Biology subjects

Rorrer, L.

Publications and source records attributed to Rorrer, L..

3 recordsLinked to original sources

bioRxiv2026

A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for High Performance Proteomics Analysis

Here we present a novel data independent acquisition (DIA) mass spectrometry (MS) operating mode termed parallel accumulation-mobility aligned fragmentation (PAMAF) that offers enhanced speed and sensitivity of ion fragmentation analysis for nontargeted discovery workflows such as bottom-up proteomics. This mode of operation leverages high-resolution ion mobility (HRIM) separation capabilities of the structures for lossless ion manipulation (SLIM) technology to achieve HRIM-based precursor isolation in place of traditional quadrupole filtering approaches. This PAMAF mode of operation increases the number of features that can be identified per MS1/MS2 acquisition cycle by employing mobility-based time alignment to associate fragment ions with their corresponding precursor ions. By using a high-speed, lossless separation technique for precursor isolation instead of the comparatively slow and wasteful quadrupole filtering method, we can avoid ion losses up to 99% while simultaneously increasing the rate at which precursor ions are sequentially fragmented and detected. Additionally, by storing ions in a trapping region while the previous packet of ions is being analyzed, the PAMAF mode achieves [~]100% ion utilization efficiency. Benchmarking results of LC-PAMAF-MS analysis of a whole cell protein digest showed approximately 6x more protein group identifications compared to a standard data-dependent acquisition (DDA) analysis without HRIM on the same QTOF instrument, and to over 100x improvement for low-load workflows. Quantitative evaluations demonstrated that PAMAF mode could quantify low abundance peptides, including those undetectable by DDA. Additionally, since precursor isolation in PAMAF mode is size-based rather than m/z-based, many coeluting isobars and isomers can be resolved prior to fragmentation to eliminate chimeric spectra that compromise identification accuracy. In this work we also explored the benefits of combining HRIM and quadrupole isolation to achieve improved specificity. This approach, known as DIA-PAMAF mode, further reduces the frequency of chimeric fragmentation spectra, and enabled the detection of over 8,000 protein groups from a HeLa digest analysis. PAMAF mode brings a powerful new technique to the field of proteomics that has the potential to improve the sensitivity and selectivity of mass spectrometry-based proteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/704896v1_ufig1.gif" ALT="Figure 1"> View larger version (114K): org.highwire.dtl.DTLVardef@7984c3org.highwire.dtl.DTLVardef@1fb2fe3org.highwire.dtl.DTLVardef@50d35org.highwire.dtl.DTLVardef@1a62926_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Novel high-resolution ion mobility mass spectrometry for site-specific quantification of the sirtuin-5 regulated kidney succinylome

Protein post-translational modifications (PTMs) dynamically regulate essential biological and cellular processes. Lysine succinylation changes the amino acid charge, potentially affecting protein structures and functions, and dysregulation of protein succinylation may lead to metabolic disorders. Proteome-wide succinylation quantification using proteomic tools remains challenging, especially due to the low abundance of succinylated peptides and the frequent presence of isomeric PTM forms. Ion mobility spectrometry workflows that can differentiate peptidoforms with different PTM distributions represent a powerful strategy to alleviate these challenges. Recently, a new Parallel Accumulation with Mobility Aligned Fragmentation (PAMAF) operating mode for high-resolution ion mobility-mass spectrometry (HRIM-MS) analysis based on the structures for lossless ion manipulation (SLIM) technology was introduced. Here, we first assessed the performance of PAMAF mode for protein succinylation analysis using synthetic succinylated peptides, demonstrating residue-level differentiation of co-eluting isomers and isobars and precise PTM site localization. We leveraged this novel approach to investigate succinylome remodeling in kidney tissues from wild-type and Sirtuin-5 (Sirt5) knock-out mice, a NAD+-dependent lysine de-succinylase. PAMAF acquisitions yielded [~]1,000 confidently identified and accurately quantified succinylated peptides and sites from mouse kidney. Sirt5 regulated succinylation of mitochondrial proteins involved in metabolic processes, including fatty acid oxidation, the tricarboxylic acid cycle, and propionate metabolism.

cell biology↗

bioRxiv2024

Abandoning the Quadrupole for Mass Spectrometry Fragmentation Analysis: Towards 1,000 Hz Speeds with 100% Ion Utilization Using High Resolution Ion Mobility Precursor Isolation

In todays fast-evolving landscape of omics research, tandem mass spectrometry has become a cornerstone for uncovering the complexities of biological systems. Yet, despite its essential role, the technology remains bound by the inherent limitations of quadrupole filters, which throw away up to 99% of the useful ion signal and caps the speed at which fragmentation data can be generated. These deficiencies often force compromises in data depth and accuracy, hindering breakthroughs that tie genomics, proteomics, and metabolomics together. A new technology is poised to break through these performance barriers, unlocking unprecedented capabilities in precision, sensitivity and throughput. This step-change in how mass spectrometry fragmentation analysis is performed will reshape the future of scientific discovery, pushing the boundaries of whats possible. The solution is high resolution ion mobility (HRIM), which offers a means to quickly and efficiently isolate ions prior to fragmentation and detection by a high resolution mass spectrometer (HRMS) while also resolving challenging isomeric and isobaric compounds that lead to chimeric MS/MS spectra. HRIM isolates ions in time as a result of a high speed separation rather than acting as a filter that discards ion signal like the pervasive quadrupole mass analyzer, allowing higher sensitivity analysis to be achieved. Also, since HRIM eliminates the need to hop or sweep electronics control parameters, as is the case with a quadrupole, fragmentation spectral generation can occur at a much faster rate, upwards of 500 Hz. This whitepaper describes an IM/MS methodology first contemplated over twenty years ago and today being positioned as the fastest method for high resolution fragmentation analysis. Revisiting this concept using the latest generation ion mobility technology based on structures for lossless ion manipulation (SLIM), which is the only HRIM technology that delivers similar resolution and range of analysis as a quadrupole, realizes the full potential of this approach to deliver benefits in both speed and sensitivity for high performance MS/MS measurements. This new way of achieving ion fragmentation in complex samples is set to revolutionize the mass spectrometry space, starting in the growing field of proteomics where all researchers are seeking faster methods to achieve more comprehensive proteomic coverage. Due to the advantages of HRIM physics, we predict this will set the bar for high throughput -omics within the coming years and will eventually be as ubiquitous as the quadrupole is today.

biochemistry↗