Revolutionary Extension of Dynamic Range for Feature Detection

This is a workshop given by Aivett Bilbao at the Agilent IMS User Meeting and Training Workshop.<br>January 17, 2018<br>University of Utrecht, Netherlands<br><br

Systems level metabolic profiling with ion mobility

This is a presentation given by Sam Payne at the Agilent IMS user meetin

Workshop: Automating the Stepped Field Method for High Throughput CCS Database Creation

This is a workshop given by Aivett Bilbao and Sam Payne at the Agilent IMS User Meeting and Training Workshop.<br>January 17, 2018<br>University of Utrecht, Netherlands<br><br

Extending Dynamic Range and Enhancing Compound Identification for Untargeted Ion Mobility-MS Workflows

<div>This poster was presented at ASMS 2018</div><div><br></div><div>• PNNL PreProcessor is free software for</div><div>enhancing signal quality of raw spectra at the</div><div>limits of the MS detection system.</div><div>• Multidimensional moving average smoothing</div><div>improves ion peaks with poor definition at low</div><div>abundances.</div><div>• A new saturation repair algorithm reconstructs</div><div>the intensities of ion peaks at high abundances.</div

Enhancing Computational Tools for Ion Mobility-Mass Spectrometry-Based Untargeted Workflows (Cascadia Proteomics Symposium 17)

<div>Ion mobility spectrometry (IMS) is a rapid and highly reproducible molecular-shape separation technique. While IMS has shown great utility when coupled with MS for analysis of complex samples, methods for processing the complex data generated have lagged behind. In fact, the incorporation of this extra IMS separation dimension requires upgrades and optimization of existing computational pipelines and the development of new algorithmic strategies to fully exploit the advantages of the technology. Here, we investigate MS pre-processing algorithms to extend feature detection and quantification performance, as well as integration of IMS collisional cross section (CCS) libraries into data analysis tools for molecular characterization. These strategies were applied for untargeted analyzes of biofluid samples to evaluate changes in endogenous metabolites and xenobiotics. IMS-MS data files were acquired by an Agilent 6560 Ion Mobility Q-TOF MS system. A software tool for IMS data analysis and processing was developed (C#) to apply multidimensional smoothing, saturation correction and generate new raw data files. Intensity values in each frame (an IMS cycle) were smoothed first in the drift dimension followed by smoothing of the chromatographic dimension considering neighboring frames. High signal intensity values that reached beyond detector capacity were identified by the characteristic flat profile at the apex of saturated peaks. Agilent Mass Profiler was used for feature extraction and sample alignment. A CCS library was created from small molecule standards. Features were annotated using Agilent ID Browser. Statistical analyses of results were performed in R. The developed software tools were integrated to process IMS-MS data from urine samples previously analyzed using a solid-phase extraction method. To minimize the effects of low ion statistics (e.g., jagged profiles), several smoothing kernels were evaluated: Gaussian, Savitzky-Golay, moving average and weighted moving average. Among those, moving average smoothing provided the best results for retrieving low-abundance features and merging at least 40% more of the features with split profiles. Smoothing increased by a factor of 2.5 the number of high quality features (quality score ≥ 80; a 0−100 scale considering signal-to-noise, number of isotopic ions and m/z stability). More specifically, 1332 features were found in at least 20 of the 96 analyzed samples, compared to 360 features found without smoothing. These improvements were also reflected by a 3-fold increase of the number of features having abundances with less than 20% coefficient of variation. A filtering strategy was incorporated in the smoothing heuristic to reduce background noise, consequently decreasing (by half, on average) file size, memory usage and processing time for feature detection and alignment. Furthermore, saturation correction improved quantification and mass accuracy of analytes with high intensity signals. We therefore observed that the implemented strategies enhanced the dynamic range of measurement: smoothing towards the lower end and saturation correction towards the higher end of abundances.</div><div>These data quality improvements also allowed us to compute more accurate CCS values from the IM-MS measurements, which in combination with CCS libraries can help to discriminate the feature of interest. For instance, our CCS library increased the identification confidence of creatinine for a detected feature (114.0656 m/z, 122.69 CCS), distinguishing among 6 hits (0.01 mass tolerance) from the METLIN database. Work is in progress to compare and examine in detail the benefits of incorporating predicted and experimental CCS libraries in the metabolite identification workflow. </div><p></p

