A simple strategy for straightforward proteome analysis by a combination of Lys-N, strong cation exchange and electron transfer dissociation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ XL
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Hek-293 Cell
SUBMITTER: 
Nadia Taouatas
LAB HEAD: Nadia Taouatas
PROVIDER: PRD000065 | Pride | 2008-10-29
REPOSITORIES: Pride
Dataset's files
Items per page: 5 1 - 5 of 294 |
Similar Datasets
Project description:Negative mode proteome analysis offers access to unique portions of the proteome and several acidic post-translational modifications; however, traditional collision-based fragmentation methods fail to reliably provide sequence information for peptide anions. Negative electron transfer dissociation (NETD), on the other hand, can sequence precursor anions in a high-throughput manner. Similar to other ion-ion methods, NETD is most efficient with peptides of higher charge state because of the increased electrostatic interaction between reacting molecules. Here we demonstrate that NETD performance for lower charge state precursors can be improved by altering the reagent cation. Specifically, the recombination energy of the NETD reaction-largely dictated by the ionization energy (IE) of the reagent cation-can affect the extent of fragmentation. We compare the NETD reagent cations of C16H10●+ (IE = 7.9 eV) and SF5●+ (IE = 9.6 eV) on a set of standard peptides, concluding that SF5●+ yields greater sequence ion generation. Subsequent proteome-scale nLC-MS/MS experiments comparing C16H10●+ and SF5●+ further supported this outcome: analyses using SF5●+ yielded 4637 peptide spectral matches (PSMs) and 2900 unique peptides, whereas C16H10●+ produced 3563 PSMs and 2231 peptides. The substantive gain in identification power with SF5●+ was largely driven by improved identification of doubly deprotonated precursors, indicating that increased NETD recombination energy can increase product ion yield for low charge density precursors. This work demonstrates that SF5●+ is a viable, if not favorable, reagent cation for NETD, and provides improved fragmentation over the commonly used fluoranthene reagent. Graphical Abstract ᅟ.
| S-EPMC5483201 | biostudies-literature
Project description:Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therapeutics. The use of electron transfer dissociation (ETD) facilitates this analysis because disulfide bonds are preferentially cleaved when subjected to ETD. Here, we make use of this well-characterized reaction to assign disulfide bonding networks by coupling the use of extracted ion chromatograms (XICs) of cysteine-containing peptides with ETD analysis to produce an efficient assignment approach for disulfide bonding. This method can be used to assign a disulfide pattern in a de novo fashion, to detect disulfide shuffling, and to provide information on heterogeneity, when more than one disulfide bonding pattern is present. The method was applied for assigning the disulfide-bonding network of a recombinant monomer of the HIV envelope protein gp120. It was found that one region of the protein, the V1/V2 loops, had significant heterogeneity in the disulfide bonds.
| S-EPMC3607449 | biostudies-literature
Project description:For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS(3) by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS(3) techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra, and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.
| S-EPMC3606914 | biostudies-literature
Project description:Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a well established method for the measurement of solution-phase deuterium incorporation into proteins, which can provide insight into protein conformational mobility. However, most HDX measurements are constrained to regions of the protein where pepsin proteolysis allows detection at peptide resolution. Recently, single-amide resolution deuterium incorporation has been achieved by limiting gas-phase scrambling in the mass spectrometer. This was accomplished by employing a combination of soft ionization and desolvation conditions coupled with the radical-driven fragmentation technique electron transfer dissociation (ETD). Here, a hybrid LTQ-Orbitrap XL is systematically evaluated for its utility in providing single-amide deuterium incorporation for differential HDX analysis of a nuclear receptor upon binding small molecule ligands. We are able to show that instrumental parameters can be optimized to minimize scrambling and can be incorporated into an established and fully automated HDX platform making differential single-amide HDX possible for bottom-up analysis of complex systems. We have applied this system to determine differential single amide resolution HDX data for the peroxizome proliferator activated receptor bound with two ligands of interest.
| S-EPMC3796066 | biostudies-literature
Project description:Top-down proteomics offers the potential for full protein characterization, but many challenges remain for this approach, including efficient protein separations and effective fragmentation of intact proteins. Capillary zone electrophoresis (CZE) has shown great potential for separation of intact proteins, especially for differentially modified proteoforms of the same gene product. To date, however, CZE has been used only with collision-based fragmentation methods. Here we report the first implementation of electron transfer dissociation (ETD) with online CZE separations for top-down proteomics, analyzing a mixture of four standard proteins and a complex protein mixture from the Mycobacterium marinum bacterial secretome. Using a multipurpose dissociation cell on an Orbitrap Elite system, we demonstrate that CZE is fully compatible with ETD as well as higher energy collisional dissociation (HCD), and that the two complementary fragmentation methods can be used in tandem on the electrophoretic time scale for improved protein characterization. Furthermore, we show that activated ion electron transfer dissociation (AI-ETD), a recently introduced method for enhanced ETD fragmentation, provides useful performance with CZE separations to greatly increase protein characterization. When combined with HCD, AI-ETD improved the protein sequence coverage by more than 200% for proteins from both standard and complex mixtures, highlighting the benefits electron-driven dissociation methods can add to CZE separations.
