We describe the implementation and characterization of activated ion electron transfer dissociation (AI-ETD) on a hybrid QLT-Orbitrap mass spectrometer. AI-ETD was performed using a collision cell that was modified to enable ETD reactions, in addition to normal collisional activation. The instrument manifold was modified to enable irradiation of ions along the axis of this modified cell with IR photons from a CO2 laser. Laser power settings were optimized for both charge (z) and mass to charge (m/z) and the instrument control firmware was updated to allow for automated adjustments to the level of irradiation. This implementation of AI-ETD yielded 1.6-fold more unique identifications than ETD in an nLC-MS/MS analysis of tryptic yeast peptides. Furthermore, we investigated the application of AI-ETD on large scale analysis of phosphopeptides, where laser power aids ETD, but can produce b- and y-type ions because of the phosphoryl moiety’s high IR adsorption. nLC-MS/MS analysis of phosphopeptides derived from human embryonic stem cells using AI-ETD yielded 2.4-fold more unique identifications than ETD alone, demonstrating a promising advance in ETD sequencing of PTM containing peptides.
Precise localization of post-translational modifications (PTMs) on proteins and peptides is an outstanding challenge in proteomics. While electron transfer dissociation (ETD) has dramatically advanced PTM analyses, mixtures of localization variants that commonly coexist in cells often require prior separation. Although differential or field asymmetric waveform ion mobility spectrometry (FAIMS) achieves broad variant resolution, the need for standards to identify the features has limited the utility of approach. Here we demonstrate full a priori characterization of variant mixtures by high-resolution FAIMS coupled to ETD and the procedures to systematically extract the FAIMS spectra for all variants from such data.
The development of activation/dissociation techniques such as ultraviolet photodissociation (UVPD), infrared multiphoton dissociation (IRMPD), and electron transfer dissociation (ETD) as alternatives to collision induced dissociation (CID) has extended the range of strategies for characterizing biologically relevant molecules. Here, we describe a comprehensive comparison of CID, IRMPD, UVPD, ETD, and hybrid processes termed ETcaD and ET-IRMPD (and analogous hybrid methods in the negative mode NETcaD and NET-IRMPD) for generating sequence-specific fragment ions and allowing adduction sites to be pinpointed for DNA/cisplatin adducts. Among the six MS/MS methods, the numerous products generated by the IRMPD and UVPD techniques resulted in the most specific and extensive backbone cleavages. We conclude that IRMPD and UVPD methods generally offer the best characteristics for pinpointing the cisplatin adduction sites in the fragment-rich spectra. 相似文献
We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion–ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.
Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS3 (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138–Cys154, Cys143–Cys150, Cys282–Cys396, Cys470–Cys482, Cys471–Cys484, and Cys475–Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70 %). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability. 相似文献
Electron transfer dissociation (ETD) has attracted increasing interest due to its complementarity to collision-induced dissociation (CID). ETD allows the direct localization of labile post-translational modifications, which is of main interest in proteomics where differences and similarities between ETD and CID have been widely studied. However, due to the fact that ETD requires precursor ions to carry at least two charges, little is known about differences in ETD and CID of small molecules such as metabolites. In this work, ETD and CID of desmosine (DES) and isodesmosine (IDS), two isomers that due to the presence of a pyridinium group can carry two charges after protonation, are studied and compared. In addition, the influence of DES/IDS derivatization with propionic anhydride and polyethyleneglycol (PEG) reagents on ETD and CID was studied, since this is a common strategy to increase sensitivity and to facilitate the analysis by reversed-phase chromatography. Clear differences between ETD and CID of non-derivatized and derivatized-DES/IDS were observed. While CID is mainly attributable to charge-directed fragmentation, ETD is initiated by the generation of a hydrogen atom at the initial protonation site and its subsequent transfer to the pyridinium ring of DES/IDS. These differences are reflected in the generation of complex CID spectra dominated by the loss of small, noninformative molecules (NH3, CO, H2O), while ETD spectra are simpler and dominated by characteristic side-chain losses. This constitutes a potential advantage of ETD in comparison to CID when employed for the targeted analysis of DES/IDS in biological samples.
