Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ion Mobility-Assisted Free Radical Initiated Peptide Sequencing v2

ABSTRACT: Free radical-initiated peptide sequencing (FRIPS) is a tandem mass spectrometry technique (MS/MS) that enables radical-based dissociation on instruments only capable of collisional activation. In FRIPS, peptides are chemically-derivatized with a compound that undergoes homolytic cleavage and generates radicals upon collisional activation. These radicals then propagate through the peptide backbone enabling the sequencing of peptide ions. This MS/MS technique has shown promise in sequencing post-translationally modified peptides, but it is typically performed in an MS3 workflow and single-step MS/MS approaches result in the generation of both collisional- and radical-driven dissociation products and highly complex spectra. Recently, our group developed a method to dissociate peptide ions prior to ion mobility analysis within a trapped-ion mobility spectrometry (TIMS) device. In this work, we examine if this CIDtims technique is capable of initiating the homolytic cleavage of the FRIPS precursor. We then examine if the resultant ion mobility separation results in additional assignments of product ions and improved sequence coverage. We demonstrate that activation within the TIMS device does indeed promote robust radical initiation and fragmentation and that the generated product ions are mobility separated enabling facile assignment and increased sequence coverage.

INSTRUMENT(S): timsTOF

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

SUBMITTER: Nicholas Borotto

PROVIDER: MSV000096102 | MassIVE | Tue Oct 15 13:56:00 BST 2024

REPOSITORIES: MassIVE

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Ion Mobility-Assisted Free Radical Initiated Peptide Sequencing

Project description:Free radical-initiated peptide sequencing (FRIPS), a TEMPO-assisted mass spectrometry, has shown potential as a peptide tandem mass spectrometry (MS/MS) tool that can be an alternative to electron-based mass spectrometry and the traditional collision-based methods. However, a couple of instrumentation barriers need to be overcome before this approach can be a practical tool. One of the barriers is the limitation of the FRIPS-based technique to ion trap mass spectrometry or an orbitrap due to the MS3 workflow. To overcome this challenge, the work introduces a recently developed activation within the trapped ion mobility mass spectrometry (TIMS) device that was found to initiate the radical species. More importantly, the generated product ion was mobility separated, which increased the sequence coverage. Overall, coupling the activation within the TIMS cell with the para-TEMPO-Bz-based FRIPS mass spectrometry offers another practical approach that can be utilized for peptide MS/MS analysis

| MSV000095935 | MassIVE

Improved Performance of Positive-ion mode Free Radical-Initiated Peptide Sequencing with p-TEMPO-Bz

Project description:Free radical-initiated peptide sequencing (FRIPS) is a tandem mass spectrometry (MS/MS) technique that generates sequence informative ions via collisionally-initiated radical chemistry. Collision activation (CA) homolytically cleaves an installed radical precursor, initiates radical formation, extensive hydrogen atom transfer, and peptide backbone dissociation. While FRIPS technique shows great promise, when applied to multiply charged derivatized peptide ions, a series of high abundance mass losses are observed which syphon ion abundance from radically generated sequence ions. This loss of ion abundance reduces the sequence coverage generated by FRIPS fragmentation. In this work, we hypothesized that these mass losses were instigated by the ortho-orientation of the radical precursor undergoing facile conversion into five- or six-membered intermediates and that the para-precursor would not undergo this chemistry. To test this assertion, we synthesized para-TEMPO-Bz, conjugated it to these peptides, and collisionally activated each. And indeed, we see the complete elimination of these undesired collisional processes and the significant increase in radical precursor ion abundance. The increase in ion abundance leads to a significant increase in the sequence coverage generated. These results demonstrate that p-TEMPO-Bz significantly improves the performance of positive-ion mode FRIPS and may be a suitable alternative to the currently utilized ortho-TEMPO-Bz-based FRIPS.

2023-01-13 | MSV000091064 | MassIVE

Deep learning the collisional cross sections of the peptide universe from a million experimental values

Project description:The size and shape of peptide ions in the gas phase are an under-explored dimension for mass spectrometry-based proteomics. To explore the nature and utility of the entire peptide collisional cross section (CCS) space, we measure more than a million data points from whole-proteome digests of five organisms with trapped ion mobility spectrometry (TIMS) and parallel accumulation – serial fragmentation (PASEF). The scale and precision (CV <1%) of our data is sufficient to train a recurrent neural network that accurately predicts CCS values solely based on the peptide sequence. Cross section predictions for the synthetic ProteomeTools library validate the model within a 1.3% median relative error (R > 0.99). Hydrophobicity, position of prolines and histidines are main determinants of the cross sections in addition to sequence-specific interactions. CCS values can now be predicted for any peptide and organism, forming a basis for advanced proteomics workflows that make full use of the additional information.

2021-01-18 | PXD019086 | Pride

capsPASEF – Collisional Cross Section Assisted Precursor Selection Applied to Cross-linking Mass Spectrometry

Project description:Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, HCD cell and a time-of-flight mass spectrometer, to interrogate ions at high speeds with on-the-fly fragmentation. Ion mobility is perfectly suited for cross-linking mass spectrometry, which aims to uncover spatial restraints for proteins. The used cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. This technique however suffers in terms of the low abundance of cross-linked peptides, which is partially resolved with enrichable cross-linking reagents. This class of reagents enables selective enrichment of cross-linking reagent modified peptides, resulting in selection of the cross-linked peptide pairs and peptides connected to a partially hydrolyzed reagent – termed mono-links. Even though mono-links carry limited structural information, for experiments aiming to uncover protein interactions in an unbiased manner they are unwanted byproducts. Gas-phase separation by IM has the potential to separate the two products and, indeed, we find separation between mono-links and cross-linked peptide pairs for PhoX cross-linked proteins. Moreover, an easy-to-interpret division at a CCS of 500 Å and a mono-isotopic mass of 2 kDa is present, which can be used for precursor selection. From our experiments on low complexity protein systems, sequencing of 50 - 70% of the mono-links is prevented, allowing the mass spectrometer the focus on sequencing the relevant cross-links. Application to a highly complex lysate focusing provides a 10-50 % increase in detected cross-links.

