Solving the Unsolved in Mass Spec; Agilent Innovation and Collaboration For Today's Lab

The ultimate goal of targeted metabolomics and lipidomics is to report absolute metabolite and lipid concentrations. The Agilent Metabolomics dMRM Database and Method is a supported solution for 219 central carbon metabolites in negative ion mode.  It is a fully tested rugged method that provides incredibly stable retention times across large numbers of samples with excellent inter-day and extended (over 4000 biological samples) retention-time reproducibility.  
 
In this presentation we show absolute quantitation of fragile metabolites using the dMRM method and incorporating isotope dilution mass spectrometry  (IDMS).  IDMS gets rid of Ionization suppression effects as well as any bias from sample preparation and instrument conditions. The only downside is the cost and availability of isotopically labeled internal standards, especially in an assay with 219 metabolites. We have circumvented the cost of purchasing individual internal standards by using 13C incorporated yeast from Cambridge Isotope Labs.  These cells are grown in 13C enriched media and the resulting metabolites in the extract have >99% isotopic enrichment.  Using this technique, we show linearity for fragile metabolites over six orders of magnitude ranging in most cases from attomole to nanomoles concentrations on column.
 
We will also present the method and results for targeted lipidomics with the use of deuterated internal standards that covers a wide range of lipid classes.

For Research Use Only. Not for use in diagnostic procedures.

Agilent’s LCMSD iQ is a small but powerful mass spectrometer designed for the chromatographer.  Join us to learn about the newest single quad in Agilent’s portfolio and how enhanced hardware and software features reduce the stress when migrating UHPLC UV Methods to MS detection.  

For Research Use Only. Not for use in diagnostic procedures.

Here we showcase the reproducibility, robustness, and analytical sensitivity of MRM-based LC/MS analysis of human plasma proteins using the Evosep One LC system and Agilent 6495 triple quadrupole LC/MS with an Agilent nanospray source. Results demonstrated excellent retention time reproducibility over a 12-day analysis period, and equivalent sensitivity before and after robustness testing, underlining the suitability of the system for high-throughput protein quantification when sample amounts were limited.

For Research Use Only. Not for use in diagnostic procedures.

The process of neurodegeneration is characterized by an accumulation of proteins in aggregated form. This aggregation suggests impairment of protein production or turnover. Hallmarks of Alzheimer’s disease (AD), Parkinson’s disease (PD) and frontotemporal dementia (FTD) include accumulation of these protein aggregates. Multiple factors contribute to the accumulation of aggregated proteins in these diseases including genetic mutations, dysfunctional proteostasis, and other disease related risk factors. The goal of this assay is to characterize changes in lysosomal function across a broad set of samples and disease types utilizing dysregulated proteins identified through discovery proteomics, genomics, and metabolomics/lipidomics. In this assay we use mass spectrometry (MS) based relative quantitation to determine changes in the concentrations of specific proteins over time or treatment. The multiplexed panel measures 77 peptides from 33 proteins involved in the lysosomal biology pathway.

For In Vitro Diagnostic Use. 
 

Per and polyfluoroalkyl substances (PFAS) are chemicals widely used in consumer products and industry due to the unique and desirable chemical properties.  Due to widespread usage and environmental persistence, legacy PFAS have been found in drinking and non-potable water in the United States and around the world. These substances, particularly with carbon-chain lengths >7 are bioaccumulative and have been linked to adverse health effects in humans and wildlife.  Regulatory lists of PFAS compounds are growing, as are state and local monitoring efforts. Targeted detection techniques for quantifying PFAS in water are important and ideally  would both meet the necessary sensitivity and capacity required.  We will explore use of the latest QQQ 6495C  technology and its ability to push sensitivity  lower with more difficult  PFAS such as GenX. Methods will be discussed with an emphasis on sensitive high-throughput  techniques.

For Research Use Only. Not for use in diagnostic procedures.

Since the sanctioning of recreational cannabis use in various U.S. States in recent years, respective lawmakers have introduced their own unique State legislation. These State legislations detail minimum acceptable levels of specified pesticide and mycotoxin content allowed in potential retail material.  This presentation will detail an LC/MS/MS analytical workflow developed by Agilent for the accurate measurement of combined pesticide and mycotoxin action lists according to the requirements of U.S.

Agilent products and solutions are intended to be used for cannabis quality control and safety testing in laboratories where such use is permitted under state/country law.

In a rapidly changing environment with new synthetic drugs appearing almost weekly, there is an analytical need to confidently identify these analytes in a timely manner.  The use of high-resolution accurate mass technology coupled with a novel sample preparation using Agilent’s Captiva EMR-Lipid allows for highly specific, selective and comprehensive screening for the identification of these emerging fentanyl analogues in serum matrix. Captiva EMR–Lipid provides highly selective and efficient lipid/matrix removal without unwanted analyte loss. The novel EMR-Lipid technology removes lipids based on a combination of size exclusion and hydrophobic interaction. Effective lipid removal assures minimal ion suppression of target analytes, which significantly improves method reliability and ruggedness.  This novel sample preparation approach allows for lower limits of detection and quantitation as well as better linearity across the entire dynamic range. 

Immunoassay-based techniques have historically been the methods of choice for drug screening. Positive presumptive drug screen results are reflexed to more specific, confirmatory testing using gas or liquid chromatography coupled to mass spectrometry. False positives and false negatives with immunoassay techniques are common problems that have substantial down-stream consequences for inaccurate results, laboratory operations, and total costs.  Thus, the use of high-resolution accurate mass liquid chromatography mass spectrometry (LC-QTOF-MS) is ideally suited for rapid analysis of emerging drugs without the drawbacks associated with legacy techniques and methodology.  Herein, a targeted and untargeted screening workflow by LC/MS/MS using Agilent’s 6546 QTOF will be presented for approximately 150 fentanyl analogues.
 
