Middle-down Approach Research Articles

Top/Middle-Down Characterization of α-Synuclein Glycoforms.

α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

Structural Analysis of Monoclonal Antibodies with Top-down and Middle-down Electron Transfer Dissociation Mass Spectrometry: The First Decade

Monoclonal antibodies (mAbs) are protein biotherapeutics with a proven efficacy toward fighting life-threatening diseases. Their exceptional healing potential drives the annual increase in the number of novel mAbs and other antibody-like molecules entering clinical trials and the number of approved mAb-based drugs. Mass spectrometry (MS) offers high selectivity and specificity for the potentially unambiguous identification and comprehensive structural characterization of proteins, including at the proteoform level. It is thus not surprising that MS-based approaches are playing a central role in the biopharma laboratories, complementing and advancing traditional biotherapeutics characterization workflows. A combination of MS approaches is required to comprehensively characterize mAbs’ structures: the commonly employed bottom-up MS approaches are efficiently complemented with mass measurements at the intact and subunit (middle-up) levels, together with product ion analysis following gas-phase fragmentation of precursor ions performed at the intact (top-down) and subunit (middle-down) levels. Here we overview our group’s contribution to increasing the efficiency of these approaches and the development of the novel strategies over the past decade. Our particular focus has been on the top-down and middle-down MS methods that utilize electron transfer dissociation (ETD) for gas-phase protein ion fragmentation. Several approaches pioneered by our group, particularly an ETD-based middle-down approach, constitute a part of commercial software solutions for the mAb’s characterization workflows.

Mimicking LysC Proteolysis by 'Arginine Modification-cum-Trypsin Digestion': Comparison of Bottom-up & Middle-down Proteomic Approaches by ESI Q-TOF MS.

Middle-down (MD) proteomics is an emerging approach for reliable identification of post-translational modifications and isoforms, as this approach focuses on proteolytic peptides containing > 25-30 amino acid residues (a.a.r.), which are longer than typical tryptic peptides. Such longer peptides can be obtained by AspN, GluC, and LysC proteases. Additionally, some special proteases were developed specifically to effect MD approach, e.g., OmpT, Sap9, etc. However, these proteases are expensive. Herein we report a cost-effective strategy 'arginine modification- cum trypsin digestion', which can produce longer tryptic peptides resembling LysC peptides derived from proteins. The aim of this study is to obtain proteolytic peptides that resemble LysC peptides by using 'trypsin', which is a less expensive protease. This strategy is based on the simple principle that trypsin cannot act at the C-termini of those arginines in proteins, whose sidechain guanidine groups are modified by 1,2-cyclohexanedione or phenylglyoxal. As a proof of concept, we demonstrate this strategy on four models: β-casein (bovine), β- lactoglobulin (bovine), ovalbumin (chick) and transferrin (human), by electrospray ionization-mass spectrometry (ESI-MS) involving hybrid quadrupole time-of-flight. From the ESI-MS of these models, we obtained several arginine modified tryptic peptides, whose lengths are in the range of 30-60 a.a.r. The collision induced dissociation MS/MS characteristics of some of the arginine modified longer tryptic peptides are compared with the unmodified standard tryptic peptides. The strategy demonstrated in this proof-of-concept study is not only useful to obtain longer tryptic peptides that mimic LysC proteolytic peptides, but also facilitates in enhancing the probability of missed cleavages by the trypsin. Hence, this method aids in evading the possibility of obtaining very short peptides that are <5-10 a.a.r. Therefore, this is indeed a cost-effective alternative/ substitute for LysC proteolysis and, in turn, for those MD proteomic studies that utilize LysC. Additionally, this methodology can be fruitful for mass spectrometry-based de novo protein and peptide sequencing.

Histone Post‐Translational Modifications Detected in Mozambique Tilapia ( Oreochromis mossambicus ) Using Three Digestion Conditions

To analyze histone post-translational modifications (PTMs) through mass spectrometry, both bottom-up and middle-down approaches to proteomics are commonly employed. In this study, we compared the histone PTMs that can be detected in Mozambique tilapia (Oreochromis mossambicus) using three different digestion conditions. First, we tested a bottom-up approach where histone proteins are propionylated then digested with trypsin for cleavage on the carboxyl end of arginine. Second, we tested a middle-down approach where histones are digested with the V8 protease in the buffer ammonium bicarbonate. This leads to cleavage on the carboxyl end of glutamate. We tested another middle-down approach using V8 in the buffer sodium phosphate for the third digestion condition. This leads to cleavage on the carboxyl end of both glutamate and aspartate. Our results indicate which histone PTMs can be identified under each digestion condition on samples of gills, kidney, and testes of Mozambique tilapia. We specify which PTMs are only detected using one method, and which are detected through multiple methods.

