Increasing peptide identification in tandem mass spectrometry through automatic function switching optimization

Comprehensive proteomic studies that employ MS directed peptide sequencing are limited by optimal peptide separation and MS and tandem MS data acquisition routines. To identify the optimal parameters for data acquisition, we developed a system that models the automatic function switching behavior of a mass spectrometer using an MS-only dataset. Simulations were conducted to characterize the number and the quality of simulated fragmentation as a function of the data acquisition routines and used to construct operating curves defining tandem mass spectra quality and the number of peptides fragmented. Results demonstrated that one could optimize for quality or quantity, with the number of peptides fragmented decreasing as quality increased. The predicted optimal operating curve indicated that significant improvements can be realized by selecting the appropriate data acquisition parameters. The simulation results were confirmed experimentally by testing 10 LC MS/MS data acquisition parameter sets on an LC-Q-TOF-MS. Database matching of the experimental fragmentation returned peptide scores consistent with the predictions of the model. The results of the simulations of mass spectrometer data acquisition routines reveal an inverse relationship between the quality and the quantity of peptide identifications and predict an optimal operating curve that can be used to select an optimal data acquisition parameter for a given (or any) sample.     

Communication: Phase incremented echo train acquisition in NMR spectroscopy

We present an improved and general approach for implementing echo train acquisition (ETA) in magnetic resonance spectroscopy, particularly where the conventional approach of Carr-Purcell-Meiboom-Gill (CPMG) acquisition would produce numerous artifacts. Generally, adding ETA to any N-dimensional experiment creates an N + 1 dimensional experiment, with an additional dimension associated with the echo count, n, or an evolution time that is an integer multiple of the spacing between echo maxima. Here we present a modified approach, called phase incremented echo train acquisition (PIETA), where the phase of the mixing pulse and every other refocusing pulse, φ(P), is incremented as a single variable, creating an additional phase dimension in what becomes an N + 2 dimensional experiment. A Fourier transform with respect to the PIETA phase, φ(P), converts the φ(P) dimension into a Δp dimension where desired signals can be easily separated from undesired coherence transfer pathway signals, thereby avoiding cumbersome or intractable phase cycling schemes where the receiver phase must follow a master equation. This simple modification eliminates numerous artifacts present in NMR experiments employing CPMG acquisition and allows "single-scan" measurements of transverse relaxation and J-couplings. Additionally, unlike CPMG, we show how PIETA can be appended to experiments with phase modulated signals after the mixing pulse.     

Exploration of continuous-flow benchtop NMR acquisition parameters and considerations for reaction monitoring

This study focused on fundamental data acquisition parameter selection for a benchtop nuclear magnetic resonance (NMR) system with continuous flow, applicable for reaction monitoring. The effect of flow rate on the mixing behaviors within a flow cell was observed, along with an exponential decay relationship between flow rate and the apparent spin–lattice relaxation time (T1*) of benzaldehyde. We also monitored sensitivity (as determined by signal-to-noise ratios; SNRs) under various flow rates, analyte concentrations, and temperatures of the analyte flask. Results suggest that a maximum SNR can be achieved with low to medium flow rates and higher analyte concentrations. This was consistent with data collected with parameters that promote either slow or fast data acquisition. We further consider the effect of these conditions on the analyte's residence time, T1*, and magnetic field inhomogeneity that is a product of continuous flow. Altogether, our results demonstrate how fundamental acquisition parameters can be manipulated to achieve optimal data acquisition in continuous-flow NMR systems.     

SpinStudioJ: A cross-platform NMR data acquisition and processing workbench based on a plug-in architecture

Flexibility and extensibility are important issues in the design of nuclear magnetic resonance (NMR) software, as these determine the ability to integrate a variety of continuously evolving data acquisition and processing methods. Here, SpinStudioJ is introduced. It is an NMR data acquisition and processing workbench with a plug-in-based architecture. The workbench is based on Eclipse Rich Client Platform, which provides a plug-and-play runtime mechanism and rich graphical user interface functionality. New data acquisition methods and processing algorithms can be easily integrated into the SpinStudioJ workbench by defining extension points, without the need to redistribute existing modules. The software is independent of operating systems, as it leverages the cross-platform feature of the Java virtual machine.     

