Enhancement of Proteome Coverage by Ion Mobility Fractionation Coupled to PASEF on a TIMS–QTOF Instrument

Guergues, Jennifer; Wohlfahrt, Jessica; Stevens, Stanley M.

doi:10.1021/acs.jproteome.2c00336

Cited by 22 publications

(24 citation statements)

References 20 publications

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“…In the course of preparing this report Guergues et al. described a method to increase proteome coverage for label‐free quantification using ddaPASEF analysis by partitioning the IM domain over four separate injections that aims to achieve a similar outcome [29]. However, we suggest that our method may have a conceptual advantage for library generation in diaPASEF analysis because the IM‐GPF strategy uses a constant IM range avoiding any potential issues with alignment or calibration in the IM domain.…”

Section: Discussionmentioning

confidence: 99%

A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation

et al. 2023

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Data independent acquisition (DIA/SWATH) MS is a primary strategy in quantitative proteomics. diaPASEF is a recent adaptation using trapped ion mobility spectrometry (TIMS) to improve selectivity/sensitivity. Complex DIA spectra are typically analyzed with reference to spectral libraries. The best‐established method for generating libraries uses offline fractionation to increase depth of coverage. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow DIA windows that cover different mass ranges of the complete precursor space, have been introduced that performed comparably to deep offline fractionation‐based libraries. We investigated whether an analogous GPF‐based approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data. We developed a rapid library generation approach using an IM‐GPF acquisition scheme in the m/z versus 1/K0 space requiring seven injections of a representative sample and compared this with libraries generated by direct deconvolution‐based analysis of diaPASEF data or by deep offline fractionation. We found that library generation by IM‐GPF outperformed direct library generation from diaPASEF and had performance approaching that of the deep library. This establishes the IM‐GPF scheme as a pragmatic approach to rapid library generation for analysis of diaPASEF data.

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Section: Discussionmentioning

confidence: 99%

A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation

et al. 2023

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“…With the availability of advanced data analysis tools, IMS has been utilized to add another dimension to LC‐MS/MS‐based proteomics workflow, thus allowing higher proteomic coverage (Prianichnikov et al, 2020). Very recently, gas phase fractionation via trapped IMS (TIMS) followed by parallel accumulation–serial fragmentation (PASEF) using a quadrupole time‐of‐flight instrument was used to obtain enhanced proteomic coverage using a small sample amount of 10–200 ng (Guergues et al, 2022; Meier et al, 2018). TIMS fractionation coupled to DDA‐PASEF improved protein identifications by ~20% and peptide identifications by ~30% using a 90‐min LC gradient compared to DDA‐PASEF alone (Guergues et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

“…Very recently, gas phase fractionation via trapped IMS (TIMS) followed by parallel accumulation–serial fragmentation (PASEF) using a quadrupole time‐of‐flight instrument was used to obtain enhanced proteomic coverage using a small sample amount of 10–200 ng (Guergues et al, 2022; Meier et al, 2018). TIMS fractionation coupled to DDA‐PASEF improved protein identifications by ~20% and peptide identifications by ~30% using a 90‐min LC gradient compared to DDA‐PASEF alone (Guergues et al, 2022). Therefore, gas‐phase fractionation using a TIMS device offers a new concept for the detection of very low abundant PTMs from complex biological mixtures.…”

Section: Discussionmentioning

confidence: 99%

Uncommon posttranslational modifications in proteomics: ADP‐ribosylation, tyrosine nitration, and tyrosine sulfation

Bashyal

Brodbelt

2022

Mass Spectrometry Reviews

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Posttranslational modifications (PTMs) are covalent modifications of proteins that modulate the structure and functions of proteins and regulate biological processes. The development of various mass spectrometry‐based proteomics workflows has facilitated the identification of hundreds of PTMs and aided the understanding of biological significance in a high throughput manner. Improvements in sample preparation and PTM enrichment techniques, instrumentation for liquid chromatography‐tandem mass spectrometry (LC‐MS/MS), and advanced data analysis tools enhance the specificity and sensitivity of PTM identification. Highly prevalent PTMs like phosphorylation, glycosylation, acetylation, ubiquitinylation, and methylation are extensively studied. However, the functions and impact of less abundant PTMs are not as well understood and underscore the need for analytical methods that aim to characterize these PTMs. This review focuses on the advancement and analytical challenges associated with the characterization of three less common but biologically relevant PTMs, specifically, adenosine diphosphate‐ribosylation, tyrosine sulfation, and tyrosine nitration. The advantages and disadvantages of various enrichment, separation, and MS/MS techniques utilized to identify and localize these PTMs are described.

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“…All raw files were analyzed with DIA-NN (version 1.8.1) , using an in-house spectral library. PQ500 peptide precursors were searched with Spectronaut (version 18.1) against a PQ500 spectral library provided by Biognosys AG.…”

Section: Methodsmentioning

confidence: 99%

timsTOF HT Improves Protein Identification and Quantitative Reproducibility for Deep Unbiased Plasma Protein Biomarker Discovery

Vitko,

Chou,

Nouri Golmaei

et al. 2024

J. Proteome Res.

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Mass spectrometry (MS) is a valuable tool for plasma proteome profiling and disease biomarker discovery. However, wide-ranging plasma protein concentrations, along with technical and biological variabilities, present significant challenges for deep and reproducible protein quantitation. Here, we evaluated the qualitative and quantitative performance of timsTOF HT and timsTOF Pro 2 mass spectrometers for analysis of neat plasma samples (unfractionated) and plasma samples processed using the Proteograph Product Suite (Proteograph) that enables robust deep proteomics sampling prior to mass spectrometry. Samples were evaluated across a wide range of peptide loading masses and liquid chromatography (LC) gradients. We observed up to a 76% increase in total plasma peptide precursors identified and a >2-fold boost in quantifiable plasma peptide precursors (CV < 20%) with timsTOF HT compared to Pro 2. Additionally, approximately 4.5 fold more plasma peptide precursors were detected by both timsTOF HT and timsTOF Pro 2 in the Proteograph analyzed plasma vs neat plasma. In an exploratory analysis of 20 late-stage lung cancer and 20 control plasma samples with the Proteograph, which were expected to exhibit distinct proteomes, an approximate 50% increase in total and statistically significant plasma peptide precursors (q < 0.05) was observed with timsTOF HT compared to Pro 2. Our data demonstrate the superior performance of timsTOF HT for identifying and quantifying differences between biologically diverse samples, allowing for improved disease biomarker discovery in large cohort studies. Moreover, researchers can leverage data sets from this study to optimize their liquid chromatography−mass spectrometry (LC−MS) workflows for plasma protein profiling and biomarker discovery. (ProteomeXchange identifier: PXD047854 and PXD047839).

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Enhancement of Proteome Coverage by Ion Mobility Fractionation Coupled to PASEF on a TIMS–QTOF Instrument

Cited by 22 publications

References 20 publications

A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation

A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation

Uncommon posttranslational modifications in proteomics: ADP‐ribosylation, tyrosine nitration, and tyrosine sulfation

timsTOF HT Improves Protein Identification and Quantitative Reproducibility for Deep Unbiased Plasma Protein Biomarker Discovery

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