Electron Capture Dissociation Mass Spectrometry in Characterization of Peptides and Proteins

Electron capture dissociation (ECD) represents one of the most recent and significant advancements in tandem mass spectrometry (MS/MS) for the identification and characterization of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally activated dissociation (CAD), ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation—an important attribute for characterizing post-translational modifications. Herein, we present a brief overview of the ECD technique as well as selected applications in characterization of peptides and proteins. Case studies including characterization and localization of amino acid glycosylation, methionine oxidation, acylation, and “top–down” protein mass spectrometry using ECD will be presented. A recent technique, coined as electron transfer dissociation (ETD), will be also discussed briefly.     

Metal oxide-based enrichment combined with gas-phase ion-electron reactions for improved mass spectrometric characterization of protein phosphorylation

Gas-phase ion-electron reactions, including electron capture dissociation (ECD) and electron detachment dissociation (EDD), are advantageous for characterization of protein posttranslational modifications (PTMs), because labile modifications are not lost during the fragmentation process. However, at least two positive charges and relatively abundant precursor ions are required for ECD due to charge reduction and lower fragmentation efficiency compared to conventional gas-phase fragmentation techniques. Both these criteria are difficult to fulfill for phosphopeptides due to their acidic character. The negative ion mode operation of EDD is more compatible with phosphopeptide ionization, but EDD suffers from a fragmentation efficiency even lower than that of ECD. Recently, metal oxides such as ZrO 2 and TiO 2 have been shown to provide selective enrichment of phosphopeptides from proteolytic digests. Here, we utilize this enrichment strategy to improve ECD and EDD of phosphopeptides. This approach allowed determination of the locations of phosphorylation sites in highly acidic, multiply phosphorylated peptides from complex peptide mixtures by ECD. For singly phosphorylated peptides, EDD provided complementary sequence information compared to ECD.     

Application of different fragmentation techniques for the analysis of phosphopeptides using a hybrid linear ion trap-FTICR mass spectrometer

Electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) present complementary techniques for the fragmentation of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in addition to the commonly used collisionally activated dissociation (CAD). Both IRMPD and ECD have been shown to be applicable for an efficient sequencing of peptides and proteins, whereas ECD has proven especially valuable for mapping labile posttranslational modifications (PTMs), such as phosphorylations. In this work, we compare the different fragmentation techniques and MS detection in a linear ion trap and the ICR cell with respect to their abilities to efficiently identify and characterize phosphorylated peptides. For optimizing fragmentation parameters, sets of synthetic peptides with molecular weights ranging from approximately 1 to 4 kDa and different levels of phosphorylation were analyzed. The influence of spectrum averaging for obtaining high-quality spectra was investigated. Our results show that the fragmentation methods CAD and ECD allow for a facilitated analysis of phosphopeptides; however, their general applicability for analyzing phosphopeptides has to be evaluated in each specific case with respect to the given analytical task. The major advantage of complementary peptide cleavages by combining different fragmentation methods is the increased amount of information that is obtained during MS/MS analysis of modified peptides. On the basis of the obtained results, we are planning to design LC time-scale compatible, data-dependent MS/MS methods using the different fragmentation techniques in order to improve the identification and characterization of phosphopeptides.     

Electron-capture dissociation tandem mass spectrometry

Electron capture dissociation (ECD) is a new fragmentation technique used in Fourier transform ion cyclotron resonance mass spectrometry and is complementary to traditional tandem mass spectrometry techniques. Disulfide bonds, normally stable to vibrational excitation, are preferentially cleaved in ECD. Fragmentation is fast and specific and labile post-translational modifications and non-covalent bonds often remain intact after backbone bond dissociation. ECD provides more extensive sequence coverage in polypeptides, and at higher electron energies even isoleucine and leucine are distinguishable. In biotechnology, the main area of ECD application is expected to be the top-down verification of DNA-predicted protein sequences, de novo sequencing, disulfide bond analysis and the combined top-down/bottom-up analysis of post-translational modifications.     

Protein primary structure using orthogonal fragmentation techniques in Fourier transform mass spectrometry

Proteomics analysis using tandem mass spectrometry requires informative backbone fragmentation of peptide ions. Collision-activated dissociation (CAD) of cations alone is not sufficiently informative to satisfy all requirements. Thus, there is a need to supplement CAD with a complementary fragmentation technique. Electron capture dissociation (ECD) is complementary to collisional excitation in terms of the cleavage of a different bond (N-Calpha versus C-N bond) and other properties. CAD-ECD combination improves protein identification and enables high-throughput de novo sequencing of peptides. ECD and its variants are also useful in mapping labile post-translational modifications in proteins and isomer differentiation; for example, distinguishing Ile from Leu, iso-Asp from Asp and even D- from L-amino acid residues.     