Figure
A mechanistic study of electron transfer dissociation (ETD) and collision-induced dissociation (CID) of labeled and free desmosine and isodesmosine provides evidence that CID is mainly due to charge-directed fragmentation while ETD is initiated by the generation of a hydrogen atom at the initial protonation site, and its subsequent transfer to the pyridinium ring. 相似文献
As the resolution of analytical methods improves, further progress tends to be increasingly limited by instrumental parameter instabilities that were previously inconsequential. This is now the case with differential ion mobility spectrometry (FAIMS), where fluctuations of the voltages and gas pressure have become critical. A new high-definition generator for FAIMS compensation voltage reported here provides a stable and accurate output than can be scanned with negligible steps. This reduces the spectral drift and peak width, thus improving the resolving power (R) and resolution. The gain for multiply-charged peptides that have narrowest peaks is up to ~40 %, and R ~400–500 is achievable using He/N2 or H2/N2 gas mixtures. 相似文献
The small ubiquitin-like modifier (SUMO) is a member of the family of ubiquitin-like modifiers (UBLs) and is involved in important cellular processes, including DNA damage response, meiosis and cellular trafficking. The large-scale identification of SUMO peptides in a site-specific manner is challenging not only because of the low abundance and dynamic nature of this modification, but also due to the branched structure of the corresponding peptides that further complicate their identification using conventional search engines. Here, we exploited the unusual structure of SUMO peptides to facilitate their separation by high-field asymmetric waveform ion mobility spectrometry (FAIMS) and increase the coverage of SUMO proteome analysis. Upon trypsin digestion, branched peptides contain a SUMO remnant side chain and predominantly form triply protonated ions that facilitate their gas-phase separation using FAIMS. We evaluated the mobility characteristics of synthetic SUMO peptides and further demonstrated the application of FAIMS to profile the changes in protein SUMOylation of HEK293 cells following heat shock, a condition known to affect this modification. FAIMS typically provided a 10-fold improvement of detection limit of SUMO peptides, and enabled a 36% increase in SUMO proteome coverage compared to the same LC-MS/MS analyses performed without FAIMS.
Electron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately 3-fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase.
High field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility spectrometry, is emerging as a tool for biomolecular analysis. In this article, the benefits and limitations of FAIMS for protein analysis are discussed. The principles and mechanisms of FAIMS separation of ions are described, and the differences between FAIMS and conventional ion mobility spectrometry are detailed. Protein analysis is considered from both the top-down (intact proteins) and the bottom-up (proteolytic peptides) perspective. The roles of FAIMS in the analysis of complex mixtures of multiple intact proteins and in the analysis of multiple conformers of a single protein are assessed. Similarly, the application of FAIMS in proteomics and targeted analysis of peptides are considered.
High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., ~60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed.
Screening of bead-based split and pool combinatorial chemistry libraries is a powerful approach to aid the discovery of new chemical compounds able to interact with, and modulate the activities of, protein targets of interest. Split and pool synthesis provides for large and well diversified chemical libraries, in this case comprised of oligomers generated from a well-defined starting set. At the end of the synthesis, each bead in the library displays many copies of a unique oligomer sequence. Because the sequence of the oligomer is not known at the time of screening, methods for decoding of the sequence of each screening “hit” are essential. Here we describe an electron-transfer dissociation (ETD) based tandem mass spectrometry approach for the decoding of mass-encoded split and pool libraries. We demonstrate that the newly described “chiral oligomers of pentenoic amides (COPAs)” yield non-sequence-specific product ions upon collisional activated dissociation; however, complete sequence information can be obtained with ETD. To aid in the decoding of libraries from MS and MS/MS data, we have incorporated 79Br/81Br isotope “tags” to differentiate N- and C-terminal product ions. In addition, we have created “Hit-Find,” a software program that allows users to generate libraries in silico. The user can then search all possible members of the chemical library for those that fall within a user-defined mass error. 相似文献
Differential mobility spectrometry (DMS) has been employed to separate isomeric species in several studies. Under the right conditions, factors such as separation voltage, temperature, the presence of chemical modifiers, and residence time can combine to provide unique signal channels for isomeric species. In this study, we examined a set of glycopeptide isomers, MUC5AC-3 and MUC5AC-13, which bear an N-acetyl-galactosamine (GalNAc) group on either threonine-3 or threonine-13. When analyzed as a mixture, the resulting MS and MS/MS spectra yield fragmentation patterns that cannot discern these convolved species. However, when DMS is implemented during the analysis of this mixture, two features emerge in the DMS ionogram representing the two glycopeptide isomers. In addition, by locking in DMS parameters at each feature, we could observe several low intensity CID fragments that contain the GalNAc functionality-specific amino acid residues – identifying the DMS separation of each isomer without standards. Besides conventional CID MS/MS, we also implemented electron-capture dissociation (ECD) after DMS separation, and clearly resolved both isomers with this fragmentation method, as well. The electron energy used in these ECD experiments could be tuned to obtain maximum sequence coverage for these glycopeptides; this was critical as these ions were present as doubly protonated species, which are much more difficult to fragment efficiently via electron-transfer dissociation (ETD). Overall, the combination of DMS with electron- or collision-based MS/MS methods provided enhanced separation and sequence coverage for these glycopeptide isomers.
