Slice-PASEF: fragmenting all ions for maximum sensitivity in proteomics

Szyrwiel, Łukasz; Sinn, Ludwig; Ralser, Markus; Demichev, Vadim

doi:10.1101/2022.10.31.514544

Cited by 41 publications

(50 citation statements)

References 33 publications

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“…10ng), which can drastically boost the number of quantified proteins. So far, either diluted bulk cell population digests or samples containing multiple cells have been used for this purpose 3,35,39,40 . However, the exact impact of using such high-load (HL) ID transfer approaches remains unclear, especially in terms of quantification accuracy, as peptides that are potentially lost during single-cell processing might be erroneously quantified.…”

Section: Resultsmentioning

confidence: 99%

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Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

Petrosius

Aragon-Fernandez

Üresin

et al. 2022

Preprint

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Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, mass spectrometry-based single-cell proteomics (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carried out comprehensive analysis of orbitrap-based data independent acquisition (DIA) for limited material proteomics. Notably, we found a fundamental difference between optimal DIA methods for high- and low-load samples. We further improved our low-input DIA method by relying on high-resolution MS1 quantification, thus more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we were able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we establish a complete experimental scp-MS workflow, combining DIA with accessible single-cell sample preparation and the latest chromatographic and computational advances and showcase our developments by profiling real single cells.

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Section: Resultsmentioning

confidence: 99%

Section: Quantification Quality Of Additional Proteins Gained By High...mentioning

confidence: 99%

Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

Petrosius

Aragon-Fernandez

Üresin

et al. 2022

Preprint

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“…DIA-NN 1.8.2 beta 11 version 18 was used for the targeted extraction of three replicates of each injection amount for both Slice-PASEF and dia-PASEF measurements. The comprehensive HeLa assay spectral library was provided to DIA-NN and library precursors were annotated with the human FASTA database.…”

Section: Dia-nnmentioning

confidence: 99%

“…Szyrwiel et al . benchmarked the Slice-PASEF method using analytical flow (500 μL/min) coupled with a new Vacuum Insulated Probe Heated ElectroSpray Ionization (VIP-HESI) source and timsTOF MS 18 ; however, to achieve the balance between performance and robustness, μLC–MS/MS could be more suitable for proteomics applications 7 . This led us to evaluate the performance of microflow chromatography for quantitative proteome analysis in Slice-PASEF and dia-PASEF modes using a sensitive timsTOF ion mobility MS. For most proteomics applications, the Bruker timsTOF MS is coupled with nanoflow rate chromatography through the CaptiveSpray (CS) ion source.…”

Section: Introductionmentioning

confidence: 99%

Vacuum Insulated Probe Heated ElectroSpray Ionization source (VIP-HESI) enhances micro flow rate chromatography signals in the Bruker timsTOF mass spectrometer

Midha

Morrone

Kapil

et al. 2023

Preprint

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By far the largest contribution to ion detectability in liquid chromatography-driven mass spectrometry-based proteomics is the efficient generation of peptide ions by the electrospray source. To maximize the transfer of peptides from liquid to a gaseous phase to allow molecular ions to enter the mass spectrometer at micro-spray flow rates, an efficient electrospray process is required. Here we describe superior performance of new Vacuum-Insulated-Probe-Heated-ElectroSpray-Ionization source (VIP-HESI) coupled with micro-spray flow rate chromatography and Bruker timsTOF PRO mass spectrometer. VIP-HESI significantly improves chromatography signals in comparison to nano-spray ionization using the CaptiveSpray source and provides increased protein detection with higher quantitative precision, enhancing reproducibility of sample injection amounts. Protein quantitation of human K562 lymphoblast samples displayed excellent chromatographic retention time reproducibility (<10% coefficient-of-variation (CV)) with no signal degradation over extended periods of time, and a mouse plasma proteome analysis identified 12% more plasma protein groups allowing confident large-scale analysis to proceed (1,267 proteins at 0.4% CV). We show that Slice-PASEF mode with VIP-HESI setup is sensitive in identifying low peptide amounts without losing the precision in the quantitation. We demonstrate that VIP-HESI coupled with micro-flow-rate chromatography achieves higher depth of coverage and run-to-run reproducibility for a broad range of proteomic applications.

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“…This approach negatively impacts sensitivity as not all precursors within the mass range of interest are selected for fragmentation during a TIMS scan. Narrowing down each window in the mobility dimension 1/K0 range (0.03-0.045) while concomitantly increasing quadrupole isolation window width as suggested by slicePASEF reduces the number of TIMS cycles required for precursor fragmentation and enhances sensitivity [12]. Recently, Amodei et al demonstrated that a marked improvement in precursor selectivity can be obtained by offsetting successive cycles of isolation windows with respect to each other [13].…”

Section: Introductionmentioning

confidence: 99%

midiaPASEF maximizes information content in data-independent acquisition proteomics

Distler

Łącki

Startek

et al. 2023

Preprint

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Data-independent acquisition (DIA) approaches provide comprehensive records of all detectable precursor and fragment ions. Here we introduce midiaPASEF, a novel DIA scan mode using mobility-specific micro-encoding of overlapping quadrupole windows to optimally cover the ion population in the ion mobility-mass to charge plane. Using overlapping ion mobility-encoded quadrupole windows, midiaPASEF maximizes information content in DIA acquisitions which enables the determination of the precursor m/z of each fragment ion with a precision of less than 2 Th. The MIDIAlyzer pipeline integrates algorithms for multidimensional peak detection and for machine-learning-based classification of precursor-fragment relationships. MIDIAlyzer enables fully automated processing and multidimensional deconvolution of midia-PASEF files and exports highly specific DDA-like MSMS spectra which are suitable for de novo sequencing and can be searched directly with established tools including PEAKS, FragPipe and Mascot. midiaPASEF acquisition identifies over 40 unique peptides per second and provides powerful library-free DIA analyses including phosphopeptidome and immunopeptidome samples.

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Slice-PASEF: fragmenting all ions for maximum sensitivity in proteomics

Cited by 41 publications

References 33 publications

Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

Vacuum Insulated Probe Heated ElectroSpray Ionization source (VIP-HESI) enhances micro flow rate chromatography signals in the Bruker timsTOF mass spectrometer

midiaPASEF maximizes information content in data-independent acquisition proteomics

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