Events

Webinars Available On-Demand

Reasons to be excited about current efforts in glycoproteomics

Nick Riley, PhD - Dept. of Chemistry, Stanford University
Zoom webinar, noon-1 pm
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Glycoproteomics is a rapidly developing field, driven by improvements in sample preparation, instrumentation, and post-acquisition software. Recent years have ushered in a wave of new glycoproteomics studies, both as mass spectrometrists learn how to best apply their expertise to the needs of glyco-analysis and as MS methods become more democratized for glycobiologists to use. Here we will cover new developments in 1) preparation of glycoproteomic samples, 2) quantitation strategies used for glycopeptide characterization, 3) the most useful data acquisition strategies for various types of glycopeptides, 4) software for interpretation of glycopeptide spectra, and 5) tools for data visualization and meta-analysis. It will be difficult for this overview to be truly comprehensive or thoroughly detailed, but the hope is that this discussion will provide a springboard to generate more interest in glycoproteomic advances and will point interested parties to relevant starting points for continued learning.

The role of host arginases in murine malaria

Nicole Davis, PhD - Dept. of Microbiology & Immunology, Stanford University School of Medicine
Zoom webinar, noon-1 pm
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Malaria, a deadly disease caused by Plasmodium parasites, remains a global health threat. Vascular distress in malaria is thought to be caused by depletion of the vasodilator nitric oxide and its amino acid precursor arginine. Arginine is depleted in the plasma of malaria patients, but the causes of hypoargininemia remain incompletely understood. We sought to determine the cause(s) of arginine deletion in a Plasmodium chabaudi murine model of malaria. In a metabolic survey of P. chabaudi-infected mice, we noted an inverse relationship between plasma arginine and alanine aminotransferase (ALT), a plasma marker for hepatocellular injury. Injured hepatocytes also release arginine-consuming arginase-1 (Arg1) into circulation, which suggested that hepatic Arg1 could deplete arginine following malaria-induced liver injury. We used computational and host genetic tools in combination with LC-MS to test the extent to which hepatic Arg1 and other host arginases deplete plasma arginine during malaria. We found that hepatic Arg1 provided a partial explanation for arginine depletion in P. chabaudi infection, and it may explain arginine depletion in some human populations. Collectively, our work motivates increased attention to the role of malarial liver damage in disrupting host arginine metabolism.

Fundamentals: An introduction to MS-based glycoproteomics

Nick Riley, PhD - Dept. of Chemistry, Stanford University
Zoom webinar, noon-1 pm
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Protein glycosylation is a prevalent, yet heterogeneous co- and post-translational modification (PTM). Glycosylation mediates biophysical and biochemical interactions both intra- and extracellularly, with roles ranging from structural stability, immune regulation, cell proliferation, and intercellular dynamics (to name a few). Similar to other PTMs, e.g., phosphorylation and acetylation, mass spectrometry is the premier method to map site of glycosylation, but challenges inherent to the heterogeneity of glycosylation (in both the glycans that modify proteins and the sites that are modified) make glycoproteomics significantly more difficult. Here we will discuss fundamental mental characteristics of several classes of protein glycosylation, including N-glycosylation and two different types of O-glycosylation (O-GlcNAc vs. mucin-type/O-GalNAc). We will also cover standard workflows used in the field, covering protease considerations, enrichment options, LC-MS/MS methods, and well-established analysis tools. Importantly, we will address shortcomings and challenges that remain in glycoproteomics, too. Glycoproteomics data will be discussed in context with other -omics efforts, e.g., glycomics and standard proteomics, with the ultimate goal that MS researchers unfamiliar with the glyco world will be able to appreciate nuances required in glycosylation analysis.

Mass Spec Fundamentals: what you didn’t know you needed to know

Speaker: Ryan Leib, PhD
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An encore performance of the inaugural seminar of our Fundamentals Series!  In case you missed it in-person, we are streaming and recording this essential presentation.  This seminar will cover the basic foundations of ion generation, manipulation, and detection in a typical LC/MS experiment, and why it matters to your research.  This is a great jumping on point for scientists new to mass spectrometery who are excited to learn a bit about the underlying physical processes that make these experiments possible.

Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

Momei Zhou, PhD - Dept. of Pediatrics, Stanford University School of Medicine
Zoom webinar, noon-1 pm
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Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin infection. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Calcineurin, a cellular phosphatase, was singled out for study and was demonstrated to regulate gB/gH-gL mediated cell fusion via compounds that bind to FKBP1A, which specifically inhibit calcineurin phosphatase activity, led to remarkably enhanced cell fusion. Consistent with a broad role in fusogen modulation, inhibition of calcineurin phosphatase activity enhanced both herpes simplex virus-1 and synctin-1 mediated cell fusion. Further supporting the role of calcineurin phosphatase activity, inhibitor-induced enhanced cell fusion was significantly reduced by FKBP1A knockdown. Importantly, inhibition of calcineurin phosphatase activity during VZV-infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with previous studies. Phosphopeptide enrichment and Orbitrap mass spectrometry identified seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited. This suggests that the dephosphorylation of one or more of those seven proteins is required for calcineurin-dependent fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis.

