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Orbitrap mass spectrometry and high-field asymmetric waveform ion mobility spectrometry (FAIMS) enable the indepth analysis of human serum proteoforms

ABSTRACT: In this study, we describe a straightforward protocol for intact proteoform sample preparation based on depletion of albumin and immunoglobulins followed by simplified fractionation of remaining serum proteins via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography tandem mass spectrometry data acquisition with high field asymmetric waveform ion mobility spectrometry (FAIMS), which served as an additional separation dimension to further simplify serum proteoforms mixtures

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Luca Fornelli

PROVIDER: MSV000092574 | MassIVE | Wed Aug 02 17:57:00 BST 2023

REPOSITORIES: MassIVE

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Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.

Kline Jake T JT Belford Michael W MW Boeser Cornelia L CL Huguet Romain R Fellers Ryan T RT Greer Joseph B JB Greer Sylvester M SM Horn David M DM Durbin Kenneth R KR Dunyach Jean-Jacques JJ Ahsan Nagib N Fornelli Luca L

Journal of proteome research 20230929 11

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sour ...[more]

PMID: 37774690

Similar Datasets

Orbitrap mass spectrometry and high-field asymmetric waveform ion mobility spectrometry (FAIMS) enable the in-depth analysis of human serum proteoforms

Project description:After molecular weight-based fractionation, we supplemented the traditional liquid chromatography tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS), which served as an additional separation dimension to further simplify serum proteoforms mixtures.

2023-02-16 | MSV000091309 | MassIVE

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

Accurate quantitative proteomic analyses using metabolic labeling and High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS)

Project description:Stable isotope labeling by amino acids in cell culture (SILAC) is routinely used to profile changes in protein and peptide abundance across different experimental paradigms. As with other quantitative proteomic approaches, the detection of peptide isotopomers can be limited by the presence of interference ions that ultimately affect the quality of quantitative measurements. Here, we evaluate high field asymmetric waveform ion mobility spectrometry (FAIMS) to improve the accuracy and dynamic range of quantitative proteomic analyses using SILAC. We compared quantitative measurements for tryptic digests of isotopically labeled protein extracts mixed in different ratios using LC-MS/MS with and without FAIMS. To further reduce sample complexity, we also examined the improvement in quantitative measurements when combining strong cation exchange (SCX) fractionation prior to LC-MS/MS analyses. Using the same amount of sample consumed, analyses performed using FAIMS provided more than 30% and 200% increase in the number of quantifiable peptides compared LC-MS/MS performed with and without SCX fractionation, respectively. Furthermore, FAIMS reduced the occurrence of interfering isobaric ions and improved the accuracy of quantitative measurements. We leveraged the application of FAIMS in phosphoproteomic analyses to profile dynamic changes in protein phosphorylation in HEK293 cells subjected to heat shock for periods up to 20 min. In addition to the enhanced phosphoproteomic coverage, FAIMS also provided the ability to separate phosphopeptide isomers that often co-elute and can be misassigned in conventional LC-MS/MS experiments.

2019-05-28 | PXD012924 | Pride

A high voltage asymmetric waveform generator for FAIMS.

Project description:High field asymmetric waveform ion mobility spectrometry (FAIMS) has been used increasingly in recent years as an additional method of ion separation and selection before mass spectrometry. The FAIMS electrodes are relatively simple to design and fabricate for laboratories wishing to implement their own FAIMS designs. However, construction of the electronics apparatus needed to produce the required high magnitude asymmetric electric field oscillating at a frequency of several hundred kilohertz is not trivial. Here we present an entirely custom-built electronics setup capable of supplying the required waveforms and voltages. The apparatus is relatively simple and inexpensive to implement. We also present data acquired on this system demonstrating the use of FAIMS as a gas-phase ion filter interface to an ion trap mass spectrometer.

| S-EPMC2900502 | biostudies-literature

pFAIMS: An optimized workflow for quantitative multiplexed phosphorylation analysis using high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS)