Enabling Massive Peptide Library Search Using GPU-FLASH (Cascadia Proteomics Symposium 2017)

Microbiome research has opened new frontiers in public health and environmental stewardship. However, protein sequence database for these complex microbial communities are often incomplete or unavailable, which limits options for spectral annotation. Spectral library search is an efficient method for MS/MS identification, but library sizes can be prohibitively large for microbiome research. Standard techniques apply a precursor ion window to filter candidates for an exact match, which can easily overlook many possible homologous matches. Emerging open library search methods appear promising, but have yet to be tested at the scale necessary for microbial communities. This calls for an efficient open spectral library search approach which can perform open search across a spectral library within a reasonable time frame. As a solution we present a GPU-accelerated, highly efficient pairwise similarity algorithm which can shortlist candidate spectra from a spectral library after performing all to all comparison. Our preliminary results show that open search for 35,000 spectra against a library of 1.18 million spectra takes approximately 45 mins, which is similar to a database search for a small bacterial organism

Effective coupling of CE with nanoESI MS via a true sheathless metal-coated emitter interface for robust and high sensitivity sample quantification (ASMS 2016)

<p>Capillary electrophoresis (CE) coupled with mass spectrometry (MS) is a promising alternative to conventional liquid chromatography (LC) MS in chemical and biological sample analysis due to its high resolving power and fast separation speed. Reproducibility and ruggedness problems, suffered to a certain degree by almost all the CE-MS interfaces, limit its broad applications. We present the development of a new sheathless CE-MS interface aiming at overcoming these problems and pushing CE-MS suitable to routine sample analysis with high sensitivity. A systematic evaluation of the new interface was performed using a hybrid capillary isotachophoresis (CITP) and capillary zone electrophoresis (CZE) separation coupled with electrospray ionization (ESI) MS for its achievable sensitivity and reproducibility in sample quantification. </p

Mass spectrometry profiling of pentosan polysulfate sodium (PPS) (ASMS 2017)

Pentosan polysulfate (PPS) is a semisynthetic heterogenous sulfated polysaccharide derived from xylan, the β-1,4-linked polymer of xylose. PPS sold by the brand name Elmiron in United States is taken orally to alleviate pain associated with interstitial cystitis. PPS is a mixture of hundreds or more discrete molecules built from a range of oligoxylose lengths modified with different combinations of functional group modifications, including sulfation, 4-O-methyl-glucuronidylation, acetylation, and others. The overall goal of our research is to develop an approach using MS together with other methods such as NMR to profile PPS at the molecular level. Profiling PPS according to its molecular composition would be invaluable for understanding biological activity, bioavailability, and pharmacokinetics, as well as for quality control.One Elmiron (100 mg PPS) capsule was extracted with 1 ml of HPLC-grade water, and further dilutions were made with this stock solution. Diluted PPS at a concentration of 0.5mg/ml was treated with an ion exchange resin for few hours, centrifuged and the supernatant collected. To this supernatant butylamine (15mM) and hexafluoroisopropanol (60mM) were added as an ion-pair reagent (final pH ~8.5). The treated sample was fractionated on C18 SPE cartridge using acetonitrile (ACN) starting from concentration of 10% up to 100% ACN. Each fraction was individually analyzed by FTICR and IMS-MS both in positive and negative mode. Agilent drift tube-IMS-QTOF MS and home-built drift tube IMS-MS were used to characterize PPS from different lots and locations of production.The mass spectrum obtained from PPS directly dissolved in water is complex and difficult to interpret due in-source fragmentation of sulfated oligosaccharides and presence of multiple metal ion adducts [M+Na]. We have explored the potential of ion-pair reversed phase chromatography to extract and analyze PPS using C18-SPE followed by MS detection using FTICR and IMS. When each eluate was injected directly in FTICR without any chromatographic separation, most of the PPS eluted in fraction containing 10% and 20% ACN. Analysis of mass spectra revealed presence of multiply charged state species, mostly +2, +3 and +4 for data collected in positive mode. Analysis of deconvulated peaks in positive mode displayed abundant neutral loss of 171.03 across the entire MS1 spectrum. This neutral loss of 171.03 units is most likely coming from the group –OSO₃NH₂(CH₂)₃CH₃ from PPS backbone. IMS-MS is capable of separating molecules that have the same mass-to-charge (m/z) ratio but different sizes, shapes or conformations. Therefore it is appealing for separating PPS with different polymerized sizes and different charge states and for reducing the complexity of mass spectra. Low-molecular-weight heparin, another sulfated oligosaccharide, was used as a standard to develop IMS-MS method. Heparin DP10 which has molecular weight around 3000 Da has shown a 2D IMS-MS spectrum with trend lines for charge +2 and +3 and m/z range from 1000 to 2000. Preliminary data of PPS showed 2D IMS-MS profiles with charge states from +1 to +5 and m/z range from 300 to 2500. These results show that IMS-MS can reduce the complexity of sulfated polysaccharide spectra by additional separation of different charge states and polysaccharide sizes. However the spectra are still complex for peak assignment without any pre-treatment. The uses of ion exchange resin and ion-pairs have shown improved sensitivity and separation in IMS-MS.<p></p