Application of middle-down approach in quantitation and catabolite identification of protein by LC-high-resolution mass spectrometry.

Aim: To further enhance the detection sensitivity and increase resolving power of top-down intact protein bioanalysis, middle-down approach was explored. Materials & methods: An monoclonal antibody (mAb) was used as a model protein to evaluate quantitative bioanalytical assay performance and a disulfide linked dimer protein was investigated for its pharmacokinetics properties and catabolism in vivo by middle-down approach. Results & Conclusion: For quantitation of the mAb, different subunits generated by middle-down approach provided different level of signal improvement in biological samples with Lc and half Fc giving five-times better sensitivity than intact mAb. For the dimer protein, middle-down analysis by reduction enabled effective differentiation of the unchanged protein and its oxidized form, and clearly elucidated their respective proteolytic catabolites.

Electron Transfer Dissociation Parameter Optimization Using Design of Experiments Increases Sequence Coverage of Monoclonal Antibodies.

Middle-down analysis of monoclonal antibodies (mAbs) by tandem mass spectrometry (MS2) can provide detailed insight into their primary structure with minimal sample preparation. The middle-down approach uses an enzyme to cleave mAbs into Fc/2, LC, and Fd subunits that are then analyzed by reversed phase liquid chromatography tandem mass spectrometry (RPLC-MS2). As maximum sequence coverage is desired to obtain meaningful structural information at the subunit level, a host of dissociation methods have been developed, and sometimes combined, to bolster fragmentation and increase the number of identified fragments. Here, we present a design of experiments (DOE) approach to optimize MS2 parameters, in particular those that may influence electron transfer dissociation (ETD) efficiency to increase the sequence coverage of antibody subunits. Applying this approach to the NIST monoclonal antibody standard (NISTmAb) using three RPLC-MS2 runs resulted in high sequence coverages of 67%, 67%, and 52% for Fc/2, LC, and Fd subunits, respectively. In addition, we apply this DOE strategy to model the parameters required to maximize the number of fragments produced in "low", "medium", and "high" mass ranges, which ultimately resulted in even higher sequence coverages of NISTmAb subunits (75%, 78%, and 64% for Fc/2, LC, and Fd subunits, respectively). The DOE approach provides high sequence coverage percentages utilizing only one fragmentation method, ETD, and could be extended to other state-of-the-art techniques that combine multiple fragmentation mechanisms to increase coverage.

Intact and middle-down CIEF of commercial therapeutic monoclonal antibody products under non-denaturing conditions.

A two‐step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non‐denaturing separation conditions. CIEF of the intact mAb was combined with a middle‐down approach analyzing Fc/2 and F(ab´)2 fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8–10.5 with PL 3–10 for variants of intact rituximab and of F(ab´)2 fragments, respectively, whereas PL 6.7–7.7 in combination with PL 3–10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)2. In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C‐terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.

All Ion Differential Analysis Refines the Detection of Terminal and Internal Diagnostic Fragment Ions for the Characterization of Biologics Product-Related Variants and Impurities by Middle-down Mass Spectrometry.

Characterization and monitoring of post-translational modifications (PTMs) are key analytical requirements during the development of biologics. Top and middle-down (MD) approaches aim at capturing a direct snapshot of all proteoforms with their combinatorial distribution. However, classical MD data analysis is predominantly limited to the interpretation of terminal ion series and PTMs matched by mass. In this study, time-resolved deconvolution (TRD) maps were produced to detect variants and impurities in Fd, Fc/2, and LC subunits of an IgG1 consistently across multiple samples. Classical MD analysis retrieved terminal ions, suggesting a deamidation at a NN motif for a LC+1 Da species, and inconclusive information for a LC+40 Da species. Additionally, we performed differential analysis of all MS2 ions across unmodified and variant subunit spectra to focus data analysis on spectral differences and reveal diagnostic ions (present, absent, enriched, or depleted ions) before fragment assignment. This sensitive methodology was able to detect diagnostic ions in a chimeric spectrum pointing at a proline-to-histidine sequence variant (+40 Da) missed by classical MD analysis. This methodology was pivotal to unravel relevant terminal ions and internal fragments N-terminal to proline as diagnostic ions to confirm the deamidation site. Moreover, different cleavage propensities were revealed at the deamidated DN site compared to the native NN motif for terminal and internal fragments, which may be tracked as a diagnostic behavior. Differential analysis may refine the detection of novel diagnostic ions and leverage the sequence information on internal fragments for the characterization of product-related variants and impurities by MD mass spectrometry.

