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Deep proteome analysis by DIA-MS with FAIMS

ABSTRACT: We aimed to expand the proteome coverage by single-shot measurements using optimizing high-field asymmetric waveform ion mobility spectrometry (FAIMS) parameters in DIA-MS. In addition, we analyzed host proteins in the feces of control and repeated social defeat stress (R-SDS) model mice with optimal FAIMS parameters in DIA-MS.

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Yusuke Kawashima

PROVIDER: PXD029853 | JPOST Repository | Thu Apr 28 00:00:00 BST 2022

REPOSITORIES: jPOST

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			Action	DRS
	60min_oDIA-FAIMS_HEK293_200ng_CV-40V_IT100OT100_1.raw	Raw
	60min_oDIA-FAIMS_HEK293_200ng_CV-40V_IT100OT100_2.raw	Raw
	60min_oDIA-FAIMS_HEK293_200ng_CV-40V_IT100OT100_3.raw	Raw
	60min_oDIA-FAIMS_HEK293_200ng_CV-45V_IT100OT100_1.raw	Raw
	60min_oDIA-FAIMS_HEK293_200ng_CV-45V_IT100OT100_2.raw	Raw

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Publications

Single-Shot 10K Proteome Approach: Over 10,000 Protein Identifications by Data-Independent Acquisition-Based Single-Shot Proteomics with Ion Mobility Spectrometry.

Kawashima Yusuke Y Nagai Hirotaka H Konno Ryo R Ishikawa Masaki M Nakajima Daisuke D Sato Hironori H Nakamura Ren R Furuyashiki Tomoyuki T Ohara Osamu O

Journal of proteome research 20220506 6

The evolution of mass spectrometry (MS) and analytical techniques has led to the demand for proteome analysis with high proteome coverage in single-shot measurements. Focus has been placed on data-independent acquisition (DIA)-MS and ion mobility spectrometry as techniques for deep proteome analysis. We aimed to expand the proteome coverage by single-shot measurements using optimizing high-field asymmetric waveform ion mobility spectrometry parameters in DIA-MS. With our established proteome ana ...[more]

PMID: 35522919

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Deep proteome analysis by DIA-MS

Project description:To expand proteome coverage, the MS range in DIA-MS was examined. In addition, we analyzed host proteins in the feces of control and repeated social defeat stress (R-SDS) model mice by DIA-MS with an optimal MS range.

2022-04-28 | PXD032698 | JPOST Repository

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients

Project description:State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 minute LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

2020-02-17 | PXD016662 | Pride

A fully automated FAIMS-DIA mass spectrometry-based proteomic pipeline

Project description:Here, we present a standardized, “off-the-shelf” proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high throughput and scalability. This integrated pipeline streamlining a fully automated sample preparation platform, data independent acquisition (DIA) coupled with high field asymmetric waveform ion mobility spectrometer (FAIMS) interface, and an optimized library-free DIA database search strategy. Our systematic evaluation of FAIMS-DIA showed single compensation voltage (CV) at -35V not only yields deepest proteome coverage but also best correlates with DIA without FAIMS. Our in-depth comparison of direct-DIA database search engines showed Spectronaut outperforms others, providing highest quantifiable proteins. Next, we apply three common DIA strategies in characterizing human induced pluripotent stem cell (iPSC)-derived neurons and show single-shot MS using single CV(-35V)-FAIMS-DIA results in >9,000 quantifiable proteins with < 10% missing values, as well as superior reproducibility and accuracy compared to other existing DIA methods.

2023-08-22 | PXD029902 | Pride

AGP nLC-MS/MS and nLC-FAIMS-MS/MS

Project description:Bottom-up nLC-MS/MS glycoprotein mass spectrometry workflows rely on the generation of a mixture of unmodified and glycosylated peptides via proteolysis. Such methods are challenged by suppression of hydrophilic glycopeptide ions by more abundant, hydrophobic, and readily ionizable unmodified peptides. Commercially available high-field asymmetric waveform ion mobility spectrometry (FAIMS) devices have recently been introduced and present an opportunity for orthogonal separation following chromatic graphic separation and prior to MS/MS analysis, with potential benefits for glycoproteomic workflows. However, knowledge is lacking regarding the optimal FAIMS conditions for glycopeptide analysis. Here, we document optimal FAIMS compensation voltages for the transmission and analysis of human alpha-1-acid glycoprotein (AGP) tryptic N-glycopeptide ions. Further, we evaluate the effect of FAIMS on AGP glycopeptide assignments by comparing the number of assignments at different confidence intervals using a standard nLC-MS/MS method to an identical method with FAIMS.

2023-03-11 | PXD036667 | Pride

IMPROVED N- AND O-GLYCOPEPTIDE IDENTIFICATION USING HIGH-FIELD ASYMMETRIC WAVEFORM ION MOBILITY SPECTROMETRY (FAIMS)

Project description:Mass spectrometry is the premier tool for identifying and quantifying site-specific protein glycosylation globally. Analysis of intact glycopeptides often requires an enrichment step, after which the samples remain highly complex and exhibit a broad dynamic range of abundance. Here, we evaluated the analytical benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to nano-liquid chromatography mass spectrometry (nLC-MS) for analyses of intact glycopeptide devoid of any enrichment step. We compared the effects of compensation voltage on the transmission of N- and O-glycopeptides derived from heterogeneous protein mixtures using two FAIMS devices. We comprehensively demonstrate the performance characteristics of the FAIMS device for glycopeptide analysis and recommend optimal electrode temperature and compensation voltage (CV) settings for N- and O-glycopeptide analysis. Under optimal CV settings, FAIMS-assisted gas-phase fractionation in conjunction with chromatographic reverse phase separation resulted in a 31% increase in the detection of both N- and O-glycopeptide compared to control experiments without FAIMS. Overall, our results demonstrate that FAIMS provides an alternative means to access glycopeptides without any enrichment providing an unbiased global glycoproteome landscape. In addition, our work provides the framework to verify ‘difficult-to-identify’ glycopeptide features.