Identifying Chemical Protein Adducts Using a Multipronged Approach (The 2017 Northwest Regional ACS Meeting)

<div>Developed strategies for estimating and controlling false discovery rate(FDR). (implemented in R)</div><div>Screened different potential modifications</div><div>Custom software tool for validation of modification and site of modification</div><div>Confident identification of protein adducts induced by chlorpyrifos-oxon exposure</div

Simultaneous and co-located dual polarity ion confinement and mobility separation in traveling wave-based structures for lossless ion manipulations (SLIM) (ASMS 2017)

Ion mobility (IM) coupled with mass spectrometry has gained prominence as a powerful analytical tool. To advance IM technology performance to higher levels SLIM technology has recently been developed in our laboratory, and has provided the basis for large gains in IM resolution as well as sensitivity. In many applications both positive and negative ion separations provide complementary information. In this work we explore the use of traveling waves in SLIM to simultaneously confine and separate by IM co-located cations and anions. SIMION ion trajectory software was used to simulate ion confinement in SLIM, as well as ion transport. Ion-neutral collisions during ion transport simulations were modelled using the SDS collision model, which employs statistical methods to account for ion collision with buffer gas. The simulations were used to optimize the SLIM design process as well as predict possible experimental performance. MATLAB software package was also used to obtain and analyze the ion confinement potentials. Static voltages applied to guard electrodes in traditional SLIM configurations provide good lateral confinement for single ion polarity experiments, but such conditions lead to the loss of opposite polarity ions. It is well recognized that rf ion traps can simultaneously confine ions of both polarities. In this work we have explored the potential for developing instrumentation allowing the simultaneous introduction, and manipulation (including IM separation) of both positive and negative ions in a new SLIM design. Preliminary data obtained from ion trajectory simulations have shown the possibility to simultaneously confine and transport both positively and negatively charged ions. Simultaneous confinement for ions of both polarities was achieved by replacing the guard electrodes in the traditional SLIM configuration which employed static voltages (typically 5V above the travelling wave (TW) voltage) for lateral confinement of ions between the SLIM boards with RF “guards” which use dynamic voltages for the lateral confinement of the ions. Concurrent ion transport is also achieved due to the nature of the dynamic voltage profile of the TW which presents a potential minima at opposite ends of the voltage wave for each ion polarity as the wave transverses the segmented TW electrodes, and thus subsequently provide efficient ion transport. We have also shown using ion trajectory simulations, the capability to manipulate the spatial separation of ion populations in SLIM based on their polarities, by biasing the RF guards on each side of the ion conduit so as to limit the interactions between the two ion polarity populations if ion-ion interactions could lead to ion loss during transmission. This presentation will also describe our progress in experimental implementation.<p></p