoveries remains a challenge in proteomics. Developing standards for evaluating the performance of mass spectrometers and algorithms used for identification of proteins is important for proteomics studies. The current study is focused on these aspects by using synthetic peptides. A total of 599 peptides were designed from in silico tryptic digest with 1 or 2 missed cleavages from 199 human proteins, and synthetic peptides corresponding to these sequences were obtained. The peptides were mixed together, and analysis was carried out using liquid chromatography-electrospray ionization tandem mass spectrometry on a Q-Exactive HF mass spectrometer. The peptides and proteins were identified with SEQUEST program. The analysis was carried out using the proteomics workflows. A total of 573 peptides representing 196 proteins could be identified, and a spectral library was created for these peptides. Analysis parameters such as "no enzyme selection" gave the maximum number of detected peptides as compared with trypsin in the selection. False discoveries could be identified. This study highlights the limitations of peptide detection and the need for developing powerful algorithms along with tools to evaluate mass spectrometers and algorithms. It also shows the limitations of peptide detection even with high-end mass spectrometers. The mass spectral data are available in ProteomeXchange with accession no. PXD017992.

Optimization of ion trajectories in a dynamically harmonized Fourier-transform ion cyclotron resonance cell using a design of experiments strategy.

With the recent introduction of the dynamically harmonized Fourier-transform ion cyclotron resonance (FT-ICR) cell, the complexity of tuning has expanded drastically, and fine-tuning of the direct current voltages is required to optimize the ion cloud movement. As this adjustment must typically be performed manually, more reliable computational methods would be useful. Here we propose a computational method based on a design of experiments (DoE) strategy to overcome the limits of classical manual tuning. This DoE strategy was exemplarily applied on a 12 T FT-ICR instrument equipped with a dynamically harmonized ICR cell. The chemometric approach, based on a central composite face (CCF) design, was first applied to a reference material (sodium trifluoroacetate) allowing for the evaluation of the primary cell parameters. Eight factors related to shimming and gating were identified. The summed intensity of the signal corresponding to the even harmonics was defined as one quality criterion. The DoE response allowed for rapid and complete mapping of cell parameters resulting in an optimized parameter set. The new set of cell parameters was applied to the study of an ultra-complex sample: Tholins, an ultra-complex mixture that mimics the haze present on Titan, was chosen. We observed a substantial improvement in mass spectrometric performance. The sum of signals related to harmonics was decreased by a factor of three (from 4% for conventional tuning to 1.3%). Furthermore, the dynamic range was also increased, which in turn led to an increase in attributed peaks by 13%. This computational procedure based on an experimental design can be applied to any other mass spectrometric parameter optimization problem. This strategy will lead to a more transparent and data-driven method development.

Design of a Resonant Radiofrequency Driver for Ion Transmission in a Desktop Mass Spectrometer and Its Application in Volatile Organic Compound Determination

The ion transformation efficiency from a high-pressure ion source region to a low-pressure mass analysis region is one of the important indices for the mass spectrometer performance. To decrease the ion loss and improve the sensitivity of ions, a radiofrequency driver was designed in a home-made time-of-flight mass spectrometer (TOF-MS), which adopted a resistance-inductance-capacitance (RLC) resonant amplification pattern to drive the radiofrequency quadrupole (RFQ). The peak-to-peak voltage Vp-p of the radiofrequency driver was continuously adjusted from 50 to 1000 V with a relative standard deviation of 2.5‰. The performance of the radiofrequency quadrupole in the mass spectrometer was assessed by the characterization of a mixture of benzene, toluene, and p-xylene. The limits of detection for benzene, toluene, and p-xylene were 0.07, 0.05, and 0.04 ppbv, respectively. Moreover, the performance and stability of radiofrequency quadrupole were assessed by the determination of volatile organic compounds (VOCs) in newly decorated rooms and exhaled breath. The results indicated that the developed radiofrequency driver has potential for use in mass spectrometers.

Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides

Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in one-dimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dye-conjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP – IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the two-dimensional analysis can be detected at low nanogram levels, comparable to results achieved in a one-dimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques.

Oligonucleotide analysis by hydrophilic interaction liquid chromatography-mass spectrometry in the absence of ion-pair reagents

Improving our understanding of nucleic acids, both in biological and synthetic applications, remains a bustling area of research for both academic and industrial laboratories. As nucleic acids research evolves, so must the analytical techniques used to characterize nucleic acids. One powerful analytical technique has been coupled liquid chromatography – tandem mass spectrometry (LC–MS/MS). To date, the most successful chromatographic mode has been ion-pairing reversed-phase liquid chromatography. Hydrophilic interaction liquid chromatography (HILIC), in the absence of ion-pair reagents, has been investigated here as an alternative chromatographic approach to the analysis of oligonucleotides. By combining a mobile phase system using commonly employed in liquid chromatography-mass spectrometry (LC–MS) - i.e., water, acetonitrile, and ammonium acetate - and a new, commercially available diol-based HILIC column, high chromatographic and mass spectrometric performance for a wide range of oligonucleotides is demonstrated. Particular applications of HILIC-MS for the analysis of deoxynucleic acid (DNA) oligomers, modified and unmodified oligoribonucleotides, and phosphorothioate DNA oligonucleotides are presented. Based on the LC–MS performance, this HILIC-based approach provides an attractive, sensitive and robust alternative to prior ion-pairing dependent methods with potential utility for both qualitative and quantitative analyses of oligonucleotides without compromising chromatographic or mass spectrometric performance.

