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SUMS Seminar Series

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Thursday, March 7th, 2024

Pioneering the Next Generation of Mass Spectrometry-Based Plasma Proteomics Biomarker Study

Analyzing biomarkers in plasma offers a minimally invasive and accessible method for disease diagnosis, including early cancer detection and translational medicine research. Untargeted proteomic mass spectrometry (MS) utilizing data independent acquisition (DIA) provides an unbiased, precise, and accurate approach to interrogate the human plasma proteome. However, plasma proteomics has historically been challenging due to sample complexity, instrument sensitivity, depth of coverage, and scalability. Recent advances in nanoparticle sample preparation with the Seer ProteographTM and MS instrumentation (i.e. Bruker timsTOF HT, Sciex ZenoTOF and Thermo Astral) have enabled the detection of thousands of proteins at high throughput, which is enabling a new era in plasma proteomics. Through a combination of our prospectively collected multi-cancer cohort, ProteographTM, orthogonal peptide fractionation techniques and the latest MS instruments, we have generated the deepest plasma proteomics spectral libraries to date. The impact of these new libraries was demonstrated by application to one of our previous multi-cancer studies, which resulted in a 2x increase in protein groups. As a demonstration of the capabilities of these technologies, we’ve conducted an untargeted biomarker study on ~3,000 subject multi-cancer cohort using multiple EvosepOne and timsTOF HT instruments. Across 2470 subjects, we’ve detected 8,335 protein groups (PGs) and 113,634 peptides at high throughput & precision. Among those identified PGs in the study, we’ve further investigated the statistic significancy of protein group level detected across 5 nano particles between different cohort conditions.

Jimmy Yi Zeng PhD: Associate Director of Mass Spectrometry, prognomiQ

 

12 - 1 pm in Bass Biology Conference Room 122 with lunch provided

 

Thursday, February 1st, 2024

Systematic mapping of the human cell surface proteome using proximity labeling and quantitative proteomics

The human cell surface is a highly heterogenous environment that harbours proteins critical for immune cell activation, cell-cell interactions, and cellular signaling among many other tasks. Despite the importance of proteins found on the cell surface, comprehensive analyses of the cell surface proteome have been lacking. Using a combination of proximity labeling approaches and an optimized DIA mass spectrometry workflow for quantitative proteomics we have developed an approach for identifying and determining the arrangement of cell surface proteins in a systematic manner. Overall, this approach grants the opportunity to probe functional clusters on the cell surface and provides insight for informed cell surface engineering.

Brendan Floyd PhD: Postdoctoral Scholar-Bertozzi Lab, Department of Chemistry at Stanford University

 

12 - 1 pm in Bass Biology Conference Room 122 with lunch provided

 

Thursday, January 18th, 2024

Answering what and where in complex samples: Advances in Imaging mass spectrometry and the impact of high-resolution mass measurement and ion mobility

This discussion will cover the advances in Imaging mass spectrometry techniques and technology using practical examples of molecular discovery and quantitation. We’ll cover ionization techniques, analysis tools and data pipelines that match spatial features on tissue with chemical signals like drugs and metabolites and potential biomarkers.  

Roy Martin, PhD - Principal Field Marketing Manager for Biological Mass Spectrometry at Waters Corporation

12 - 1 pm in Bass Biology Conference Room 122 with lunch provided

RSVP Here by Friday, 01/12/2024

Thursday, December 7th, 2023

Structural characterization of lipids using electron-activated dissociation (EAD): From plasma analysis to improved quantitative measurements

The analysis of lipids by mass spectrometry is the foundation of modern lipidomics. Using electrospray ionization (ESI), most lipids can be readily quantified in targeted assays. However, discovery experiments, such as data-dependent acquisition (DDA) methods, have proven to be more difficult to achieve in terms of specificity and structural characterization. This challenge arises from the extensive inter- and intra-molecular isobaric overlap among lipids. Collision-induced dissociation (CID), the primary fragmentation method used for lipid analysis, generates few diagnostic ions other than the lipid head group and the acyl chains, which are fragments often shared among isobars. Consequently, discovery experiments generally identify lipids at the sum composition or fatty acid level, which lack structural specificity. Recent advances in mass spectrometry have produced an alternative fragmentation mechanism, EAD, that provides structurally diagnostic fragment ions to enable the complete characterization of a lipid molecule by mass spectrometry.

