Higher-energy Collisional Dissociation Fragmentation Research Articles

In-Depth Mass Spectrometry Study of Vanadium(IV) Complexes with Model Peptides.

Investigating the speciation of vanadium complexes in the presence of potential biomolecular targets under physiological conditions remains challenging, and further experimental techniques are needed to better understand the mechanism of action of potential metallodrugs. The interaction of two model peptides (angiotensin I and angiotensin II) with three well-known oxidovanadium(IV) compounds with antidiabetic and/or anticancer activity, [VIVO(pic)2(H2O)], [VIVO(ma)2], and [VIVO(dhp)2] (where pic, ma, and dhp are picolinate, maltolate, and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate anions, respectively), was investigated by ESI-MS/MS (electrospray ionization tandem mass spectrometry) and complemented by EPR (electron paramagnetic resonance) spectroscopy measurements and theoretical calculations at the DFT (density functional theory) level. The results demonstrated that vanadium-peptide bonds are preserved after HCD (higher energy collisional dissociation) fragmentation, allowing for the identification of binding sites through a detailed analysis of the fragmentation spectra. Angiotensin I (AT1) and angiotensin II (AT2) exhibited different coordination behaviors. AT1, with two His residues (His6, His9), prefers to form [AT1 + VOL] adducts with both histidine residues coordinated to the metal ion, while AT2, which has only His6, can bind the metal in a monodentate fashion, forming also [AT2 + VOL2] adducts. Insights from this study pave the way to ESI-MS/MS investigations of more complex systems, including target proteins and further development of vanadium-based drugs.

Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling.

Isobaric chemical tag labeling (e.g., TMT) is a commonly used approach in quantitative proteomics, and quantification is enabled through detection of low-mass reporter ions generated after MS2 fragmentation. Recently, we have introduced and optimized an intact protein-level TMT labeling platform that demonstrated >90% labeling efficiency in complex samples with top-down proteomics. Higher-energy collisional dissociation (HCD) is commonly utilized for isobaric tag-labeled peptide fragmentation because it produces accurate reporter ion intensities and avoids loss of low mass ions. HCD energies have been optimized for isobaric tag labeled-peptides but have not been systematically evaluated for isobaric tag-labeled intact proteins. In this study, we report a systematic evaluation of normalized HCD fragmentation energies (NCEs) on TMT-labeled HeLa cell lysate using top-down proteomics. Our results suggested that reporter ions often result in higher ion intensities at higher NCEs. Optimal fragmentation of intact proteins for identification, however, required relatively lower NCE. We further demonstrated that a stepped NCE scheme with energies from 30% to 50% resulted in optimal quantification and identification of TMT-labeled HeLa proteins. These parameters resulted in an average reporter ion intensity of ∼4E4 and average proteoform spectrum matches (PrSMs) of >1000 per RPLC-MS/MS run with a 1% false discovery rate (FDR) cutoff.

Structural Characterization of Daunomycin-Peptide Conjugates by Various Tandem Mass Spectrometric Techniques.

The use of peptide-drug conjugates has generated wide interest as targeted antitumor therapeutics. The anthracycline antibiotic, daunomycin, is a widely used anticancer agent and it is often conjugated to different tumor homing peptides. However, comprehensive analytical characterization of these conjugates via tandem mass spectrometry (MS/MS) is challenging due to the lability of the O-glycosidic bond and the appearance of MS/MS fragment ions with little structural information. Therefore, we aimed to investigate the optimal fragmentation conditions that suppress the prevalent dissociation of the anthracycline drug and provide good sequence coverage. In this study, we comprehensively compared the performance of common fragmentation techniques, such as higher energy collisional dissociation (HCD), electron transfer dissociation (ETD), electron-transfer higher energy collisional dissociation (EThcD) and matrix-assisted laser desorption/ionization–tandem time-of-flight (MALDI-TOF/TOF) activation methods for the structural identification of synthetic daunomycin-peptide conjugates by high-resolution tandem mass spectrometry. Our results showed that peptide backbone fragmentation was inhibited by applying electron-based dissociation methods to conjugates, most possibly due to the “electron predator” effect of the daunomycin. We found that efficient HCD fragmentation was largely influenced by several factors, such as amino acid sequences, charge states and HCD energy. High energy HCD and MALDI-TOF/TOF combined with collision induced dissociation (CID) mode are the methods of choice to unambiguously assign the sequence, localize different conjugation sites and differentiate conjugate isomers.

Semiautomated glycoproteomics data analysis workflow for maximized glycopeptide identification and reliable quantification.

