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 the selective and sensitive analysis of peptides identified in global discovery experiments. Selected reaction monitoring (SRM) is the most widely accepted clinical MS method due to its reliability and performance. However, SRM and parallel reaction monitoring (PRM) are limited in throughput and are typically used for assays with around 100 targets or fewer.

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.

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.

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.

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.

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

Data-independent acquisition (DIA) mass spectrometry has grown in popularity in recent years, because of the reproducibility and quantitative rigor of a systematic tandem mass spectrometry (MS/MS) sampling method. However, traditional DIA methods may spend valuable instrument time acquiring MS/MS spectra with no usable information in them, affecting sensitivity and quantitative performance. We developed a DIA strategy that dynamically adjusts the MS/MS windows during the chromatographic separation.

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.

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. PRM's 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.

Highly Multiplex Targeted Proteomics Enabled by Real-Time Chromatographic Alignment.

Targeted mass spectrometry methods produce high-quality quantitative data in terms of limits of detection and dynamic range, at the cost of a substantial compromise in throughput compared to methods such as data independent and data dependent acquisition. The logistical and experimental issues inherent to maintaining assays of even several hundred targets are significant. Prominent among these issues is the drift in analyte retention time as liquid chromatography (LC) columns wear, forcing targeted scheduling windows to be much larger than LC peak widths.

Combining native and 'omics' mass spectrometry to identify endogenous ligands bound to membrane proteins.

Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.

Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates.

Proteome coverage and peptide identification rates have historically advanced in line with improvements to the detection limits and acquisition rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid, the acquisition rate has been limited primarily by the duration of the ion accumulation and analysis steps. It is shown here that the spectral acquisition rate can be significantly improved through extensive parallelization of the acquisition process using a novel mass spectrometer incorporating quadrupole, Orbitrap, and linear trap analyzers.

Evaluation of front-end higher energy collision-induced dissociation on a benchtop dual-pressure linear ion trap mass spectrometer for shotgun proteomics.

We report the implementation of front-end higher energy collision-induced dissociation (fHCD) on a benchtop dual-pressure linear ion trap. Software and hardware modifications were employed, described in detail vide-infra, to allow isolated ions to undergo collisions with ambient gas molecules in an intermediate multipole (q00) of the instrument. Results comparing the performance of fHCD and resonance excitation collision-induced dissociation (RE-CID) in terms of injection time, total number of scans, efficiency, mass measurement accuracy (MMA), unique peptide identifications, and spectral quality of labile modified peptides are presented.

On the time scale of internal energy relaxation of AP-MALDI and nano-ESI ions in a quadrupole ion trap.

Recently reported results (Konn et al. [14]) on the collisional cooling of atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) and nano-electrospray ionization (nano-ESI) generated ions in a quadrupole ion trap mass spectrometer (QITMS) are inconsistent with measured collisional cooling rates. The work reported here presents a re-examination of those previous results.

Mapping the distribution of ion positions as a function of quadrupole ion trap mass spectrometer operating parameters to optimize infrared multiphoton dissociation.

Infrared multiphoton dissociation (IRMPD) combined with ion trajectory simulations has been used to obtain probability maps of ion position as a function of different operating parameters in a quadrupole ion trap mass spectrometer. The factors that contribute to the depth of the pseudopotential trapping well are analyzed, and their effects on the efficiency of IRMPD are demonstrated. Ion trajectory simulations are used to substantiate experimental results and demonstrate in greater detail the dynamic nature of the ion population's positional distribution.

Thermally assisted collision-induced dissociation in a quadrupole ion trap mass spectrometer.

Thermally assisted collision-induced dissociation (TA-CID) provides increased dissociation in comparison with CID performed at ambient temperature in a quadrupole ion trap mass spectrometer. Heating the bath/collision gas during CID increases the initial internal energy of the ions and reduces the collisional cooling rate. Thus, using the same CID parameters, the parent ion can be activated to higher levels of internal energy, increasing the efficiency of dissociation and the number of dissociation pathways.