In this seminar, the use of EAD to fully characterize lipids structurally will be demonstrated using the SCIEX ZenoTOF 7600 system. This instrument has a tunable electron beam to produce fragments from singly charged lipids that can be used to identify lipids by their class, fatty acid composition, fatty acid position, double bond position(s) and double bond stereochemistries. These data enable the complete structural identification of lipid molecular species. The results presented will demonstrate that this level of characterization can be achieved on a liquid chromatography (LC) time scale, which enables high-throughput data acquisition in samples of diverse origins. The basic mechanisms of lipid fragmentation will be discussed using human plasma as an example but the use of this technique extends beyond traditional discovery experiments to quantitative applications that leverage EAD to increase the speed and specificity of assays. The data presented will show that the ZenoTOF 7600 system with EAD is uniquely capable of the specific structural identification of lipid molecular species in simple and complex matrices. 

Paul RS Baker, PhD - Senior Staff Scientist at Sciex

12 - 1 pm in Bass Biology Conference Room 122 with lunch provided

RSVP Here by Friday, 12/01/2023

Thursday, November 2nd, 2023

Leveraging proteomics for biomarker and pathway discovery in neurodegeneration

Neurodegeneration is a broad set of complex diseases that are increasingly associated with genetic risk factors related to cellular homoeostasis, lysosomal function, and glial biology in the brain. The difficulty in developing therapeutics targeting these diseases necessitates tools to probe the increasingly novel biology and complex interplay between normal and pathological function at the protein level. Recent advances in mass spectrometry instrumentation have brought unparalleled increases in speed, sensitivity and capability that has unlocked new opportunities for proteomics in therapeutic research and biomarker discovery. I will discuss how we have set up and utilized the timsTOF platform to expand our proteomics capabilities with increased depth and reproducibility and how we support discovery biology and biomarker programs. 

Neal Gould, PhD - Principal Proteomics Scientist, Denali Therapeutics

12 - 1 pm in Bass Biology Conference Room 122 with lunch provided

RSVP here by Monday, 10/30

Thursday October 26, 2023

Next-generation Protein Sequencing™: Understanding single-molecule amino acid changes with Platinum™

Next-generation protein sequencing on Platinum enables individual amino acid interrogation with the use of semiconductor chips and fluorescently labeled-n-terminal amino acid recognizers, which leads to deeper understanding of proteins and their impact on cell function.  
David Dickey, PhD - Senior Field Applications Scientist, Quantum-Si Inc.
 
12 - 1 pm in Bass Biology Conference Room 122 with lunch provided
RSVP here by Monday 10/23
Thursday, January 18th, 2024

Answering what and where in complex samples: Advances in Imaging mass spectrometry and the impact of high-resolution mass measurement and ion mobility

This discussion will cover the advances in Imaging mass spectrometry techniques and technology using practical examples of molecular discovery and quantitation. We’ll cover ionization techniques, analysis tools and data pipelines that match spatial features on tissue with chemical signals like drugs and metabolites and potential biomarkers.  

