Biology subjects

Heil, L. R.

Publications and source records attributed to Heil, L. R..

10 recordsLinked to original sources

bioRxiv2026

Revisiting resonance-excitation collision-induced dissociation for data-independent acquisition

Data-independent acquisition (DIA) proteomics relies almost exclusively on beam-type collision-induced dissociation (HCD) because its short activation time supports fast acquisition rates required for DIA. However, HCD requires charge state calibration and is therefore imperfect for mixed-charge DIA isolation windows. Resonance-excitation collision-induced dissociation (reCID) offers a promising alternative to HCD for DIA because the ion activation is effectively independent of charge state. Historically, reCIDs longer activation time has been considered too slow for DIA. Here, we revisit reCID for DIA proteomics using a modified Orbitrap Tribrid Apex MultiOmics mass spectrometer (Apex) that recovers acquisition-matrix overhead as additional ion injection time, enabling reCID acquisition rates comparable to HCD. Using matched acquisition rate settings of tryptic HeLa digests, reCID achieved precursor and protein detections similar to HCD when used with Carafe fine-tuned, fragmentation-matched spectral libraries. Library fine-tuning improved reCID precursor detections more than HCD detections, 24% versus 5%, indicating that HCD-trained prediction models are suboptimal for reCID spectra. ReCID also maintained peptide-level quantitative performance, including ions measured per peptide, precision, and accuracy. Across seven NCI cancer cell lines and pooled mixtures, protein abundance rankings were highly conserved between the Apex reCID and HCD methods, and also across Orbitrap Astral Zoom platforms. These results support reCID as a practical fragmentation mode for DIA proteomics.

biochemistry↗

bioRxiv2024

Comet fragment-ion indexing for enhanced peptide sequencing

Fragment ion indexing has significantly improved the efficiency of proteomics database search tools. This work implements fragment ion indexing in Comet, a widely-used, open-source search engine. We demonstrate that this enhancement maintains scoring and identification accuracy while substantially increasing peptide spectral matching speeds across multiple applications, including open modification searches, immunopeptidomics, and real-time searches. Comet-FI reduced search speeds by up to 94%, enabling the rapid analysis of complex data types like immunopeptidomes. Fragment-ion indexing enables Comet to keep pace with modern instrumentation and expanding applications in proteomics, reinforcing its utility in diverse proteomics workflows and its integration with a wide range of proteomics tools and platforms.

bioinformatics↗

bioRxiv2024

Development of highly multiplex targeted proteomics assays in biofluids using the Stellar mass spectrometer

The development of targeted assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to large scale clinical studies. Targeted assays are currently unable to scale to hundreds or thousands of targets. We demonstrate the generation of large-scale assays using a novel hybrid nominal mass instrument. The scale of these assays is achievable with the StellarTM mass spectrometer through the accommodation of shifting retention times by real-time alignment, while being sensitive and fast enough to handle many concurrent targets. Assays were constructed using precursor information from gas-phase fractionated (GPF) data-independent acquisition (DIA). We demonstrate the ability to schedule methods from an orbitrap and linear ion trap acquired GPF DIA library and compare the quantification of a matrix-matched calibration curve from orbitrap DIA and linear ion trap parallel reaction monitoring (PRM). Two applications of these proposed workflows are shown with a cerebrospinal fluid (CSF) neurodegenerative disease protein PRM assay and with a Mag-Net enriched plasma extracellular vesicle (EV) protein survey PRM assay.

biochemistry↗

bioRxiv2024

Hybrid Quadrupole Mass Filter Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics

Targeted mass spectrometry (MS) methods are powerful tools for selective and sensitive analysis of peptides identified by global discovery experiments. Selected reaction monitoring (SRM) is currently the most widely accepted MS method in the clinic, due to its reliability and analytical performance. However, due to limited throughput and the difficulty in setting up and analyzing large scale assays, SRM and parallel reaction monitoring (PRM) are typically used only for very refined assays of on the order of 100 targets or less. Here we introduce a new MS platform with a quadrupole mass filter, collision cell, linear ion trap architecture that has increased acquisition rates compared to the analogous hardware found in the Orbitrap Tribrid series instruments. The platform can target more analytes than existing SRM and PRM instruments - in the range of 5000 to 8000 peptides per hour. This capability for high multiplexing is enabled by acquisition rates of 70-100 Hz for peptide applications, and the incorporation of real-time chromatogram alignment that adjusts for retention time drift and enables narrow time scheduled acquisition windows. Finally, we describe a Skyline external software tool that implements the building of targeted methods based on data independent acquisition chromatogram libraries or unscheduled analysis of heavy labeled standards. We show that the platform delivers ~10x lower LOQs than traditional SRM analysis for a highly multiplex assay and also demonstrate how analytical figures of merit change while varying method duration with a constant number of analytes, or by keeping a constant time duration while varying the number of analytes.

biochemistry↗

bioRxiv2024

A workflow for targeted proteomics assay development using a versatile linear ion trap