Multidimensional phosphorimetry

Phosphorescence analysis of multicomponent samples can be enhanced by the rapid acquisition of multiparametric data. A new approach to phosphorimetry provides a sensitive and selective analytical technique through the acquisition of phosphorescence data in the form of an emission-excitation matrix.     

Negative-ion counting techniques for gas chromatography—mass spectrometry

Negative-ion counting techniques for gas chromatography—electron-impact ionization quadrupole mass spectrometry (g.c.—m.s.) are described. Signal-to-noise performance is good and data acquisition is fast. Digital processing of the negative ion signals is done with an ion-counting device and data acquisition with a multi-channel analyzer. The negative-ion mass spectra from g.c.—m.s. measurements are shown for a mixture of tetrachloro-methane, 1,2,2-trichloroethane and 1,1,2,2-tetrachloroethane.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

Computerized system with a hard-wired data-acquisition unit for potentiometric stripping analysis

The system comprises an electrochemical module interfaced to an controlled by an Apple-IIe microcomputer. A high data-acquisition rate (660 kHz) is achieved by equipping the electrochemical module with a separate memory which is updated in hardware during recording of the stripping step. When a preset cut-off potential is exceeded, data acquisition is automatically terminated, and potentiostatic conditions are resumed. The electrochemical module also contains facilities for instrumental control. The controlling program for the microcomputer is written in assembler code, which can be linked to BASIC routines if necessary. Examples are given to illustrate the advantages of fast data acquisition in improving detection limits and reducing deposition times.     

Making Chemical Measurements Using the Lab Works Interface and a Handheld Graphing Calculator

The LabWorks learning system is a computer-controlled data acquisition interface that allows students to quickly obtain and analyze chemistry data. This article describes how the LabWorks interface can be controlled with an HP 48G hand-held calculator rather than a personal computer. The calculator provides users with the same automated data acquisition as if a computer were controlling the interface, but it costs less and requires less maintenance and laboratory bench space. The types of measurements made by the system are discussed and a sample experiment described. Student data and comments are also presented.     

Data-Dependent Acquisition with Precursor Coisolation Improves Proteome Coverage and Measurement Throughput for Label-Free Single-Cell Proteomics**

We combined efficient sample preparation and ultra-low-flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to quantify >3,000 proteins from single cells in rapid label-free analyses. WWA employs large isolation windows to intentionally co-isolate and co-fragment adjacent precursors along with the selected precursor. Optimized WWA increased the number of MS2-identified proteins by ≈40 % relative to standard data-dependent acquisition. For a 40-min LC gradient operated at ≈15 nL/min, we identified an average of 3,524 proteins per single-cell-sized aliquot of protein digest. Reducing the active gradient to 20 min resulted in a modest 10 % decrease in proteome coverage. Using this platform, we compared protein expression between single HeLa cells having an essential autophagy gene, atg9a, knocked out, with their isogenic WT parental line. Similar proteome coverage was observed, and 268 proteins were significantly up- or downregulated. Protein upregulation primarily related to innate immunity, vesicle trafficking and protein degradation.     

实时电导率法测定乙酸乙酯皂化反应速率常数

采用由电导率传感器、数据采集卡和个人计算机构成的模块化设计,以及基于LabVIEW 2013的虚拟仪器编程技术,建立了一套多通道的、并行的、实时的和自动化的电导率数据采集实验系统,使用该系统对经典的电导率法测定乙酸乙酯皂化二级反应速率常数实验进行了改革,提高了这一实验的教学效率。此项改革可以为今后其他经典化学实验改革提供参考。     