Roy Martin, PhD - Principal Field Marketing Manager for Biological Mass Spectrometry at Waters Corporation

12 - 1 pm in Bass Biology Conference Room 122 with lunch provided

RSVP Here by Friday, 01/12/2024

Thursday, December 7th, 2023

Structural characterization of lipids using electron-activated dissociation (EAD): From plasma analysis to improved quantitative measurements

The analysis of lipids by mass spectrometry is the foundation of modern lipidomics. Using electrospray ionization (ESI), most lipids can be readily quantified in targeted assays. However, discovery experiments, such as data-dependent acquisition (DDA) methods, have proven to be more difficult to achieve in terms of specificity and structural characterization. This challenge arises from the extensive inter- and intra-molecular isobaric overlap among lipids. Collision-induced dissociation (CID), the primary fragmentation method used for lipid analysis, generates few diagnostic ions other than the lipid head group and the acyl chains, which are fragments often shared among isobars. Consequently, discovery experiments generally identify lipids at the sum composition or fatty acid level, which lack structural specificity. Recent advances in mass spectrometry have produced an alternative fragmentation mechanism, EAD, that provides structurally diagnostic fragment ions to enable the complete characterization of a lipid molecule by mass spectrometry.

In this seminar, the use of EAD to fully characterize lipids structurally will be demonstrated using the SCIEX ZenoTOF 7600 system. This instrument has a tunable electron beam to produce fragments from singly charged lipids that can be used to identify lipids by their class, fatty acid composition, fatty acid position, double bond position(s) and double bond stereochemistries. These data enable the complete structural identification of lipid molecular species. The results presented will demonstrate that this level of characterization can be achieved on a liquid chromatography (LC) time scale, which enables high-throughput data acquisition in samples of diverse origins. The basic mechanisms of lipid fragmentation will be discussed using human plasma as an example but the use of this technique extends beyond traditional discovery experiments to quantitative applications that leverage EAD to increase the speed and specificity of assays. The data presented will show that the ZenoTOF 7600 system with EAD is uniquely capable of the specific structural identification of lipid molecular species in simple and complex matrices. 

Paul RS Baker, PhD - Senior Staff Scientist at Sciex

12 - 1 pm in Bass Biology Conference Room 122 with lunch provided

RSVP Here by Friday, 12/01/2023

Thursday, November 2nd, 2023

Leveraging proteomics for biomarker and pathway discovery in neurodegeneration

Neurodegeneration is a broad set of complex diseases that are increasingly associated with genetic risk factors related to cellular homoeostasis, lysosomal function, and glial biology in the brain. The difficulty in developing therapeutics targeting these diseases necessitates tools to probe the increasingly novel biology and complex interplay between normal and pathological function at the protein level. Recent advances in mass spectrometry instrumentation have brought unparalleled increases in speed, sensitivity and capability that has unlocked new opportunities for proteomics in therapeutic research and biomarker discovery. I will discuss how we have set up and utilized the timsTOF platform to expand our proteomics capabilities with increased depth and reproducibility and how we support discovery biology and biomarker programs. 

Neal Gould, PhD - Principal Proteomics Scientist, Denali Therapeutics

12 - 1 pm in Bass Biology Conference Room 122 with lunch provided

RSVP here by Monday, 10/30

Thursday October 26, 2023

Next-generation Protein Sequencing™: Understanding single-molecule amino acid changes with Platinum™

Next-generation protein sequencing on Platinum enables individual amino acid interrogation with the use of semiconductor chips and fluorescently labeled-n-terminal amino acid recognizers, which leads to deeper understanding of proteins and their impact on cell function.  
David Dickey, PhD - Senior Field Applications Scientist, Quantum-Si Inc.
 
12 - 1 pm in Bass Biology Conference Room 122 with lunch provided
RSVP here by Monday 10/23

Past Seminars

Thursday July 20, 2023
Noon - 1 pm
Bass Biology Conference Room 122

Daniel Hornburg, PhD - Vice President of R&D and Tech Development, Seer Inc.