A laser desorption ionization/matrix-assisted laser desorption ionization target system applicable for three distinct types of instruments (LinTOF/curved field RTOF, LinTOF/RTOF and QqRTOF) with different performance characteristics from three vendors.

RationaleWe have developed a target system which enables the use of only one target (i.e. target preparation set) for three different laser desorption ionization (LDI)/matrix‐assisted laser desorption ionization (MALDI) mass spectrometric instruments. The focus was on analysing small biomolecules with LDI for future use of the system for the study of meteorite samples (carbonaceous chondrites) using devices with different mass spectrometric performance characteristics.MethodsThree compounds were selected due to their potential presence in meteoritic chondrites: tryptophan, 2‐deoxy‐d‐ribose and triphenylene. They were prepared (with and without MALDI matrix, i.e. MALDI and LDI) and analysed with three different mass spectrometers (LinTOF/curved field RTOF, LinTOF/RTOF and QqRTOF). The ion sources of two of the instruments were run at high vacuum, and one at intermediate pressure. Two devices used a laser wavelength of 355 nm and one a wavelength of 337 nm.ResultsThe developed target system operated smoothly with all devices. Tryptophan, 2‐deoxy‐d‐ribose and triphenylene showed similar desorption/ionization behaviour for all instruments using the LDI mode. Interestingly, protonated tryptophan could be observed only with the LinTOF/curved field RTOF device in LDI and MALDI mode, while sodiated molecules were observed with all three instruments (in both ion modes). Deprotonated tryptophan was almost completely obscured by matrix ions in the MALDI mode whereas LDI yielded abundant deprotonated molecules.ConclusionsThe presented target system allowed successful analyses of the three compounds using instruments from different vendors with only one preparation showing different analyser performance characteristics. The elemental composition with the QqRTOF analyser and the high‐energy 20 keV collision‐induced dissociation fragmentation will be important in identifying unknown compounds in chondrites.

Supercharging Proteins: How Many Charges Can a Protein Carry?

Very highly charged proteins, so-called "supercharged" ions, can lose (excess) protons to background gases like N2 . It is remarkable that such extremely acidic species can be generated in electrospray ionization, in the presence of not just N2 but also much higher-basicity solvents. What mechanism(s) can explain such high charging, and what is the ultimate limit?

Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry.

Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases-casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6-along with 16O4- and 18O4-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%-40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency. Graphical Abstract ᅟ.

Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins

BackgroundGiven the excellent performance of modern mass spectrometers, their clinical application for the analysis of macromolecules is a growing field of interest. This principle is explored by hemoglobin analysis, which is a representative example by its molecular weight and clinical relevance in e.g. screening programs for thalassemia and hemoglobin variants. Considering its abundance and cellular containment, pre-analysis is significantly reduced allowing for essential rapid acquisitions. MethodsBy parallel analysis of routine diagnostics for hemoglobin variants and thalassemia, we acquired samples of adults who were consented for hemoglobinopathy screening in our clinical laboratory. The pre-analytical process comprised of red cell lysis only; without further digestion and purification steps, the samples were directly injected in an electrospray ionization quadrupole time-of-flight setup and the intact proteins were analyzed by flow injection analysis. After optimization of process parameters, the deconvoluted mass spectra revealed the presence of α- and β-globulins. The reference ranges for the average mass of both globulins and their intensity ratio (α/β-ratio) were deduced from a disease-free subgroup and patients with a hemoglobinopathy were compared. ResultsThe α/β-ratio is a poor marker for thalassemia patients, yet deviant α/β-ratios are found for patients with a hemoglobin variant. Mass deviations down to 1Da can be resolved; even if the patient suffers from a heterozygotic disorder, the average mass is found outside the established reference interval. ConclusionsAlthough subjects with mild thalassemia were not detected, all patients with a hemoglobin variant were resolved by top-down mass spectrometry using the average globulin mass and the α/β-ratio as screening parameters.

Surface acoustic wave nebulization device with dual interdigitated transducers improves SAWN-MS performance.

We compared mass spectrometric (MS) performance of surface acoustic wave nebulization (SAWN) generated by a single interdigitated transducer (IDT) designed to produce a progressive wave (PW) to one with a dual IDT that can in theory generate standing waves (SW). Given that devices using dual IDTs had been shown to produce fewer large size droplets on average, we hypothesized they would improve MS performance by improving the efficiency of desolvation. Indeed, the SW-SAWN chip provided an improved limit of detection of 1 femtomole of peptide placed on chip making it 100× more sensitive than the PW design. However, as measured by high-speed image recording and phase Doppler particle analyzer measurements, there was only a 26% increase in the small diameter (1-10 µm) droplets produced from the new device, precluding a conclusion that the decrease in droplet size was solely responsible for the improvement in MS signal/noise. Given that the dual IDT design produced a more instantaneous plume than the PW design, the more likely contributor to improved MS signal/noise was concluded to be a higher ion flux entering the mass spectrometer for the dual IDT designs. Notably, the dual IDT device allowed production of much higher quality protein mass spectra up to about 20 kDa, compared with the single IDT device. Copyright © 2016 John Wiley & Sons, Ltd.