Ultrafast two-dimensional NMR spectroscopy using constant acquisition gradients

Multidimensional NMR spectroscopy plays an important role in the characterization of molecular structure and dynamics. A new methodology for acquiring this kind of spectra has been recently demonstrated, endowed with the potential to compress arbitrary multidimensional NMR acquisitions into a single scan. This "ultrafast" nD acquisition protocol is based on a spatiotemporal encoding of the indirect-domain spin evolution, followed by a repetitive decoding and re-encoding of the information thus stored employing a train of alternating-sign gradients. Such train of switching gradients extending throughout the course of the data acquisition may pose extreme demands on a magnetic resonance system, particularly when dealing with nonshielded gradients, strong eddy currents, or rapidly relaxing spin systems. Limits to the in vivo applicability of such fast-switching scheme may also arise due to gradient-induced perineural stimulation. The present study describes a new approach to ultrafast nD NMR that reduces the number of gradient switchings during the acquisition period to zero, leading in essence to a constant-gradient acquisition scheme. This approach operates on the basis of a novel spatiotemporal encoding including discrete, temporally overlapping, frequency-shifted pulses. Principles and examples of this new approach are given; sensitivity limitations and signal-enhancing prospects of such constant-gradient acquisitions are also discussed and exemplified.     

色谱工作站采样时间的迭代计算

采用迭代计算确定色谱工作站采样时间,建立了连续变速采样的方法。方法具有采样点数少,节省内存空间,数据处理速度快及误差小的优点,可用于恒温及等梯度洗脱分析。     

"In-house likeness": comparison of large compound collections using artificial neural networks

Binary classification models able to discriminate between data sets of compounds are useful tools in a range of applications from compound acquisition to library design. In this paper we investigate the ability of artificial neural networks to discriminate between compound collections from various sources aiming at developing an "in-house likeness" scoring scheme (i.e. in-house vs external compounds) for compound acquisition. Our analysis shows atom-type based Ghose-Crippen fingerprints in combination with artificial neural networks to be an efficient way to construct such filters. A simple measure of the chemical overlap between different compound collections can be derived using the output scores from the neural net models.     

Raman line mapping as a fast method for analyzing pharmaceutical bead formulations

This paper describes the use of principal component analysis (PCA) to de-noise Raman spectra and considerably shorten data acquisition time in Raman mapping experiments. A solid dosage pharmaceutical material (bead) is mapped by a Raman line-mapping system. The mapping acquisition time was varied from 30 s (usually employed in practice) to only 3 s. Apparently excessive noise in the maps measured for 3 s is removed by PCA and the maps of all three components of the bead are then binarized and compared. It is found that spatial difference is negligible despite the remarkably different acquisition times employed. The spectra acquired for 3 s and reconstructed via PCA are found to largely overlap with the spectra acquired for 30 s. The signal to noise ratio of the Raman mapping spectra does not obey the expected root t dependence, thereby preventing straightforward estimation of the shortest usable acquisition time (which is to a lesser extent also a function of the binarization threshold). The results reveal that Raman microscopy can be considered a fast method for mapping some materials, in contrast to the established opinion that it is an inherently slow technique.     

Polarized Raman confocal microscopy of single gallium nitride nanowires

Polarized Raman spectra and corresponding Raman scattering intensity images of an isolated gallium nitride nanowire with a diameter of 170 nm are presented. The sensitivity of the confocal microscope combined with a high-resolution piezoelectric stage enables analysis of the crystalline phase and crystallographic orientation of an individual nanowire with an excellent spatial and spectral resolution in a short acquisition time.     

An intelligent data acquisition system for simultaneous screening of microsomal stability and metabolite profiling by liquid chromatography/mass spectrometry

This paper describes the development of a mass spectrometer-based, intelligent, programmable, sample-selection data acquisition system with two unique features. One is that the system allows automatic determination of the mass to charge ratio (m/z) of an unknown compound and the utilization of the molecular ion information to perform selective ion monitoring (SIM) experiments for quantitation. The other is its decision-making capability to select intelligently different samples and perform different experiments during data acquisition. These features were demonstrated by the application of the system to simultaneous screening for the microsomal stability and metabolite profiling of adatanserin. In this application, the data acquisition system continuously calculated the peak areas of adatanserin from SIM analyses of a batch of microsomal incubates stopped at various time points. Once the peak area of adatanserin had dropped to an arbitrarily predefined 60% of the initial value, the system made a decision to perform metabolite profiling of the sample. This decision initiated a series of automated operations, such as selecting a sample for re-analysis, changing the data acquisition time and liquid chromatographic gradient and switching the SIM mode to the data-dependent product ion scanning mode. The completed analysis of the batch of samples provided information both on the microsomal stability and on the metabolic profile of adatanserin. This simultaneous approach to investigating microsomal stability and metabolite profiling significantly increases the throughput for drug discovery support.     