Advances in proteomics and mass spectrometry have enabled the study of limited cell populations, such as single-cell proteomics, where high-mass accuracy instruments are typically required. While triple quadrupoles offer fast and sensitive nominal resolution measurements, these instruments are effectively limited to targeted proteomics. Linear ion traps (LITs) offer a versatile, cost-effective alternative capable of both targeted and global proteomics. We demonstrate a workflow using a newly released, hybrid quadrupole-LIT instrument for developing targeted proteomics assays from global data-independent acquisition (DIA) measurements without needing high-mass accuracy. Gas-phase fraction-based DIA enables rapid target library generation in the same background chemical matrix as each quantitative injection. Using a new software tool embedded within EncyclopeDIA for scheduling parallel reaction monitoring assays, we show consistent quantification across three orders of magnitude of input material. Using this approach, we demonstrate measuring peptide quantitative linearity down to 25x dilution in a background of only a 1 ng proteome without requiring stable isotope labeled standards. At 1 ng total protein on column, we found clear consistency between immune cell populations measured using flow cytometry and immune markers measured using LIT-based proteomics. We believe hybrid quadrupole-LIT instruments represent an economic solution to democratizing mass spectrometry in a wide variety of laboratory settings.

biochemistry↗

bioRxiv2023

Evaluating the performance of the Astral mass analyzer for quantitative proteomics using data independent acquisition

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

biochemistry↗

bioRxiv2022

Dynamic Data Independent Acquisition Mass Spectrometry with Real-Time Retrospective Alignment

We report a data independent acquisition (DIA) strategy that dynamically adjusts the tandem mass spectrometry (MS/MS) windows during the chromatographic separation. The method focuses MS/MS acquisition on the most relevant mass range at each point in time - improving the quantitative sensitivity by increasing the time spent on each DIA window. We demonstrate an improved lower limit of quantification, on average, without sacrificing the number of peptides detected.

bioinformatics↗

bioRxiv2021

A flexible workflow for building spectral libraries from narrow window data independent acquisition mass spectrometry data

Advances in library-based methods for peptide detection from data independent acquisition (DIA) mass spectrometry have made it possible to detect and quantify tens of thousands of peptides in a single mass spectrometry run. However, many of these methods rely on a comprehensive, high quality spectral library containing information about the expected retention time and fragmentation patterns of peptides in the sample. Empirical spectral libraries are often generated through data-dependent acquisition and may suffer from biases as a result. Spectral libraries can be generated in silico but these models are not trained to handle all possible post-translational modifications. Here, we propose a false discovery rate controlled spectrum-centric search workflow to generate spectral libraries directly from gas-phase fractionated DIA tandem mass spectrometry data. We demonstrate that this strategy is able to detect phosphorylated peptides and can be used to generate a spectral library for accurate peptide detection and quantitation in wide window DIA data. We compare the results of this search workflow to other library-free approaches and demonstrate that our search is competitive in terms of accuracy and sensitivity. These results demonstrate that the proposed workflow has the capacity to generate spectral libraries while avoiding the limitations of other methods.

bioinformatics↗

bioRxiv2021

Comparison of unit resolution versus high-resolution accurate mass for parallel reaction monitoring

Parallel reaction monitoring (PRM) is an increasingly popular alternative to selected reaction monitoring (SRM) for targeted proteomics. PRMs strengths over SRM are that it monitors all product ions in a single spectrum, thus eliminating the need to select interference-free product ions prior to data acquisition, and that it is most frequently performed on high-resolution instruments, such as quadrupole-orbitrap and quadrupole-time of flight instruments. Here, we show that the primary advantage of PRM is the ability to monitor all transitions in parallel, and that high-resolution data are not necessary to obtain high quality quantitative data. We run the same scheduled PRM assay, measuring 432 peptides from 126 plasma proteins, multiple times on a Orbitrap Eclipse Tribrid mass spectrometer, alternating separate liquid chromatography-tandem mass spectrometry runs between the high resolution Orbitrap and the unit resolution linear ion trap for PRM. We find that both mass analyzers have similar technical precision, and that the linear ion traps superior sensitivity gives it better lower limits of quantitation on over 62% of peptides in the assay. Abstract graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/442680v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@225f7dorg.highwire.dtl.DTLVardef@ab09a9org.highwire.dtl.DTLVardef@1076dfborg.highwire.dtl.DTLVardef@c5a34c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Spontaneous Isomerization of Asp387 in Tau is Diagnostic for Alzheimer's Disease: An Endogenous Indicator of Reduced Autophagic Flux

Amino acid isomerization is a spontaneous chemical modification potentially related to the underlying causes of Alzheimers disease (AD). We demonstrate that data-independent acquisition mass spectrometry can be used to characterize isomerization in complex protein mixtures. Examination of a large cohort of brain tissue samples revealed a striking relationship between isomerization of tau and AD status. Surprisingly, isomerization was found to be more abundant in both autosomal dominant and sporadic AD samples relative to controls. We hypothesize that lower autophagic flux in AD brains accounts for these results. Additional data, including quantitative analysis of proteins related to autophagy, strongly support this hypothesis. For example, isomerization of tau is positively correlated with levels of p62, a recognized indicator of autophagic inhibition. In sum, the data suggest strong ties between isomerization and autophagic flux, which may therefore represent a promising target for future investigations into the therapy and prevention of AD.

neuroscience↗