2020-07-22 | PXD018189 | Pride

Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics

Project description:The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear extracts. We identified a total of 623 unique cross-linking sites for BSA, 670 for the E. coli ribosome, and 1,617 unique cross-links for nuclear extracts, corresponding to 1,088 intra- and 529 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies.

2022-03-17 | PXD027234 | Pride

Ion mobility fractionation coupled to PASEF for enhanced proteome coverage

Project description:Data provided report the use of trapped ion mobility spectrometry (TIMS) to fractionate ions in the gas phase based on their ion mobility (V⋅s/cm2) followed by parallel accumulation serial fragmentation (PASEF) in a quadrupole time-of-flight instrument. TIMS fractionation coupled to DDA-PASEF allowed for the detection of approximately 7,000 proteins and over 70,000 peptides per overall run from 200ng of human (HeLa) cell lysate per injection using a commercial UPLC column with a 90-minute gradient. This project also explored the utility of TIMS fractionation to generate a DDA library for downstream DIA analysis using shorter LC gradients (20 minutes) as well as lower sample input. Using a 20min gradient, we identified 4,092 and 6,654 proteins on average per run, respectively, from 10ng and 200ng of human (HeLa) cell lysate input based on a TIMS-fractionated library consisting of 82,214 peptides derived from 7,615 proteins.

2022-08-12 | PXD033129 | Pride

Effect of Phosphorylation on the Collision Cross Sections of Peptide Ions in Ion Mobility Spectrometry

Project description:The insertion of ion mobility spectrometry (IMS) between LC and MS can improve peptide identification in both proteomics and phosphoproteomics by providing structural information that is complementary to LC and MS, because IMS separates ions on the basis of differences in their shapes and charge states. However, it is necessary to know how phosphate groups affect the peptide collision cross sections (CCS) in order to accurately predict phosphopeptide CCS values and to maximize the usefulness of IMS. In this work, we systematically characterized the CCS values of 4,433 pairs of mono-phosphopeptide and corresponding unphosphorylated peptide ions using trapped ion mobility spectrometry (TIMS). Nearly one-third of the mono-phosphopeptide ions evaluated here showed smaller CCS values than their unphosphorylated counterparts, even though phosphorylation results in a mass increase of 80 Da. Significant changes of CCS upon phosphorylation occurred mainly in structurally extended peptides with large numbers of basic groups, possibly reflecting intramolecular interactions between phosphate and basic groups.

2021-01-14 | PXD019746 | JPOST Repository

MaxQuant software for ion mobility enhanced shotgun proteomics

Project description:Ion mobility can add a dimension to LC-MS based shotgun proteomics which has the potential to boost proteome coverage, quantification accuracy and dynamic range. Required for this is suitable software that extracts the information contained in the four-dimensional (4D) data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification of peptides, proteins and posttranslational modification sites in LC-IMS-MS/MS shotgun proteomics data. We apply it to trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer. A highly parallelizable 4D feature detection algorithm extracts peaks which are assembled to isotope patterns. Masses are recalibrated with a non-linear m/z, retention time, ion mobility and signal intensity dependent model, based on peptides from the sample. A new matching between runs (MBR) algorithm that utilizes collisional cross section (CCS) values of MS1 features in the matching process significantly gains specificity from the extra dimension. Prerequisite for using CCS values in MBR is a relative alignment of the ion mobility values between the runs. The missing value problem in protein quantification over many samples is greatly reduced by CCS aware MBR.MS1 level label-free quantification is also implemented which proves to be highly precise and accurate on a benchmark dataset with known ground truth. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org.

2020-04-23 | PXD014777 | Pride

Online parallel accumulation – serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer

Project description:In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, while mass spectra are acquired in about 100 microseconds with time-of-fight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive due to its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation – serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 800,000 fragmentation spectra in standard 120 min LC runs are easily achievable, which can be used for near exhaustive precursor selection in complex mixtures or re-sequencing weak precursors. MaxQuant identified more than 6,000 proteins in single run HeLa analyses without matching to a library, and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,000 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

2018-11-06 | PXD010012 | Pride

Extending the Separation Space with Trapped Ion Mobility Spectrometry Improves the Accuracy of Isobaric Tag-based Quantitation in Proteomic LC/MS/MS

Project description:Two-dimensional separation by nano-LC and trapped ion mobility spectrometry (TIMS) prior to Q-TOF tandem mass spectrometry significantly improves the accuracy of isobaric tag-based quantitation in proteome analysis without the need for additional measurement time for TIMS insertion. The obtained peak capacity of up to 3,300 per hour in LC/TIMS reduced the co-isolation of precursor ions at the quadrupole analyzer, resulting in more accurate ratios of reporter ions derived from isobaric tags in product ion spectra obtained at the TOF analyzer. We also found that TIMS with a narrow quadrupole isolation window could reduce the ratio compression effect at least as effectively as the synchronous precursor selection method using MS3 scans without compromising sensitivity or coverage. Our results suggest that the short-gradient LC/TIMS/Q/TOF system is an excellent platform for high-throughput proteomics studies.

2020-05-22 | PXD018650 | JPOST Repository

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data