Glycoproteomic data are often very complex, reflecting the high structural diversity of peptide and glycan portions. The use of glycopeptide-centered glycoproteomics by mass spectrometry is rapidly evolving in many research areas, leading to a demand in reliable data analysis tools. In recent years, several bioinformatic tools were developed to facilitate and improve both the identification and quantification of glycopeptides. Here, a selection of these tools was combined and evaluated with the aim of establishing a robust glycopeptide detection and quantification workflow targeting enriched glycoproteins. For this purpose, a tryptic digest from affinity-purified immunoglobulins G and A was analyzed on a nano-reversed-phase liquid chromatography–tandem mass spectrometry platform with a high-resolution mass analyzer and higher-energy collisional dissociation fragmentation. Initial glycopeptide identification based on MS/MS data was aided by the Byonic software. Additional MS1-based glycopeptide identification relying on accurate mass and retention time differences using GlycopeptideGraphMS considerably expanded the set of confidently annotated glycopeptides. For glycopeptide quantification, the performance of LaCyTools was compared to Skyline, and GlycopeptideGraphMS. All quantification packages resulted in comparable glycosylation profiles but featured differences in terms of robustness and data quality control. Partial cysteine oxidation was identified as an unexpectedly abundant peptide modification and impaired the automated processing of several IgA glycopeptides. Finally, this study presents a semiautomated workflow for reliable glycoproteomic data analysis by the combination of software packages for MS/MS- and MS1-based glycopeptide identification as well as the integration of analyte quality control and quantification.

Ultraviolet Photodissociation for Non-Target Screening-Based Identification of Organic Micro-Pollutants in Water Samples.

Non-target screening (NTS) based on the combination of liquid chromatography coupled to high-resolution mass spectrometry has become the key method to identify organic micro-pollutants (OMPs) in water samples. However, a large number of compounds remains unidentified with current NTS approaches due to poor quality fragmentation spectra generated by suboptimal fragmentation methods. Here, the potential of the alternative fragmentation technique ultraviolet photodissociation (UVPD) to improve identification of OMPs in water samples was investigated. A diverse set of water-relevant OMPs was selected based on k-means clustering and unsupervised artificial neural networks. The selected OMPs were analyzed using an Orbitrap Fusion Lumos equipped with UVPD. Therewith, information-rich MS2 fragmentation spectra of compounds that fragment poorly with higher-energy collisional dissociation (HCD) could be attained. Development of an R-based data analysis workflow and user interface facilitated the characterization and comparison of HCD and UVPD fragmentation patterns. UVPD and HCD generated both unique and common fragments, demonstrating that some fragmentation pathways are specific to the respective fragmentation method, while others seem more generic. Application of UVPD fragmentation to the analysis of surface water enabled OMP identification using existing HCD spectral libraries. However, high-throughput applications still require optimization of informatics workflows and spectral libraries tailored to UVPD.

Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics

Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an ad hoc fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.

Optimized Workflow for Multiplexed Phosphorylation Analysis of TMT-Labeled Peptides Using High-Field Asymmetric Waveform Ion Mobility Spectrometry.

Phosphorylation is a post-translational modification with a vital role in cellular signaling. Isobaric labeling-based strategies, such as tandem mass tags (TMT), can measure the relative phosphorylation states of peptides in a multiplexed format. However, the low stoichiometry of protein phosphorylation constrains the depth of phosphopeptide analysis by mass spectrometry. As such, robust and sensitive workflows are required. Here we evaluate and optimize high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid mass spectrometers for the analysis of TMT-labeled phosphopeptides. We determined that using FAIMS-MS3 with three compensation voltages (CV) in a single method (e.g., CV = -40/-60/-80 V) maximizes phosphopeptide coverage while minimizing inter-CV overlap. Furthermore, consecutive analyses using MSA-CID (multistage activation collision-induced dissociation) and HCD (higher-energy collisional dissociation) fragmentation at the MS2 stage increases the depth of phosphorylation analysis. The methodology and results outlined herein provide a template for tailoring optimized FAIMS-based methods.

HCD Fragmentation of Glycated Peptides.

Protein glycation is a concentration-dependent nonenzymatic reaction of reducing sugars with amine groups of proteins to form early as well as advanced glycation (end-) products (AGEs). Glycation is a highly disease-relevant modification but is typically only studied on a few blood proteins. To complement our blood proteomics studies in diabetics, we here investigate protein glycation by higher energy collisional dissociation (HCD) fragmentation on Orbitrap mass spectrometers. We established parameters to most efficiently fragment and identify early glycation products on in vitro glycated model proteins. Retaining standard collision energies does not degrade performance if the most dominant neutral loss of H6O3 is included into the database search strategy. Glycation analysis of the entire HeLa proteome revealed an unexpected intracellular preponderance for arginine over lysine modification in early and advanced glycation (end-) products. Single-run analysis from 1 μL of undepleted and unenriched blood plasma identified 101 early glycation sites as well as numerous AGE sites on diverse plasma proteins. We conclude that HCD fragmentation is well-suited for analyzing glycated peptides and that the diabetic status of patients can be directly diagnosed from single-run plasma proteomics measurements.