For Forensic Use. 
 

Algorithms to increase duty cycle for the acquisition of and increase selectivity for the processing of data containing unknown , but related compounds of interest will be presented.  In most cases, related compounds of interest are not present in anywhere near the intensity of the most abundant components in a sample.  This means that a purely automated data-dependent MSMS experiment may often fail to target these components for an autoMSMS experiment.  Once data are acquired unbiased compound searching algorithms are very efficient at finding compounds, but means that our compounds of interest are but a few among the housands of compounds in a sample mixture.  In addition, if the autoMSMS experiment failed to target the compound of interest, then confidence will require a second targeted acquisition.  The MassHunter acquisition concept of a Preferred MSMS list will be presented and how it can dramatically increase the duty cycle for compounds of interest.  The MassHunter concept of Mass Defect, MSMS pseudo Constant Neutral Loss, and MSMS Reporter Ions will also be presented as time and labor saving for sample data processing.

For Research Use Only. Not for use in diagnostic procedures.

Native mode proteins and protein complexes typically are analyzed using nanospray techniques or capillary LC flow rates with gentle ionization conditions to achieve best responses and preserve the native conformations.  However, a few recent publications suggest that higher flow LC/MS techniques perform acceptably well for native mode protein and protein complexes.  Here we explore the feasibility of high flow approaches for native mode analysis of non-covalent complexes. 

For Research Use Only. Not for use in diagnostic procedures.

Recent advancements in commercial instrumentation have allowed for more routine analysis of large biomolecules (>100 kDa) with extended mass range modes on Orbitrap and Q-TOF mass spectrometers. These improvements in both the hardware and software have also been coupled with the maturation of static nanoelectrospray ionization (nESI) to improve sensitivity and minimize sample consumption. The combination of these improvements has enabled the analysis of large, native proteins and protein complexes with high mass-to-charge (approaching 30,000 m/z) values without significant losses in sensitivity or mass resolution. Herein, we will describe simple modifications to the Agilent electrospray source to accommodate static nanoelectrospray ionization (nESI). This modification in tandem with the improved instrumentation, allow for MS and IM-MS analyses of large protein complexes (>100 kDa) on both the Agilent 6545XT Q-TOF and Agilent 6560 IM-Q-TOF mass spectrometers.
 
For Research Use Only. Not for use in diagnostic procedures.
 
 

Mass spectrometry plays a vital role in the measurement and characterization of synthetic DNA and RNA oligonucleotides (oligos), which are used widely in molecular biology and in the development of therapeutics.  This webinar will highlight (1) the quantitative analysis of oligos, both by QQQ and HRAM technologies, to achieve the best combination of sensitivity and speed, (2) impurities determinations using HRAM, multiple analysis techniques, and purpose-built software, and (3) high-throughput methods that enable the acquisition, analysis, and purity review of over 5,000 samples a day.  Join us to push the speed, sensitivity, and dynamic range of oligo measurements to new heights.

For Research Use Only. Not for use in diagnostic procedures.

Multidimensional measurements integrating liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) provide valuable polarity, structural and mass information simultaneously for lipidomic analyses and show tremendous power for attaining more confident lipid identifications. For all of their advantages, LC-IMS-MS measurements are highly complex and result in huge datasets which are difficult to process in a timely fashion. Thus, developing a data analysis workflow that is capable of accurate and rapid molecular analyses is essential. The freely available, open-source software Skyline offers targeted processing of lipid data which ultimately allows for confident identification of diverse lipid species. We have developed sample-specific lipid spectral libraries which include over 700 target lipids from multiple lipid categories. Each target lipid is populated with m/z values, normalized retention times, ion mobility collision cross section (CCS) values, and known fragmentation patterns. These values were manually extracted from LC-IMS-MS experimental data and verified using existing literature. Recently developed aspects of the Skyline small molecule interface are utilized in this workflow including IMS spectrum filtering and retention time prediction (iRT) using a set of ~20 endogenous lipids for gradient correction and LC alignment. Application of lipid CCS value filtering further increased lipid annotation confidence and greatly improved the signal to noise ratio for the target species. These lipid spectral libraries will be made publically available through Skyline’s online repository Panorama after additional validation studies.

For Research Use Only. Not for use in diagnostic procedures.

Over the last two decades, more than one hundred mAbs have been approved or are in regulatory review in the US and EU. This growth has led to an increasing need for developing rapid technologies to support the characterization of mAbs to secure drug product safety, quality, and efficacy. In this presentation we present several mass spectrometry-based technologies used in a stage appropriate manner to dramatically shorten timelines and improve throughput in biologics research. Examples include; automated and rapid sample preparation, multiplexed workflows, high-resolution SLIM-IMS-MS, RapidFire-MS, and our labs ongoing efforts for an end-to-end automated process from sample preparation to reporting.

For Research Use Only. Not for use in diagnostic procedures.

The talk will present on two specific areas of developments in Ion Mobility - Mass Spectrometry. The first topic is on the development and application of Collision Induced Activation and Unfolding as applied to uncovering structural differences in native proteins. The second topic focuses on the achievements in high resolution ion mobility analysis including Structures for Lossless Ion Manipulation (SLIM) and the use of multiplexed data acquisition and post-acquisition maximum likelihood deconvolution enabled Hadamard transform demultiplexing (HRdm) and its application to biologically relevant small molecules.    

For Research Use Only. Not for use in diagnostic procedures.