Deep and Scalable Discoveries in the Human Plasma Proteome

Profile picture of Daniel Hornburg

 

Thursday March 16, 2023
Noon - 1 pm

Bass Biology Conference Room 122
Lunch provided

Roy Martin, PhD - Senior Manager for Biological Mass Spectrometry, Waters Corporation

Advances in Imaging Mass Spectrometry: enabling faster and in-depth analysis of lipids and metabolites using MALDI and DESI

profile photo of Roy Martin, PhD

Imaging mass spectrometry is driven by the need to spatially locate molecules of interest in biologically relevant materials such as biopsies and tissue. Rapid development in the sample preparation, ionization techniques and methodologies and new Mass Spec technology have opened up many new avenues of analysis. Both MALDI and DESI have long been available as ionization modes for imaging and direct sample analysis and now DESI has only been recently developed to match the robustness and simplicity of MALDI methods. This presentation will investigate the state of the art of these two ionization techniques, how they differ and where they might be most applicable. With recent advances similarities in sensitivity (compound specific) and spatial resolution the choice between these can distill down to the consideration of specific chemistry required, the speed of the analysis and the tissue or matrix type.

Dr. LeRoy B. Martin, III is a Senior Manager for Biological Mass Spectrometry at Waters Corporation.  He received a BS in Chemistry from Davidson College in Davidson, NC, and a PhD in Analytical Chemistry from North Carolina State University in Raleigh, NC. At Waters, Roy has been part of the development of the Omics and biological research program since starting in 1992. Currently, his primary role is developing and implementing advanced mass spectrometry solutions in the Americas.  He is currently managing the Biomolecular Research marketing effort in the Americas. He is also charged with evaluating and integrating new technologies and planning for their inclusion either as Waters Corp. offerings or in combinations of instrumentation platforms. Recently he’s been particularly focused on the ion mobility, imaging, and direct analysis platforms and integrating these technologies into a molecular discovery platform.

Thursday May 12, 2022 - SUMS Seminar Series

Daojing Wang, PhD - Founder & CEO, Newomics Inc.

Silicon Microfluidic Chip Platform for Proteomics

Daojing Wang, PhD

Proteomics of small volumes of biological samples down to single cells has progressed rapidly. However, simultaneous achievement of sensitivity, reproducibility, and throughput for LC-MS-based proteomics remains a challenge. Newomics silicon-microfluidic-chip platform has been demostrated to provide novel solutions to address the challenge. Two products will be introduced in this seminar. The first is the M3 emitter, a multinozzle emitter that enables microflow LC-nanospray ESI-MS, which achieves the robustness and throughput with microflow LC, while maintaining the sensitivity of nanoflow ESI-MS. The second is the MEA chip that monothically integrates the M3 emitter with an on-chip LC column, thereby reducing the dead volume and simplifying the plumbing and connection for nanoflow LC-MS. Applications in bottom-up, targeted, and top-down proteomics, as well as native MS analysis of proteins and protein complexes, will be showcased.

View recording
 
Thursday May 5, 2022 - SUMS Seminar Series

Daniel Hornburg - Senior Director, Discovery, Research and Tech Development, Seer Inc.

Optimized nanoparticle-based plasma proteomics with enhanced scale, precision, and depth of coverage for low abundant protein biomarkers

Daniel Hornburg

Intriguingly, most FDA approved biomarkers in blood are high-abundance proteins. Considering that in biology, the utility of quantifying a protein is not expected to be a function of its abundance, this suggests that many biomarkers are yet to be discovered among hard-to-detect, low-abundance proteins. To overcome the current limitation of a deep, unbiased access to the plasma proteome at population scale, we developed a fast and scalable technology that leverages protein-nano interactions. Introducing nanoparticles (NPs) into a biofluid such as blood plasma leads to the formation of selective, specific, and reproducible protein coronas driven by protein-NP affinities, protein abundances, and protein-protein interactions. Importantly, in contrast to targeted approaches, NPs leverage combinations of multiple generic affinities, which across a panel of engineered NPs compress the entire dynamic range enabling quantitative detection of known as well as novel protein variants. We will discuss how combining nanotechnology, biochemistry, mass spectrometry and data science will advance our understanding of the molecular landscape of health and disease and how machine learning can dissect nano-bio interactions to guide the NP engineering process.