| S-EPMC4439324 | biostudies-literature
Project description:Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique which has proven to be an excellent tool for the elucidation of labile post-translational modifications such as phosphorylation and O-GlcNAcylation of serine and threonine residues. However, unlike collision induced dissociation (CID), which has been studied for decades, the intricacies of ETD-based fragmentation have not yet been firmly established or systematically addressed. In this analysis, we have systematically compared the CID and ETD fragmentation patterns for the large majority of the peptides that do not contain such labile modifications. Using a standard 48 protein mix, we were able to measure false-positive rates for the experiments and also assess a large number of peptides for a detailed comparison of CID and ETD fragmentation pattern. Analysis of approximately 19,000 peptides derived from both standard proteins and complex protein samples revealed that (i) CID identified 50% more peptides than ETD; (ii) ETD resulted in approximately 20% increase in amino acid sequence coverage over CID; and (iii) combining CID and ETD fragmentation increased the sequence coverage for an average tryptic peptide to 92%. Interestingly, our analysis revealed that nearly 60% of all ETD-identified peptides carried two positive charges, which is in sharp contrast to what has been generally accepted. We also present a novel strategy for automatic validation of peptide assignments based on identification of a peptide by consecutive CID and ETD fragmentation in an alternating mode.
| S-EPMC2664833 | biostudies-literature
Project description:Mass spectrometry analysis of protein-nucleic acid cross-links is challenging due to the dramatically different chemical properties of the two components. Identifying specific sites of attachment between proteins and nucleic acids requires methods that enable sequencing of both the peptide and oligonucleotide component of the heteroconjugate cross-link. While collision-induced dissociation (CID) has previously been used for sequencing such heteroconjugates, CID generates fragmentation along the phosphodiester backbone of the oligonucleotide preferentially. The result is a reduction in peptide fragmentation within the heteroconjugate. In this work, we have examined the effectiveness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) for sequencing heteroconjugates. Both methods were found to yield preferential fragmentation of the peptide component of a peptide:oligonucleotide heteroconjugate, with minimal differences in sequence coverage between these two electron-induced dissociation methods. Sequence coverage was found to increase with increasing charge state of the heteroconjugate, but decreases with increasing size of the oligonucleotide component. To overcome potential intermolecular interactions between the two components of the heteroconjugate, supplemental activation with ETD was explored. The addition of a supplemental activation step was found to increase peptide sequence coverage over ETD alone, suggesting that electrostatic interactions between the peptide and oligonucleotide components are one limiting factor in sequence coverage by these two approaches. These results show that ECD/ETD methods can be used for the tandem mass spectrometry sequencing of peptide:oligonucleotide heteroconjugates, and these methods are complementary to existing CID methods already used for sequencing of protein-nucleic acid cross-links.
| S-EPMC3638748 | biostudies-literature
Project description:The ability to localize phosphosites to specific amino acid residues is crucial to translating phosphoproteomic data into biological meaningful contexts. In a companion manuscript ( Anal. Chem. 2017 , DOI: 10.1021/acs.analchem.7b00213 ), we described a new implementation of activated ion electron transfer dissociation (AI-ETD) on a quadrupole-Orbitrap-linear ion trap hybrid MS system (Orbitrap Fusion Lumos), which greatly improved peptide fragmentation and identification over ETD and other supplemental activation methods. Here we present the performance of AI-ETD for identifying and localizing sites of phosphorylation in both phosphopeptides and intact phosphoproteins. Using 90 min analyses we show that AI-ETD can identify 24,503 localized phosphopeptide spectral matches enriched from mouse brain lysates, which more than triples identifications from standard ETD experiments and outperforms ETcaD and EThcD as well. AI-ETD achieves these gains through improved quality of fragmentation and MS/MS success rates for all precursor charge states, especially for doubly protonated species. We also evaluate the degree to which phosphate neutral loss occurs from phosphopeptide product ions due to the infrared photoactivation of AI-ETD and show that modifying phosphoRS (a phosphosite localization algorithm) to include phosphate neutral losses can significantly improve localization in AI-ETD spectra. Finally, we demonstrate the utility of AI-ETD in localizing phosphosites in α-casein, an ∼23.5 kDa phosphoprotein that showed eight of nine known phosphorylation sites occupied upon intact mass analysis. AI-ETD provided the greatest sequence coverage for all five charge states investigated and was the only fragmentation method to localize all eight phosphosites for each precursor. Overall, this work highlights the analytical value AI-ETD can bring to both bottom-up and top-down phosphoproteomics.
| S-EPMC5555596 | biostudies-literature
Project description:Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissociation, electron-transfer dissociation, and electron-capture dissociation combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.
| S-EPMC9311227 | biostudies-literature
Project description:Secondary fragmentations of three synthetic peptides (human alphaA crystallin peptide 1-11, the deamidated form of human betaB2 crystallin peptide 4-14, and amyloid beta peptide 25-35) were studied in both electron capture dissociation (ECD) and electron-transfer dissociation (ETD) mode. In ECD, in addition to c and z. ion formations, charge remote fragmentations (CRF) of z. ions were abundant, resulting in internal fragment formation or partial/entire side-chain losses from amino acids, sometimes several residues away from the backbone cleavage site, and to some extent multiple side-chain losses. The internal fragments were observed in peptides with basic residues located in the middle of the sequences, which was different from most tryptic peptides with basic residues located at the C-terminus. These secondary cleavages were initiated by hydrogen abstraction at the alpha-, beta-, or gamma-position of the amino acid side chain. In comparison, ETD generates fewer CRF fragments than ECD. This secondary cleavage study will facilitate ECD/ETD spectra interpretation, and help de novo sequencing and database searching.
| S-EPMC2882803 | biostudies-literature