IMFrag: A Tool to Recognize In-Source Fragmentation in Ion Mobility-Enabled Data-Independent Acquisition Workflows

High-resolution mass spectrometry is a powerful tool for untargeted analysis. However, in-source fragmentation (ISF) could lead to the misidentification of compounds in untargeted metabolomics or exp...

pubs.acs.org

Bimodal Peptide Collision Cross Section Distribution Reflects Two Stable Conformations in the Gas Phase

Recent high-throughput applications to shotgun proteomics have shown great benefits of coupling ion mobility spectrometry (IMS) to mass spectrometry. IMS adds a separation dimension by differentiating...

pubs.acs.org

From Congestion to Clarity: On the Complementarity of Resolving Power and Spectral Simplification for Intact Protein Characterization

Top-down mass spectrometry (TDMS) is a powerful platform for the structural and functional analysis of intact proteins, enabling the detailed characterization of proteoforms and precise localization of post-translational modifications. The incorporation of alternative fragmentation techniques, such as electron transfer dissociation, electron transfer higher-energy collisional dissociation, and ultraviolet photodissociation, in instruments such as Tribrid Orbitrap mass spectrometers enhances sequence coverage and improves the confidence in PTM assignment. However, tandem mass spectrometry of intact proteins >30 kDa presents substantial challenges. The resulting spectra are often highly complex with overlapping product ion signals that complicate spectral interpretation. Although increasing the mass resolution can help resolve closely spaced product ions, it is often insufficient to fully alleviate spectral congestion for large proteins. In such cases, proton transfer charge reduction (PTCR) can simplify mass spectra by dispersing product ions across a wider mass-over-charge (m/z) range. In this study, we evaluated the impact on TDMS of increasing resolving power and PTCR-enabled spectral simplification using four intact proteins: enolase (46.6 kDa), carbonic anhydrase (29 kDa), myoglobin (16.9 kDa), and ubiquitin (8.6 kDa). For carbonic anhydrase, combined MS2 fragmentation at low resolving power (60,000 at m/z 200) yielded 50.5% sequence coverage, which increased to 92.6% at high resolving power (480,000 at m/z 200) and further to 97.7% when PTCR was applied. This approach was applied to the characterization of biopharmaceuticals by analyzing the three digested and disulfide-reduced ∼25 kDa subunits of the NIST monoclonal antibody (mAb) on a liquid chromatography time scale.

pubs.acs.org

Use of Ion Oscillation Phase Values for Peak Classification and Noise Filtering Efficiency Estimation in Orbitrap Mass Spectra of Natural Organic Matter

An unprocessed high-resolution mass spectrum of natural organic matter contains tens of thousands of peaks, many of which are uninformative and associated with noise. Several noise filtering algorithm...

pubs.acs.org

Enhancing Sensitivity in Targeted Single-Cell Proteomics by Coupling a Dual Ion Funnel Interface with Triple Quadrupole Mass Spectrometer

Single-cell proteomics (SCP) has emerged as a powerful approach for understanding cellular heterogeneity and biological processes at unprecedented resolution. However, the extremely limited protein co...

pubs.acs.org

Zero-Dalton Resolution in Nanopore Peptide Recognition

Achieving zero-dalton resolution─the discrimination of biomolecules with identical mass─remains a fundamental challenge in molecular analysis. Nanopore sensing can resolve short peptide isomers, but w...

pubs.acs.org

Lessons Learned in Orbitrap MS-Based Isotope Ratio Analysis of Organic Acid Mixtures

Stable isotope analysis is a vital tool across chemistry, geology, and environmental science, but conventional Isotope Ratio Mass Spectrometry (IRMS) techniques have limited capabilities for site-spec...

pubs.acs.org

Most mass spectrometers can process just a few molecules at once: Reengineered prototype does a billion simultaneously

Mass spectrometry is already a powerful tool for determining what kind and how many molecules are present in a given sample. But most instruments still analyze their molecules one or just a few at a t...

phys.org

Spatial selection of ions separated in a multi-reflection time-of-flight mass analyzer

Multi-reflection time-of-flight mass analyzers (MR TOF MAs) have become widely used in nuclear physics research due to their combination of high mass …

sciencedirect.com

Pioneer and Altimeter: Fast Analysis of DIA Proteomics Data Optimized for Narrow Isolation Windows

Advances in mass spectrometry have enabled increasingly fast data-independent acquisition (DIA) experiments, producing datasets whose scale and complexity challenge existing analysis tools. Those same advances have also led to the use of narrow isolation windows, which alter MS2 spectra via fragment isotope effects and give rise to systematic deviations from spectral libraries. Here we introduce Pioneer and Altimeter, open-source tools for fast DIA analysis with explicit modeling of isolation-window effects. Altimeter predicts deisotoped fragment intensity as a continuous function of collision energy, allowing a single spectral library to be reused across datasets. Pioneer re-isotopes predicted spectra per scan and combines an intensity-aware fragment index, spectral deconvolution, and dual-window quantification for fast, spectrum-centric DIA analysis. Across instruments, experimental designs, and sample inputs, Pioneer enables high-confidence identification and precise quantification at scale, completing analyses 2-6x faster and maintaining conservative false-discovery rate control.

dlvr.it

Regarding Emitter Positioning for Nanoflow Electrospray Ionization with a High-Capacity Inlet Capillary

Nanoflow electrospray ionization is commonly used for proteomics due to its high sensitivity. Signal intensity, however, is dependent on optimal emitter positioning relative to the mass spectrometer i...

pubs.acs.org

Quantifying the ∼75–95% of Peptides in DIA-MS Data Sets that Were Not Previously Quantified

We have developed a novel algorithm termed GoldenHaystack (GH) that was designed for enhanced peptide quantification of data-independent acquisition liquid-mass spectrometry (DIA-LC-MS) data files reg...

dx.doi.org

A Novel Compact Multi-Reflecting Time-of-Flight Mass Spectrometer

Mass spectrometry is an indispensable tool for the rapid and in-depth analysis of complex mixtures across diverse biologically important fields including metabolomics, lipidomics, and proteomics. Thes...

pubs.acs.org

Characterization and Manipulation of Ion Clouds Inside an FT-ICR Instrument: A Laser Tomography Perspective

The efficiency of many tandem mass spectrometry (MS/MS) techniques depends on the position of the trapped ion cloud and its overlap with a beam of photons or electrons. Here, we report photoactivated ...

pubs.acs.org

Boosting the Speed and Accuracy of Protein Quantification Algorithms in Mass Spectrometry-Based Proteomics

Protein quantification is a crucial data processing step that combines quantitative values at the peptide or fragment level into protein levels in mass spectrometry-based proteomics. However, many of ...

dx.doi.org

dx.doi.org

A Systematic Approach to Selecting the Right Tool for Mass Spectrometry Data Analysis in Protein and Peptide Bioanalysis

Mass spectrometry (MS) has emerged as an imperative technology in protein and peptide bioanalysis, offering unparalleled sensitivity, specificity, and dynamic range. However, the complexity of MS dat...

analyticalsciencejournals.onlinelibrary.wiley.com

Native Charge Detection Mass Spectrometry of Kilobase-Scale Messenger RNAs

Charge detection mass spectrometry (CDMS) enables direct measurement of mass and charge for individual ions and overcomes fundamental limitations of conventional electrospray mass spectrometry for lar...

chemrxiv.org