One-Pot Quantitative Top- and Middle-Down Analysis of GluC-Digested Histone H4.

Histone post-translational modifications (PTMs) have been intensively investigated due to their essential function in eukaryotic genome regulation. Histone modifications have been effectively studied using modified bottom-up proteomics approaches; however, the methods often do not capture single-molecule combinations of PTMs (proteoforms) that mediate known and expected biochemical mechanisms. Both middle-down mass spectrometry (MS) and top-down MS quantitation of H4 proteoforms present viable access to this important information. Histone H4 middle-down has previously avoided GluC digestion due to complex digestion products and interferences; however, the common AspN digestion cleaves at amino acid 23, disconnecting K31ac from other PTMs. Here, we demonstrate the effective use of GluC-based middle-down quantitation and compare it to top-down-based quantitation of proteoforms. Despite potential interferences in the m/z space, the proteoforms arising from all three GluC products (E52, E53, and E63) and intact H4 are chromatographically resolved and successfully analyzed in a single LC-MS analysis. Quantitative results and associated analytical metrics are compared between the different analytes of a single sample digested to different extents to reveal general concordance as well as the relative biases and complementarity of each approach. There is moderate proteoform discordance between digestion products (e.g., E52 and E53); however, each digestion product exhibits high concordance, regardless of digestion time. Under the conditions used, the GluC products are better chromatographically resolved yet show greater variance than the top-down quantitation that are more extensively sampled for MS2. GluC-based middle-down of H4 is thus viable. Both top-down and middle-down approaches have comparable quantitation capacity and are complementary.

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry.

Owing to rapid growth in the elucidation of genome sequences of various organisms, deducing proteome sequences has become imperative, in order to have an improved understanding of biological processes. Since the traditional Edman method was unsuitable for high-throughput sequencing and also for N-terminus modified proteins, mass spectrometry (MS) based methods, mainly based on soft ionization modes: electrospray ionization and matrix-assisted laser desorption/ionization, began to gain significance. MS based methods were adaptable for high-throughput studies and applicable for sequencing N-terminus blocked proteins/peptides too. Consequently, over the last decade a new discipline called ‘proteomics’ has emerged, which encompasses the attributes necessary for high-throughput identification of proteins. ‘Proteomics’ may also be regarded as an offshoot of the classic field, ‘biochemistry’. Many protein sequencing and proteomic investigations were successfully accomplished through MS dependent sequence elucidation of ‘short proteolytic peptides (typically: 7–20 amino acid residues), which is called the ‘shotgun’ or ‘bottom-up (BU)’ approach. While the BU approach continues as a workhorse for proteomics/protein sequencing, attempts to sequence intact proteins without proteolysis, called the ‘top-down (TD)’ approach started, due to ambiguities in the BU approach, e.g., protein inference problem, identification of proteoforms and the discovery of posttranslational modifications (PTMs). The high-throughput TD approach (TD proteomics) is yet in its infancy. Nevertheless, TD characterization of purified intact proteins has been useful for detecting PTMs. With the hope to overcome the pitfalls of BU and TD strategies, another concept called the ‘middle-down (MD)’ approach was put forward. Similar to BU, the MD approach also involves proteolysis, but in a restricted manner, to produce ‘longer’ proteolytic peptides than the ones usually obtained in BU studies, thereby providing better sequence coverage. In this regard, special proteases (OmpT, Sap9, IdeS) have been used, which can cleave proteins to produce longer proteolytic peptides. By reviewing ample evidences currently existing in the literature that is predominantly on PTM characterization of histones and antibodies, herein we highlight salient features of the MD approach. Consequently, we are inclined to claim that the MD concept might have widespread applications in future for various research areas, such as clinical, biopharmaceuticals (including PTM analysis) and even for general/routine characterization of proteins including therapeutic proteins, but not just limited to analysis of histones or antibodies.