2023-10-24 | PXD038673 | Pride

Deep proteomics of HEK293 cells by DIA-MS with FAIMS

Project description:HEK293 cell digest was analyzed by overlapping DIA-MS with FAIMS.

2022-05-26 | PXD026271 | JPOST Repository

Improved identification of proteoforms using FAIMS with internal CV stepping in top-down proteomics

Project description:In top-down (TD) proteomics, fractionation prior to mass spectrometric (MS) analysis is a crucial step for the high confidence identification of proteoforms and increased sample depth. In addition to liquid phase separations, gas-phase fractionation strategies such as field asymmetric ion mobility spectrometry (FAIMS) have been shown to be highly beneficial in TD proteomics. However, the need for multiple injections using different compensation voltages (CV) leads to a huge increase in measurement time and the amount of sample required. Therefore, we here investigated for the first time the use of internal CV stepping for single shot TD analysis, i.e., the application of multiple CVs per acquisition. In addition, MS parameters were optimized for the individual CVs since different CVs target certain mass ranges. For example, small proteoforms identified mainly with lower CVs can be identified with a lower resolution and number of microscans than larger proteins identified mainly with higher CVs. We investigated the optimal combination and number of CVs for different gradient lengths and validated the optimized settings with the low molecular weight proteome of CaCo 2 cells obtained by different sample preparation techniques. Compared to measurements without FAIMS, both the number of identified protein groups (+60-94%) and proteoforms (+46-127%), and their confidence were significantly increased, while the measurement time remained identical. In total, we identified 684 protein groups and 2,675 proteoforms from CaCo-2 cells in less than 24 hours using the optimized multi-CV method.

2022-02-28 | PXD029792 | Pride

Evaluation of disposable-trap column nanoLC-FAIMS-MS/MS for the proteomic analysis of FFPE tissue

Project description:Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving high proteomic depth and limiting overall analysis time. To this end, we evaluated the merits of online coupling of disposable trap column nano-flow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS) and tandem mass spectrometry (nLC-FAIMS-MS/MS). The data shows that ≤ 600 ng of peptide digest should be loaded onto the chromatographic part of the system. Careful characterization of the FAIMS settings enabled the choice of optimal combinations of compensation voltages (CV) as a function of the employed LC gradient time. We found nLC-FAIMS-MS/MS to be on par with stage tip-based off-line high pH reversed phase fractionation in terms of proteomic depth and reproducibility of protein quantification (coefficient of variation ≤ 15% for 90 % of all proteins) but requiring 50% less sample and substantially reducing sample handling. Using FFPE material from lymph node, lung and prostate tissue as examples, we show that nLC-FAIMS-MS/MS can identify 5-6,000 proteins from the respective tissue within a total of 3 hours of analysis time.

2021-11-02 | PXD028136 | Pride

Improved Proteoform Characterization using FAIMS in Top-down Proteomic Analysis of Heterogeneous Samples.

Project description:Field Asymmetric Ion Mobility Spectrometry (FAIMS) used in proteomics studies provides superior selectivity and enables more proteins to be identified by providing orthogonal gas phase separation. We sought to test the performance of cylindrical FAIMS in the identification of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 60% increase in protein identifications and 18% increase in proteoform identifications as compared to samples analyzed without FAIMS. This increase was attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the analytes molecular weight. Collectively these results suggest that FAIMS can enhance top-down proteomics for both discovery and targeted applications.

2021-01-22 | MSV000086748 | MassIVE

Combining FAIMS based Glycoproteomic and DIA Proteomic of reveals widespread proteome alteration in response to changes in N. gonorrhoeae glycosylation.- FAIMS pglO alleles

Project description:Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within members of the Neisseria genus O-linked protein glycosylation plays important roles in virulence and antigenic variation yet our understanding of the substrates of glycosylation are limited. Recently it was identified that even closely related Neisserial species can possess O-oligosaccharyltransferases, pglOs, that possess varying glycosylation specificities suggesting that distinct targeting activities may impact both the glycoprotome as well as the proteome of Neisserial species. Within this work we explore this concept using of Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of differences in the glycoproteomes and proteomes within N. gonorrhoeae strains expressing differing pglO alleles. We demonstrate the utility of FAIMS to expand the known glycoproteome of N. gonorrhoeae and enable comparative glycoproteomics of a recently reported panel of N. gonorrhoeae strains expressing different pglO allelic chimeras (15 pglO enzymes) with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae yet lead to minimal effects on the abundance of glycoproteins. Additionally, while DIA analysis can allow occupancy to be inferred by the absence or presence of peptides known to be modified, we observe a poor correlation between DIA measurements of non-modified versions of glycopeptides and glycoproteomic analysis. Combined this work expands our understanding of the N. gonorrhoeae glycoproteome and supports that the expression of different pglO alleles appears to drive proteomic changes independent of the glycoproteins targeted for glycosylation.

2024-02-01 | PXD044666 | Pride

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