Publications

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Advancements in NETD

We continue to refine the laser alignment and implementation of the IRMPD on the Orbitrap Ascend. Recently we have leveraged these improvements to better sequence modified RNA and RNA therapeutics. Our study highlighting the improved sequencing of phosphorothioate using negative electron-transfer dissociation (NETD) alongside complementary photodissociation methods (IRMPD) was recently published in Molecular and Cellular Proteomics.

Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry

iDiLeu labeling for absolute quantification of metabolites

Quantitative measurement of metabolites is essential to understand biological and disease processes. Absolute quantification is a key method to determine the concentration of metabolites in biological samples. However, MS-based absolute quantification is often challenged by the commercial availability and high costs of isotope-labeled internal standards. Absolute quantification of metabolites with excellent accuracy and precision can be achieved with five-plex iDiLeu labeling without the need of isotope-labeled internal standards.

Absolute quantification of amine metabolites in human cerebrospinal fluid via MS1-centric isotopic N,N-dimethyl leucine (iDiLeu) labeling

An in-depth review of proteomics tools and instrumentation

As our appreciation for the complexity of the proteome has evolved, so have the technologies we use to interrogate its composition. This paper focuses on innovations within the past 5 years in MS-based instrumentation that continue to expand our ability to survey the proteome with ever increasing sensitivity, speed, and flexibility. Because instrument development is a vibrant and active field within proteomics, we focus on tools used widely in the proteomics community.

Instrumentation at the leading edge of proteomics

Exploring mitochondrial stress recovery

Mitochondria are central to many biochemical processes, therefore even moderate impairment could have drastic cellular consequences. To explore cellular strategies for surmounting mitochondrial stress, we conducted a series of chemical and genetic perturbations to Saccharomyces cerevisiae and analysed the cellular responses using deep multiomic mass spectrometry profiling. Collectively, this work reveals a key component of mitochondrial stress recovery and offers a rich resource for further exploration of the broad cellular responses to mitochondrial dysfunction.

Triacylglycerol mobilization underpins mitochondrial stress recovery

Advancing fast protein post-translation modification (PTM) analysis

Following the success using the Orbitrap Astral for rapid protein analysis, we have been exploring it’s use for deep and rapid PTM mapping. In an article published in Nature Communications, we describe the use of a data-independent acquisition (DIA) approach to identify over 29,000 phosphorylation sites within 30 minutes. We applied this rapid phosphoproteomic method to generate a comprehensive mouse phosphoproteome atlas, detecting 81,120 unique phosphorylation sites in 12 hours across 12 tissues. We have continued to expand our PTM efforts to include glycopeptide analysis. In 30 min of analysis time we can identify over 4,000 unique glycopeptides from a sample of glycosylation-enriched mouse brain.

Fast and deep phosphoproteome analysis with the Orbitrap Astral mass spectrometer

High-flow LC separations for faster throughput quantitative proteomics

Classical proteomics experiments offer high-throughput protein quantification but lack direct evidence of the spatial organization of the proteome, including protein–protein interaction (PPIs) networks. While affinity purification mass spectrometry (AP-MS) is the method of choice for generating these networks, technological impediments have stymied the throughput of AP-MS sample collection and therefore constrained the rate and scale of experiments that can be performed. Here, we build on advances in mass spectrometry hardware that have rendered high-flow liquid chromatography separations a viable solution for faster throughput quantitative proteomics using the Orbitrap–Astral mass spectrometer.

Affinity Purification Mass Spectrometry on the Orbitrap−Astral Mass Spectrometer Enables High-Throughput Protein−Protein Interaction Mapping.

Significant improvements in multi-omics analysis

This work highlights our significant efforts in transition of multi-omics single shot technology (MOST) to the nano-flow regime to improve the sensitive of the analysis. This project demonstrates the robustness of the nMOST platform with minimal change in biomolecule quantification across >350 analyses. To further develop the MOST platform we have been exploring chromatographic conditions that would enable the addition of metabolites.

Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST.

Discoveries in deep proteome sequencing

Deeper proteome sequencing is required for the global discovery of protein isoforms. Using six different human cell lines, six proteases, deep fractionation and three tandem mass spectrometry fragmentation methods, we identify a million unique peptides from 17,717 protein groups. Direct comparison with RNA expression data provides evidence for the translation of most nonsynonymous variants. This dataset represents a resource for proteoform discovery and provides direct evidence that most frame-preserving alternatively spliced isoforms are translated.

Read the article: Global Detection of Human Variants and Isoforms by Deep Proteome Sequencing

Achieving deep analysis of the human proteome in less than an hour

In this study, deep analysis of the human proteome in less than 1 h was achieved by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer.

Read the article: The one hour human proteome

Comprehensive quantification of lipids using high-throughput MS with isobaric labelling

Mass spectrometry-based quantitative lipidomics is an emerging field aimed at understanding the relationships between lipidomes and disease development. However, quantifying lipidomes comprehensively in a high-throughput manner is challenging because of the complex structure of lipids. This study shows a diazobutanone-assisted isobaric labelling strategy that can be used as a rapid and robust platform for multiplexed quantitative lipidomics across a broad range of lipid classes, including phospholipids and glycolipids.

Read the full article: Diazobutanone-assisted isobaric labelling of phospholipids and sulfated glycolipids enables multiplexed quantitative lipidomics using tandem mass spectrometry