Project description:Protein phosphorylation is vital for the regulation of cellular signaling. Isobaric tag-based proteomic techniques, such as tandem mass tags (TMT), can measure the relative phosphorylation states of peptides in a multiplexed format. However, the overall low stoichiometry of protein phosphorylation constrains the analytical depth of phosphopeptide analysis by mass spectrometry, thereby requiring robust and sensitive workflows. Here we evaluate and optimize high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid mass spectrometers for the analysis of TMT10plex-labeled phosphopeptides. We determined that using FAIMS-SPS-MS3 with three compensation voltages (CV) in a single method minimizes inter-CV overlap and maximizes peptide coverage (e.g., CV=-40V/-60V/-80V) and that consecutive analyses using CID-MSA and HCD fragmentation at the MS2 stage increases the depth of phosphorylation analysis.

2020-01-10 | PXD014593 | Pride

Separation of cis and trans Isomers of Polyproline by FAIMS Mass Spectrometry.

Project description:High field asymmetric waveform ion mobility spectrometry (FAIMS) is well-established as a tool for separating peptide isomers (sequence inversions and post-translationally modified localization variants). Here, we demonstrate the FAIMS is able to differentiate cis and trans isomers of polyproline. Polyproline assumes an all-cis conformation-the PPI helix-in 1-propanol, and an all-trans conformation-the PPII helix-in aqueous solutions. Differentiation of these conformers may be achieved both through use of a cylindrical FAIMS device and a miniaturized ultrahigh field planar FAIMS device. Graphical Abstract ?.

| S-EPMC5088216 | biostudies-literature

Comprehensive LESA Mass Spectrometry Imaging of Intact Proteins by Integration of Cylindrical FAIMS.

Project description:The benefits of high field asymmetric waveform ion mobility spectrometry (FAIMS) for mass spectrometry imaging of intact proteins in thin tissue sections have been demonstrated previously. In those works, a planar FAIMS device coupled with a Thermo Elite mass spectrometer was employed. Here, we have evaluated a newly introduced cylindrical FAIMS device (the FAIMS Pro) coupled with a Thermo Fusion Lumos mass spectrometer for liquid extraction surface analysis mass spectrometry imaging of intact proteins in thin tissue sections from rat testes, kidney, and brain. The method makes use of multiple FAIMS compensation values at each location (pixel) of the imaging array. A total of 975 nonredundant protein species were detected in the testes imaging dataset, 981 in the kidney dataset, and 249 in the brain dataset. These numbers represent a 7-fold (brain) and over 10-fold (testes, kidney) improvement on the numbers of proteins previously detected in LESA FAIMS imaging, and a 10-fold to over 20-fold improvement on the numbers detected without FAIMS on this higher performance mass spectrometer, approaching the same order of magnitude as those obtained in top-down proteomics of cell lines. Nevertheless, high throughput identification within the LESA FAIMS imaging workflow remains a challenge.

| S-EPMC7145278 | biostudies-literature

A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol

Project description:Streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. Protocol is based on direct ligation of a fluorescently labeled adapter to the 3’end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation time from 4-5 to 2 days and increases sensitivity by 10-100-fold.

2020-12-29 | GSE163925 | GEO

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

High Field Asymmetric Waveform Ion Mobility Spectrometry in Non-targeted Bottom-up Proteomics of Dried Blood Spot

Project description:Dried Blood Spots samples are a rich source of proteins and have therefore great potential for proteomics biomarker discovery studies. Only a few articles have used DBS samples for non-targeted bottom-up protein analysis, probably due to the complexity of the DBS samples. Gas-phase separation by ion mobility spectrometry such as Field Asymmetric Waveform Ion Mobility (FAIMS) could be useful in analysis of DBS samples as FAIMS has previously shown to be advantageous for bottom-up protein analysis of complex samples. The aim of this project was therefore to evaluate FAIMS in non-targeted analysis of Dried Blood Spots.

2018-05-03 | PXD007926 | Pride

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