Enhanced acylcarnitine annotation in high-resolution mass spectrometry data: fragmentation analysis for the classification and annotation of acylcarnitines.

Metabolite annotation and identification are primary challenges in untargeted metabolomics experiments. Rigorous workflows for reliable annotation of mass features with chemical structures or compound classes are needed to enhance the power of untargeted mass spectrometry. High-resolution mass spectrometry considerably improves the confidence in assigning elemental formulas to mass features in comparison to nominal mass spectrometry, and embedding of fragmentation methods enables more reliable metabolite annotations and facilitates metabolite classification. However, the analysis of mass fragmentation spectra can be a time-consuming step and requires expert knowledge. This study demonstrates how characteristic fragmentations, specific to compound classes, can be used to systematically analyze their presence in complex biological extracts like urine that have undergone untargeted mass spectrometry combined with data dependent or targeted fragmentation. Human urine extracts were analyzed using normal phase liquid chromatography (hydrophilic interaction chromatography) coupled to an Ion Trap-Orbitrap hybrid instrument. Subsequently, mass chromatograms and collision-induced dissociation and higher-energy collisional dissociation (HCD) fragments were annotated using the freely available MAGMa software1. Acylcarnitines play a central role in energy metabolism by transporting fatty acids into the mitochondrial matrix. By filtering on a combination of a mass fragment and neutral loss designed based on the MAGMa fragment annotations, we were able to classify and annotate 50 acylcarnitines in human urine extracts, based on high-resolution mass spectrometry HCD fragmentation spectra at different energies for all of them. Of these annotated acylcarnitines, 31 are not described in HMDB yet and for only 4 annotated acylcarnitines the fragmentation spectra could be matched to reference spectra. Therefore, we conclude that the use of mass fragmentation filters within the context of untargeted metabolomics experiments is a valuable tool to enhance the annotation of small metabolites.

Bridging the gap between molecular and elemental mass spectrometry: higher energy collisional dissociation (HCD) revealing elemental information.

Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to in solution standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS.

O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry

Serine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93-639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques associated to a new software tool, demonstrate glycosylation heterogeneity of Srr1, characterize a new protein modification, and identify six glycosylation sites located in the N-terminal region of the protein.

Identification and characterization of citrulline-modified brain proteins by combining HCD and CID fragmentation.

Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited. Using RP LC and high-resolution MS, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP), neurogranin (NRGN/RC3), and myelin basic protein (MBP) and in vivo sites in brain protein extract. Human GFAP has five endogenous citrullination sites, R30, R36, R270, R406, and R416, and MBP has 14 in vivo citrullination sites. Human NRGN/RC3 was found citrullinated at residue R68. The sequence of citrullinated peptides and citrullination sites were confirmed from peptides identified in trypsin, Lys-C, and Glu-C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimer's and control brain samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6 to 9.5%. Compared to CID, higher-energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID-triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests.

PTM MarkerFinder, a software tool to detect and validate spectra from peptides carrying post‐translational modifications

Mass spectrometry (MS) analysis of peptides carrying post-translational modifications is challenging due to the instability of some modifications during MS analysis. However, glycopeptides as well as acetylated, methylated and other modified peptides release specific fragment ions during CID (collision-induced dissociation) and HCD (higher energy collisional dissociation) fragmentation. These fragment ions can be used to validate the presence of the PTM on the peptide. Here, we present PTM MarkerFinder, a software tool that takes advantage of such marker ions. PTM MarkerFinder screens the MS/MS spectra in the output of a database search (i.e., Mascot) for marker ions specific for selected PTMs. Moreover, it reports and annotates the HCD and the corresponding electron transfer dissociation (ETD) spectrum (when present), and summarizes information on the type, number, and ratios of marker ions found in the data set. In the present work, a sample containing enriched N-acetylhexosamine (HexNAc) glycopeptides from yeast has been analyzed by liquid chromatography-mass spectrometry on an LTQ Orbitrap Velos using both HCD and ETD fragmentation techniques. The identification result (Mascot .dat file) was submitted as input to PTM MarkerFinder and screened for HexNAc oxonium ions. The software output has been used for high-throughput validation of the identification results.