A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection.

Oxidation of biopharmaceuticals may affect their bioactivity, serum half-life, and (bio)chemical stability. The Fc domain of IgG monoclonal antibodies (mAbs) contains two methionine residues which are susceptible to oxidation. Here, we present a middle-down approach employing the cysteine protease IdeS under reducing conditions to obtain three mAb subunits of approximately 25 kDa: Fc/2, Fd', and LC. These subunits were separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and detected by UV spectroscopy as well as Orbitrap mass spectrometry (MS), as well as MS upon all-ion fragmentation (AIF-MS). We evaluated the feasibility of three strategies for absolute quantification of oxidation in the Fc region of hydrogen peroxide-stressed Rituximab, using a single, commercially available software platform both for data acquisition and evaluation: UV spectroscopy, full-scan MS, and monitoring of product ions obtained by AIF-MS. UV spectroscopy showed the lowest limits of quantification (LOQ) (0.96 ng μL-1) and featured the lowest relative process standard deviation (Vx0%) of 7.2% compared to MS and AIF-MS with LOQs of 1.24-4.32 ng μL-1 and relative process standard deviations of 9.0-14%, respectively. Our approach is generic in that it allows monitoring and quantification of oxidation in the Fc regions of fully human and humanized IgG1 mAbs as well as of Fc-fusion proteins. This is exemplified by limits of detection of 1.2%, 1.0%, and 1.2% of oxidation in drug products containing the biopharmaceuticals Rituximab, Adalimumab, and Etanercept, respectively. The presented method is an attractive alternative to conventional time-intensive peptide mapping which is prone to artificial oxidation due to extensive sample preparation.

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS.

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background. Graphical Abstract ᅟ.

Top-down and middle-down approach by fraction collection enrichment using off-line capillary electrophoresis – mass spectrometry coupling: Application to monoclonal antibody Fc/2 charge variants

The characterization of complex protein mixtures represents one of the biggest challenge in many research fields such as biological or biopharmaceutical sciences. Out of all categories, monoclonal antibodies (mAbs) and related products drawn the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need for analytical methods to provide comprehensive in-depth characterization of these proteins. In this work, we developed a methodology using CE-UV/MALDI-MS to perform top-down or middle-down characterization after fraction collection enrichment applied to intact protein and mAbs samples. The performance of the method was evaluated with the rapid separation of three intact protein mixture. Good robustness of CZE separation and quality of MALDI-MS spectra and MALDI-ISD spectra of each protein confirms the usefulness of sample enrichment to obtain adequate quantity of deposed protein for top-down analysis and the proof of principle of the method. In a second step, the method was applied to the middle-down characterization of Fc/2 cetuximab variants. Identification of around 9% sequence coverage of Fc/2 cetuximab fragments allows to conclude on the feasibility of the strategy for middle-down characterization of Fc/2 cetuximab variants using CE-UV/MALDI-MS. Moreover, MALDI-ISD fragmentation of Fc/2 cetuximab variants confirm separation phenomenon based on the formation of Fc/2 dimers with and without C-terminal truncation.

Cross Reactive Material 197 glycoconjugate vaccines contain privileged conjugation sites.

Production of glycoconjugate vaccines involves the chemical conjugation of glycans to an immunogenic carrier protein such as Cross-Reactive-Material-197 (CRM197). Instead of using glycans from natural sources recent vaccine development has been focusing on the use of synthetically defined minimal epitopes. While the glycan is structurally defined, the attachment sites on the protein are not. Fully characterized conjugates and batch-to-batch comparisons are the key to eventually create completely defined conjugates. A variety of glycoconjugates consisting of CRM197 and synthetic oligosaccharide epitopes was characterised using mass spectrometry techniques. The primary structure was assessed by combining intact protein MALDI-TOF-MS, LC-MALDI-TOF-MS middle-down and LC-ESI-MS bottom-up approaches. The middle-down approach on CNBr cleaved glycopeptides provided almost complete sequence coverage, facilitating rapid batch-to-batch comparisons, resolving glycan loading and identification of side products. Regions close to the N- and C-termini were most efficiently conjugated.

High resolution parallel reaction monitoring with electron transfer dissociation for middle-down proteomics.