Improved protein and PTM characterization with a practical electron-based fragmentation on Q-ToFs and ion mobility separations

Joe Beckman, PhD - Distinguished Professor of Biochemistry, Oregon State University, CEO of e-MSion, Inc.
Zoom webinar, noon-1 pm
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Electron-induced fragmentation (ExD) is well known to produce uncluttered spectra of entire proteins with labile post translational modifications preserved, but has not been practical in most mass spectrometers. We have developed an efficient ExD device that can be retrofitted into Q-ToFs and Orbitrap QE instruments.  Once the ECD cell has been optimized to maximize fragmentation of small peptides like substance P, the same parameters also work with slight adjustments for fragmenting native and unfolded proteins. Nearly complete sequence coverage was obtained with “native”-folded proteins. ECD also efficiently cleaves disulfide bonds within proteins to increase coverage.  Sequence coverage of 80-95% was obtained for small proteins like ubiquitin and a-synuclein (14 kDa) during UPLC separations from chromatographic peaks lasting 3-5 seconds. For carbonic anhydrase (29kDa), sequence coverage as 93% (half of the human proteome is smaller than 30kDa). Approximately 90% sequence coverage for each of the three subunits from 0.1 ug of an IdeS-digested antibody was obtained in a five-minute nanoflow separation. The protein spectra consisted primarily of c and z ions, though the ECD cell also produced a substantial number of d and w sidechain fragments. These side-chain fragments allow leucine/isoleucine and isoaspartate/aspartate pairs to be distinguished, which facilitates de novo sequencing. Labile post-translational modifications are also retained, including phosphorylation, glycation and deuterium incorporation. Deuterium labeling of ubiquitin enabled top-down hydrogen/deuterium exchange with residue-specific resolution at rates consistent with NMR. The copper and zinc cofactors in superoxide dismutase (17 kDa) remained bound to their respective binding sites in ECD fragments. The simplified fragmentation patterns made possible with the ExD cell allows existing mass spectrometers to characterize mid-sized proteins even using fast front-end separations including ion mobility separations.  Because the ExD cell supports collisional ion activation and unfolding, more complete sequence coverage can be achieved for large native protein complexes than previously possible on any instrument.

In-depth shotgun triacylglyceride profiling

Matias Cabruja, PhD - Dept. of Genetics, Stanford University School of Medicine
Zoom webinar, noon-1 pm
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Triacylglycerides (TAG) levels are associated with metabolic (e.g. diabetes) and cardiovascular diseases. TAG detailed analysis might be important to better understand their biological properties and their roles in health and disease states. Comprehensive TAG content characterization is methodologically challenging due to the high complexity and similarity. Current high-throughput targeted methods only inform on one fatty acid chain while single species quantification requires extensive LC separations or highly sophisticated (Tribrid) spectrometers. We have developed a high-throughput method to quantify individual TAG species using shotgun MS3.

Brought to you in conjunction with Sciex

Fundamentals: Bioanalytical LC/MS method validation -- fit for purpose

Ludmila Alexandrova, PhD
Zoom webinar, noon-1 pm
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With the increasing importance of research reproducibility and requirements of peer-reviewed scientific journals, the reliability of bioanalytical data and the value of method validation cannot be underestimated.  Unreliable results are subject to misinterpretation and can lead to project failure.  At the same time, extensive method validation is a complicated and time-consuming process and is not to be undertaken lightly.  This seminar will provide an overview of validation guidelines, requirements, approaches, workflow, and data analysis.  Case studies will illustrate the fit-for-purpose concept -- suiting the level of validation to the intended purpose of the study.  Please bring your questions and suggestions.

ASMS review: recap the reboot!

Zoom meeting, noon-1 pm
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What caught your eye at ASMS?  What are your take-homes?  Which presentations would you recommend to others?  Join us to recap the reboot -- compare notes, share highlights, and discuss developments of interest.  With recorded content online until August 31st, get the inside scoop on which sessions to watch on-demand.

Seminar series is on hiatus during the online ASMS Reboot

https://asms.org/conferences/asms-2020-reboot/

Mass spec bloopers!  And other amusing adventures

The lighter side of mass spec.  A collection of the weird and the wonderful -- we couldn't make this stuff up if we tried!

Hear from our intrepid crew:

Zoom meeting, noon-1 pm
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Fundamentals: Applications of LC/MS in small molecule drug discovery

Liquid chromatography-mass spectrometry (LC/MS) is a proven technique of choice for many assays implemented during the various stages of drug discovery and development. Join us for an overview of LC/MS applications in the drug discovery process with emphasis on metabolism and pharmacokinetic studies. We will share real-world project examples with focus on selection of suitable LC/MS instrumentation, method development approaches, sample preparation, data analysis and interpretation.