Electron transfer dissociation mass spectrometry in proteomics

Mass spectrometry has rapidly evolved to become the platform of choice for proteomic analysis. While CID remains the major fragmentation method for peptide sequencing, electron transfer dissociation (ETD) is emerging as a complementary method for the characterization of peptides and post-translational modifications (PTMs). Here, we review the evolution of ETD and some of its newer applications including characterization of PTMs, non-tryptic peptides and intact proteins. We will also discuss some of the unique features of ETD such as its complementarity with CID and the use of alternating CID/ETD along with issues pertaining to analysis of ETD data. The potential of ETD for applications such as multiple reaction monitoring and proteogenomics in the future will also be discussed.     

Protein primary structure using orthogonal fragmentation techniques in Fourier transform mass spectrometry

Proteomics analysis using tandem mass spectrometry requires informative backbone fragmentation of peptide ions. Collision-activated dissociation (CAD) of cations alone is not sufficiently informative to satisfy all requirements. Thus, there is a need to supplement CAD with a complementary fragmentation technique. Electron capture dissociation (ECD) is complementary to collisional excitation in terms of the cleavage of a different bond (N-C_α versus C-N bond) and other properties. CAD-ECD combination improves protein identification and enables high-throughput de novo sequencing of peptides. ECD and its variants are also useful in mapping labile post-translational modifications in proteins and isomer differentiation; for example, distinguishing Ile from Leu, iso-Asp from Asp and even D- from L-amino acid residues.     

Phosphorylation, but not alternative splicing or proteolytic degradation, is conserved in human and mouse cardiac troponin T

Cardiac troponin T (cTnT), the tropomyosin binding subunit of the troponin complex, plays a pivotal regulatory role in the Ca(2+)-mediated interaction between actin thin filament and myosin thick filament. The post-translational modifications (PTMs) and alternative splicing of cTnT may represent important regulatory mechanisms of cardiac contractility. However, a complete characterization of PTMs and alternatively spliced isoforms in cTnT present in vivo is lacking. Top-down protein mass spectrometry (MS) analyzes whole proteins, thus providing a global view of all types of modifications, including PTMs and sequence variants, simultaneously in one spectrum without a priori knowledge. In this study, we applied an integrated immunoaffinity chromatography and top-down MS approach to comprehensively characterize PTMs and alternatively spliced isoforms of cTnT purified from healthy human and wild-type mouse heart tissue. High-resolution Fourier transform MS revealed that human cTnT (hcTnT) and mouse cTnT (mcTnT) have similar phosphorylation patterns, whereas higher molecular heterogeneity was observed for mcTnT than hcTnT. Further MS/MS fragmentation of monophosphorylated hcTnT and mcTnT by electron capture dissociation and collisionally activated dissociation unambiguously identified Ser1 as the conserved in vivo phosphorylation site. In contrast, we identified a single spliced isoform for hcTnT but three alternatively spliced isoforms for mcTnT. Moreover, we observed distinct proteolytic degradation products for hcTnT and mcTnT. This study also demonstrates the advantage of top-down MS/MS with complementary fragmentation techniques for the identification of modification sites in the highly acidic N-terminal region of cTnT.     

The utility of ETD mass spectrometry in proteomic analysis

Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.     

Comparison of the electron capture dissociation fragmentation behavior of doubly and triply protonated peptides from trypsin, Glu-C, and chymotrypsin digestion

In bottom-up proteomics, proteolytically derived peptides from proteins of interest are analyzed to provide sequence information for protein identification and characterization. Electron capture dissociation (ECD), which provides more random cleavages compared to "slow heating" techniques such as collisional activation, can result in greater sequence coverage for peptides and proteins. Most bottom-up proteomics approaches rely on tryptic doubly protonated peptides for generating sequence information. However, the effectiveness, in terms of peptide sequence coverage, of tryptic doubly protonated peptides in ECD remains to be characterized. Herein, we examine the ECD fragmentation behavior of 64 doubly- and 64 triply protonated peptides (i.e., a total of 128 peptide ions) from trypsin, Glu-C, and chymotrypsin digestion in a Fourier transform ion cyclotron resonance mass spectrometer. Our findings indicate that when triply protonated peptides are fragmented in ECD, independent of which proteolytic enzyme was used for protein digestion, more c- and z-type product ions are observed, and the number of complementary fragment pairs increases dramatically (44%). In addition, triply protonated peptides provide an increase (26%) in peptide sequence coverage. ECD of tryptic peptides, in both charge states, resulted in higher sequence coverage compared to chymotryptic and Glu-C digest peptides. The peptide sequence coverage we obtained in ECD of tryptic doubly protonated peptides (64%) is very similar to that reported for electron transfer dissociation of the same peptide type (63%).     