Analytical performance of the various acquisition modes in Orbitrap MS and MS/MS

Quadrupole Orbitrap instruments (Q Orbitrap) permit high‐resolution mass spectrometry‐based full scan acquisitions and have a number of acquisition modes where the quadrupole isolates a particular mass range prior to a possible fragmentation and high‐resolution mass spectrometry‐based acquisition. Selecting the proper acquisition mode(s) is essential if trace analytes are to be quantified in complex matrix extracts. Depending on the particular requirements, such as sensitivity, selectivity of detection, linear dynamic range, and speed of analysis, different acquisition modes may have to be chosen. This is particularly important in the field of multi‐residue analysis (eg, pesticides or veterinary drugs in food samples) where a large number of analytes within a complex matrix have to be detected and reliably quantified. Meeting the specific detection and quantification performance criteria for every targeted compound may be challenging. It is the aim of this paper to describe the strengths and the limitations of the currently available Q Orbitrap acquisition modes. In addition, the incorporation of targeted acquisitions between full scan experiments is discussed. This approach is intended to integrate compounds that require an additional degree of sensitivity or selectivity into multi‐residue methods.     

Quadrupole mass spectrometer operating in the electron-capture negative ion mode as detector for comprehensive two-dimensional gas chromatography

A recently introduced rapid-scanning quadrupole mass spectrometer (qMS) with an electron-capture negative ion (ECNI) option, the Perkin-Elmer Clarus 500, was tested as a detector for comprehensive two-dimensional gas chromatography (GC x GC). The parameters influencing the data acquisition rate in the scan mode, such as scan time and inter-scan delay, and in the selected ion monitoring mode, such as dwell time and inter-channel delay, were evaluated. In the scan mode, good-quality mass spectra covering a range of 300 Da can be obtained at an acquisition rate of 23 Hz; in selected ion monitoring, an acquisition rate of 90Hz can be achieved when monitoring a single ion. Compared with electron ionisation, the use of electron-capture negative ionisation causes no extra peak broadening. As applications, mixtures of polychlorinated n-alkanes (PCAs), polybrominated diphenyl ethers (PBDEs) and polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were analysed. The separation of PCAs based on their number of chlorine substituents was confirmed for the first time by using GC x GC-ECNI qMS in the scan mode and a significantly improved limit of detection was achieved for BDEs (10-150 fg injected) and CDD/Fs (10-700 fg injected) in the selected ion monitoring mode.     

Real-time 2D NMR identification of analytes undergoing continuous chromatographic separation

We have recently proposed and demonstrated an approach that enables the acquisition of multidimensional nuclear magnetic resonance (NMR) spectra within a single scan. A promising application opened up by this new accelerated form of data acquisition concerns the possibility of monitoring in real time the chemical nature of analytes subject to a continuous flow. The present paper illustrates such potential, with the real-time acquisition of a series of 2D 1H NMR spectra arising from a mixture of compounds subject to a continuous liquid chromatography (LC) separation. This real-time 2D NMR identification of chemicals eluted minutes apart under usual LC-NMR conditions differs from the way in which LC-2D NMR has hitherto been carried out, which relies on stopped-flow modes of operations whereby fractions are first collected and then subject to individual, aliquot-by-aliquot analyses. The real-time LC-2D NMR experiment hereby introduced can be implemented in a straightforward manner using modern commercial LC-NMR hardware, thus opening up immediate possibilities in high-throughput characterizations of complex molecules.