Ludmila Alexandrova, PhD
Zoom webinar, noon-1 pm
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Breakout discussion topics:

• Implementing new technologies & techniques
  What new areas are looming on your horizon?  Think and talk through approaches to selecting, testing and onboarding new methods in a core lab environment.
   
• Folk wisdom: sample prep, instrument operations, data analysis
  The devil is in the (unpublished) details!  Share best practices for the various stages of a mass spec experiment, both in and out of the lab.
   
• Research restart in the COVID-19 era
  During the unfolding COVID-19 situation, universities including Stanford have ceased non-essential research activities. As we prepare to return to the lab, this forum will be an opportunity to discuss changed workflows, new laboratory safety measures, and phased research restart strategies.
   

Bring your questions, share your experiences, and learn from each other in the hosted breakout discussions; then rejoin everyone in the main meeting room for a report back on key points from each of the discussions.  Note: the breakout room discussions will not be recorded; the summary reports to the recombined group will be recorded and made available here.

Speakers: you, and many others!
Zoom meeting, noon-1 pm
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Fundamentals: Native mass spectrometry & relevant techniques

Beryl Xia, PhD
Zoom webinar, noon-1 pm
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Interested in using mass spectrometry as a tool for structural biology? Curious about emerging  approaches for protein research? This seminar will provide a broad overview of native mass spectrometry and its applications, and touch on both popular and non-commercial emerging techniques relevant to native mass spectrometry. Topics include charge detection mass spectrometry (CDMS), ion mobility spectrometry (IMS), hydrogen-deuterium exchange (HDX), and more.

Fundamentals: Peptide quantitation strategies

Fang Liu, PhD; Beryl Xia, PhD
Zoom webinar, noon-1 pm
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A deeper dive into quantitation techniques specifically for peptides, including selected reaction monitoring (SRM), parallel reaction monitoring (PRM), TOMAHAQ, SureQuant, and more.

Fundamentals: Measuring concentrations of small molecules using mass spectrometry - theory and practice, part II

Karolina Krasinska, MS
Zoom webinar, noon-1 pm
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Part II, with emphasis on the practice: How can I measure concentrations of specific molecules in my sample? What are the possible approaches and how do I determine which one is best for my application?  What do I need to consider when planning such a project? This seminar will answer these questions and more, explaining mass spectrometry-based targeted quantitation from the ground up.  Learn about common pitfalls and strategies to navigate around them. Gain practical tips on data analysis and what your data can and can’t tell you. Bring your questions, learn from your fellow researchers’ questions, and come away with a solid grounding in this core analytical technique.

Fundamentals: New proteomic approaches and essential data handling tips

Kratika Singhal, MS; Rowan Matney, BA
Zoom webinar, noon-1 pm
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Get the inside scoop on both ends of proteomic workflows: from sample prep approaches for experiments like TMTPro-16plex, phosphoproteomics and SureQuant, to what to do with your data once the results are in.

Fundamentals: Intact protein mass spectrometry - tips and best practices

Theresa McLaughlin, MS
Zoom webinar, noon-1 pm
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Mass spectrometry is a powerful tool for analyzing intact proteins.  This seminar will focus on recommendations and practical considerations for success.  Which instruments should you use? How much sample is required? What buffer components can be tolerated?  Which modifications can be detected? How is the data analyzed? We will answer these questions and cover details of the ESI-LC/MS methods used at SUMS for intact protein MW determination.

Fundamentals: Measuring concentrations of small molecules using mass spectrometry - theory and practice, Part I

Karolina Krasinska, MS
Zoom webinar, noon-1 pm
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How can I measure concentrations of specific molecules in my sample? Can I use a mass spectrometer to analyze my samples of interest?  What mass spectrometers are best suited for this purpose? What are the possible approaches and how do I determine which one is best for my application?  What do I need to consider when planning such a project? This seminar will answer these questions and more, explaining mass spectrometry-based targeted quantitation from the ground up.  Learn about common pitfalls and strategies to navigate around them. Gain practical tips on data analysis and what your data can and can’t tell you. Bring your questions, learn from your fellow researchers’ questions, and come away with a solid grounding in this core analytical technique.

Fundamentals: Quantitation strategies in mass spectrometry-based proteomics

Fang Liu, PhD
Zoom webinar, noon-1 pm
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This seminar will cover the most commonly used methods for mass spectrometry‐based quantification in proteomic applications. Quantification can be performed using either unlabeled or labeled approaches. Advantages and disadvantages, including quantification accuracy and reproducibility, will be discussed for each type of approach. This is a great opportunity for scientists who are new to mass spectrometry to learn about options available in qualitative and quantitative proteomics, and which method to choose for their research applications.

Sponsor: PharmaFluidics