Method development for fecal lipidomics profiling.

Robust methodologies for the analysis of fecal material will facilitate the understanding of gut (patho)physiology and its role in health and disease and will help improve care for individual patients, especially high-risk populations, such as premature infants. Because lipidomics offers a biologically and analytically attractive approach, we developed a simple, sensitive, and quantitatively precise method for profiling intact lipids in fecal material. The method utilizes two separate, complementary extraction chemistries, dichloromethane (DCM) and a methyl tert-butyl ether/hexafluoroisopropanol (MTBE) mixture, alone or with high pressure cycling. Extracts were assessed by liquid chromatography-high-resolution mass spectrometry-based profiling with all ion higher energy collisional dissociation fragmentation in both positive and negative ionization modes. This approach provides both class-specific and lipid-specific fragments, enhancing lipid characterization. Solvents preferentially extracted lipids based on hydrophobicity. More polar species preferred MTBE; more hydrophobic compounds preferred DCM. Pressure cycling differentially increased the yield of some lipids. The platform enabled analysis of >500 intact lipophilic species with over 300 lipids spanning 6 LIPID MAPS categories identified in the fecal matter from premature infants. No previous report exists that provides these data; thus, this study represents a new paradigm for assessing nutritional health, inflammation, and infectious disease in vulnerable populations.

Correction to Feasibility of Large-Scale Phosphoproteomics with Higher Energy Collisional Dissociation Fragmentation

Correction to Feasibility of Large-Scale Phosphoproteomics with Higher Energy Collisional Dissociation Fragmentation

Ultra High Resolution Linear Ion Trap Orbitrap Mass Spectrometer (Orbitrap Elite) Facilitates Top Down LC MS/MS and Versatile Peptide Fragmentation Modes

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm—incorporating phase information—further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s—increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

An experimental approach to enhance precursor ion fragmentation for metabolite identification studies: application of dual collision cells in an orbital trap

Recent advancements in mass spectrometry including data-dependent scanning and high-resolution mass spectrometry have aided metabolite profiling for non-radiolabeled xenobiotics. However, narrowing down a site of metabolism is often limited by the quality of the collision-induced dissociation (CID)-based precursor ion fragmentation. An alternative dissociation technique, higher energy collisional dissociation (HCD), enriches compound fragmentation and yields 'triple-quadrupole-like fragmentation'. Applying HCD along with CID and data-dependent scanning could enhance structural elucidation for small molecules. Liquid chromatography/multi-stage mass spectrometry (LC/MS(n) ) experiments with CID and HCD fragmentation were carried out for commercially available compounds on a hybrid linear ion trap orbital trap mass spectrometer equipped with accurate mass measurement capability. The developed method included stepped normalized collision energy (SNCE) parameters to enhance MS fragmentation without tuning for individual compounds. All the evaluated compounds demonstrated improved fragmentation under HCD as compared with CID. The results suggest that an LC/MS(n) method that incorporated both SNCE HCD- and CID-enabled precursor ion fragmentation afforded comprehensive structural information for the compounds under investigation. A dual collision cell approach was remarkably better than one with only CID MS(n) in an orbital trap. It is evident that such an acquisition method can augment the identification of unknown metabolites in drug discovery by improving fragmentation efficiency of both the parent compound and its putative metabolite(s).

Feasibility of Large-Scale Phosphoproteomics with Higher Energy Collisional Dissociation Fragmentation

Mass spectrometry (MS)-based proteomics now enables the analysis of thousands of phosphorylation sites in single projects. Among a wide range of analytical approaches, the combination of high resolution MS scans in an Orbitrap analyzer with low resolution MS/MS scans in a linear ion trap has proven to be particularly successful ("high-low" strategy). Here we investigate if the improved sensitivity of higher energy collisional dissociation (HCD) on an LTQ-Orbitrap Velos instrument allows a "high-high" strategy. A high resolution MS scan was followed by up to 10 HCD MS/MS scans, and we achieved cycle times of about 3 s making the method compatible with chromatographic time scales. Fragment mass accuracy increased about 50-fold compared to the "high-low" strategy. Unexpectedly, the HCD approach mapped up to 16,000 total phosphorylation sites in one day's measuring time--the same or better than the standard high-low strategy. Reducing the target values from a standard of 30,000 to 5000 ions did not severely affect identification rates but did decrease identification and localization scores for phosphorylation sites. We conclude that HCD in the new configuration is now a viable method for large-scale phosphoproteome analysis alongside collisional induced dissociation, (CID) and electron capture/transfer dissociation (ECD/ETD).