We report a comprehensive study of collision-induced dissociation (CID) and near-UV photodissociation (UVPD) of a series of tyrosine-containing peptide cation radicals of the hydrogen-rich and hydrogen-deficient types. Stable, long-lived, hydrogen-rich peptide cation radicals, such as [AAAYR + 2H]+● and several of its sequence and homology variants, were generated by electron transfer dissociation (ETD) of peptide-crown-ether complexes, and their CID-MS3 dissociations were found to be dramatically different from those upon ETD of the respective peptide dications. All of the hydrogen-rich peptide cation radicals contained major (77%–94%) fractions of species having radical chromophores created by ETD that underwent photodissociation at 355 nm. Analysis of the CID and UVPD spectra pointed to arginine guanidinium radicals as the major components of the hydrogen-rich peptide cation radical population. Hydrogen-deficient peptide cation radicals were generated by intramolecular electron transfer in CuII(2,2′:6′,2″-terpyridine) complexes and shown to contain chromophores absorbing at 355 nm and undergoing photodissociation. The CID and UVPD spectra showed major differences in fragmentation for [AAAYR]+● that diminished as the Tyr residue was moved along the peptide chain. UVPD was found to be superior to CID in localizing Cα-radical positions in peptide cation radical intermediates.
We reported the use of ion mobility (IM) combined with mass spectrometry (MS) as an analytical tool to investigate low generation polyamidoanine (PAMAM) dendrimers. This analytical approach has been employed to separate ions of defective structures with different charge state but exactly the same m/z value. Tandem mass spectrometry (MS/MS) after IM separation allowed a comprehensive structural characterization of defective dendrimers. In addition, IM was used to evaluate the collision cross-sections of ions of perfect dendrimers. They showed a good correlation with calculated collision cross-sections obtained by the trajectory method (TM) and were also consistent with dimensions reported by other established analytical methods. 相似文献
Pyteomics is a cross-platform, open-source Python library providing a rich set of tools for MS-based proteomics. It provides modules for reading LC-MS/MS data, search engine output, protein sequence databases, theoretical prediction of retention times, electrochemical properties of polypeptides, mass and m/z calculations, and sequence parsing. Pyteomics is available under Apache license; release versions are available at the Python Package Index http://pypi.python.org/pyteomics, the source code repository at http://hg.theorchromo.ru/pyteomics, documentation at http://packages.python.org/pyteomics. Pyteomics.biolccc documentation is available at http://packages.python.org/pyteomics.biolccc/. Questions on installation and usage can be addressed to pyteomics mailing list: pyteomics@googlegroups.com 相似文献
One difficult problem in the analysis of peptide modifications is quantifying isomeric modifications that differ by the position of the amino acid modified. HPLC separation using C18 reverse phase chromatography coupled with electron transfer dissociation (ETD) in tandem mass spectrometry has recently been shown to be able to relatively quantify how much of a given modification occurs at each amino acid position for isomeric mixtures; however, the resolution of reverse phase chromatography greatly complicates quantification of isomeric modifications by ETD because of the chromatographic separation of peptides with identical modifications at different sequence positions. Using peptide oxidation as a model system, we investigated the use of size exclusion chromatography coupled with ETD fragmentation to separate peptide sequences. This approach allows for the benefits of chromatographic separation of peptide sequences while ensuring co-elution of modification isomers for accurate relative quantification of modifications using standard data-dependent acquisitions. Using this method, the relative amount of modification at each amino acid can be accurately measured from single ETD MS/MS spectra in a standard data-dependent acquisition experiment.
Sulfotyrosine and phosphotyrosine are two post-translational modifications present in higher eukaryotes. A simple and direct mass spectrometry method to distinguish between these modifications is crucial to advance our understanding of the sulfoproteome. While sulfation and phosphorylation are nominally isobaric, the accurate mass of the sulfuryl moiety is 9.6 mDa less than the phosphoryl moiety. Based on this difference, we have used an Orbitrap Fusion Lumos mass spectrometer to characterize, resolve, and distinguish between sulfotyrosine and phosphotyrosine modifications using a set of model peptides. Multiple fragmentation techniques, namely HCD, CID, ETD, ETciD, and EThcD, have been used to compare the different fragmentation behaviors between peptides modified with these species. Sulfotyrosine undergoes neutral loss using HCD and CID, but the sulfuryl moiety is largely stable under ETD. In contrast, phosphotyrosine is stable during fragmentation using all these methods. This differential stability provides a mechanism to distinguish sulfopeptides from phosphopeptides. Based on the rigorous characterization presented herein, this work serves as a model for accurate identification of phosphotyrosine and, more challenging, sulfotyrosine, in complex proteomic samples.