View recording
Download .pdf of slides

Thursday April 28, 2022 - SUMS Seminar Series

Connecting LC-MS data analysis with non-MS techniques to provide deeper insights

Eric Carlson, PhD - CEO, Protein Metrics

In person and online:
Bass Biology conference room 122 / Zoom webinar, noon-1 pm

Lunch provided by Protein Metrics

View recording

Eric Carlson

LC-MS data is central to protein characterization and to proteomics research, but it never stands alone. Decisions are made and insights are gained when these data are reviewed in the context of other experimental techniques and meta data related to the sample and the experiment. In this talk, Dr Carlson will review recent updates to the Protein Metrics platform and talk about the “next steps” of informing MS-level experiments with information from biology and process, as well as what all mass spectrometrists already proclaim, that we can inform biology in reverse.

Thursday April 21, 2022 - SUMS Seminar Series

Optimizing Proteolysis for Improved Coverage of Challenging Proteins

Norah Brown, MS - Stanford University
In person and online:
Bass Biology conference room 122 / Zoom webinar, noon-1 pm

Lunch provided by Promega

View recording

Norah Brown

Proteomics mass spectrometry is a powerful tool to characterize various disease states at a molecular level, especially when biological material is in ready supply. However, many investigations remain challenging when they are limited by the types of peptides which are readily detected for proteins of interest. There is increasing need for experimental methods which are capable of detecting specific classes of proteins or post translational modifications efficiently with high throughput. For example, while trypsin remains the standard protease used for discovery proteomics, it can perform poorly depending on the amino acid composition of a target protein. Furthermore, the impact of pH and heat on the stability of trypsin creates a significant bottleneck in the sample preparation pipeline at clinical scale. ProAlanase is a protease that has high proteolytic activity in acidic conditions and digestion times under 2 hours. This enzyme has high specificity for proline and alanine, which has the capability to profile characteristics of histones where lysines and arginines are often modified, membrane-bound proteins which often lack these residues, and other proteins and protein regions which are not readily observed with trypsin. This protease can be used independently, or if sufficient sample is available as a complementary choice to trypsin, and assist in de-novo sequencing or phosphorylation site localization of proteins with proline-rich regions. Here, we optimize proteomic sample preparation to improve throughput for multi-protease analyses by decreasing incubation times for reduction, alkylation, and digestion. Furthermore we investigate the benefits of using ProAlanase as both a complementary and an alternative protease for bottom-up proteomics and peptide mapping. This approach enables rapid digestion of samples and complementary sequence coverage of proline and alanine containing proteins.

Thursday October 1, 2020 - SUMS Seminar Series

Reasons to be excited about current efforts in glycoproteomics

Nick Riley, PhD - Dept. of Chemistry, Stanford University
Zoom webinar, noon-1 pm
View recorded webinar
Download .pdf of slides

 

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.

Thursday September 24, 2020 - SUMS Seminar Series

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
View recorded webinar

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.

Thursday September 3, 2020 - SUMS Seminar Series

Fundamentals: An introduction to MS-based glycoproteomics

Nick Riley, PhD - Dept. of Chemistry, Stanford University
Zoom webinar, noon-1 pm
View recorded webinar
Download .pdf of slides 

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.

Thursday July 23, 2020 - SUMS Seminar Series

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

Speaker: Ryan Leib, PhD
View recorded webinar
Download .pdf of slides
 

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.

Thursday October 15, 2020 - SUMS Seminar Series

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
View recorded webinar

Download .pdf of slides  *** MISSING DOWNLOAD FILE ***

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.

Thursday July 9, 2020 - SUMS Seminar Series

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
View recorded webinar

Download .pdf of slides
 

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.

Thursday April 16, 2020 - SUMS Seminar Series

Fundamentals: New proteomic approaches and essential data handling tips

Kratika Singhal, MS; Rowan Matney, BA
Zoom webinar, noon-1 pm
View recorded webinar
Download .pdf of slides

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.

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