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.     

Global mapping of post-translational modifications on histone H3 variants in mouse testes

Mass spectrometry (MS)-based characterization is important in proteomic research for verification of structural features and functional understanding of gene expression. Post-translational modifications (PTMs) such as methylation and acetylation have been reported to be associated with chromatin remodeling during spermatogenesis. Although antibody- and MS-based approaches have been applied for characterization of PTMs on H3 variants during spermatogenesis, variant-specific PTMs are still underexplored. We identified several lysine modifications in H3 variants, including testis-specific histone H3 (H3t), through their successful separation with MS-based strategy, based on differences in masses, retention times, and presence of immonium ions. Besides methylation and acetylation, we detected formylation as a novel PTM on H3 variants in mouse testes. These patterns were also observed in H3t. Our data provide high-throughput structural information about PTMs on H3 variants in mouse testes and show possible applications of this strategy in future proteomic studies on histone PTMs.     

Evolution and functional cross‐talk of protein post‐translational modifications

Protein post‐translational modifications (PTMs) allow the cell to regulate protein activity and play a crucial role in the response to changes in external conditions or internal states. Advances in mass spectrometry now enable proteome wide characterization of PTMs and have revealed a broad functional role for a range of different types of modifications. Here we review advances in the study of the evolution and function of PTMs that were spurred by these technological improvements. We provide an overview of studies focusing on the origin and evolution of regulatory enzymes as well as the evolutionary dynamics of modification sites. Finally, we discuss different mechanisms of altering protein activity via post‐translational regulation and progress made in the large‐scale functional characterization of PTM function.     

Broad Ranges of Affinity and Specificity of Anti-Histone Antibodies Revealed by a Quantitative Peptide Immunoprecipitation Assay

Antibodies directed against histone posttranslational modifications (PTMs) are critical tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. However, a lack of quantitative methods for characterizing such antibodies has been a major bottleneck in accurate and reproducible analysis of histone modifications. Here, we report a simple and sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Importantly, it determines the apparent dissociation constants for the interactions of an antibody with peptides harboring cognate or off-target PTMs. Analyses of commercial antibodies revealed large ranges of affinity, specificity and binding capacity as well as substantial lot-to-lot variations, suggesting the importance of quantitatively characterizing each antibody intended to be used in ChIP experiments and optimizing experimental conditions accordingly. Furthermore, using this method, we identified additional factors potentially affecting the interpretation of ChIP experiments.     

Data-independent acquisition proteomics methods for analyzing post-translational modifications

Protein post-translational modifications (PTMs) increase the functional diversity of the cellular proteome. Accurate and high throughput identification and quantification of protein PTMs is a key task in proteomics research. Recent advancements in data-independent acquisition (DIA) mass spectrometry (MS) technology have achieved deep coverage and accurate quantification of proteins and PTMs. This review provides an overview of DIA data processing methods that cover three aspects of PTMs analysis, that is, detection of PTMs, site localization, and characterization of complex modification moieties, such as glycosylation. In addition, a survey of deep learning methods that boost DIA-based PTMs analysis is presented, including in silico spectral library generation, as well as feature scoring and error rate control. The limitations and future directions of DIA methods for PTMs analysis are also discussed. Novel data analysis methods will take advantage of advanced MS instrumentation techniques to empower DIA MS for in-depth and accurate PTMs measurements.     

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine,two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics

ABSTRACT

Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via co-chromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.     

Characterization of the iron-binding properties of pyoverdine using electron-capture dissociation-tandem mass spectrometry

Pyoverdines (PVD) are a group of siderophores produced by fluorescent Pseudomonads. Identification of PVD variants mostly relies on liquid chromatography-tandem mass spectrometry (LC–MS/MS) using collision-induced dissociation (CID). Here, both CID and the novel dissociation technique electron-capture dissociation (ECD) were applied to characterize PVD succinamide and its Fe(III)-chelated complex. The results clearly showed that ECD produced diagnostic side chain fragmentation of the PVD peptide chain and preserved the labile Fe(III) binding to the chromophore in contrast to CID. The ECD technique is therefore expected to support the understanding of strain-specific Fe(III) transport processes of PVDs.     

A Quadrupole Dalton-based multi-attribute method for product characterization,process development,and quality control of therapeutic proteins

During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.