In recent years, middle-down proteomics has emerged as a popular technique for the characterization and quantification of proteins not readily amenable to typical bottom-up approaches. So far, all high resolution middle-down approaches are done in data-dependent acquisition mode, using both collision-induced dissociation or electron capture/transfer dissociation techniques. Here, we explore middle-down proteomics with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion. As an example of a highly modified protein, we used histone H3 fractions from untreated and DMSO-treated Murine ErythroLeukemia (MEL) cells. We first determined optimized instrument parameters to obtain high sequence coverage using a synthetic standard peptide. We then setup a combined method of both MS1 scans and PRM scans of the 20 most abundant combinations of methylation and acetylation of the +10 charge state of the N-terminal tail of H3. Weak cation exchange hydrophilic interaction chromatography was used to separate the N-terminal H3 tail, primarily, by its acetylation and, to a secondary degree, by its methylation status, which aided in the interpretation of the results. After deconvolution of the highly charged ions, peaks were annotated to a minimum set of 254 H3 proteoforms in the untreated and treated samples. Upon DMSO treatment, global quantitation changes from the MS1 level show a relative decrease of 2, 3, 4, and 5 acetylations and an increase of 0 and 1 acetylations. A fragment ion map was developed to visualize specific differences between treated and untreated samples. Taken together, the data presented here show that middle-down proteomics with electron transfer dissociation using PRM is a novel, attractive method for the effective analysis and quantification of large and highly modified peptides.

High resolution data-independent acquisition with electron transfer dissociation mass spectrometry: Multiplexed analysis of post-translationally modified proteins

Data-dependent acquisition (DDA) mode is the most commonly used method in bottom-up proteomics. Recently, data-independent acquisition (DIA) modes have become popular alternatives because of their unbiased analysis, leading in general to more comprehensive, global qualitative profiling of proteome systems and also higher quantitative reproducibility in such profiling. Most of the previously established DIA methods are based on collision-induced dissociation (CID). However, when it comes to the analysis of labile post-translational modifications (PTMs), electron capture/transfer dissociation (ECD/ETD) may be better suited. In addition to the bottom-up approach, the middle-down approach, which analyzes peptides in the range of 3,000–10,000Da has emerged as an attractive alternative, including the analysis of highly modified and highly variable protein variants that exist in key system functions, such as histone signaling cascades. Here, we establish that a data-independent (DIA) middle-down ETD approach is a superior strategy in the differential characterization of PTM changes in histone H2B. We suggest that this strategy can further be used for other approaches where dynamic PTM characterization or changes due to different conditions are fundamental to accurate understanding of biological systems and function.

Middle-down electron capture dissociation and electron transfer dissociation for histone analysis

Abstract The post-translational modifications (PTMs) of histones play a major role in activating or silencing gene transcription. To gain better understanding of the interplay between the PTMs that occur on histones, they are extensively studied using mass spectrometry techniques. Due to the abundance of lysines and arginines, the typical trypsin digestion has been found less favorable and GluC-digests have been explored as an alternative to yield larger peptides amenable to middle-down approaches. In addition, the use of weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) and the use of electron-based fragmentation techniques were found to be advantageous for the in-depth characterization of histone variants containing multiple PTMs. As a test model, we used histones from MEL (murine erythroleukemia) cells treated with butyric acid or DMSO. After acid extraction, histone pellets were dried and fractionated using a reversed-phase C3 column. For middle-down analysis, selected histone fractions were digested using GluC. The digested samples were separated on a WCX-HILIC capillary column packed in-house with PolyCAT A resin, coupled to a linear trap quadrupole Fourier transformation ion cyclotron resonance (LTQFT-ICR) instrument. Raw data was acquired on the LTQFT-ICR using electron capture dissociation (ECD). After deconvolution of the raw data, we generated heatmaps to illustrate differential maps between differentially treated histone samples. We also explored the innovative use of Skyline to quantify histone tails. In addition, we report some preliminary data using a synthetic histone peptide acquired on an Orbitrap Fusion using electron transfer dissociation (ETD). Both, ECD and ETD methods are capable of comprehensively analyzing complex histone variations not accessible with conventional techniques.

Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry.

O-glycosylation of natural or recombinant proteins poses a challenge because of the lack of unambiguous consensus sites, the agglomeration of several O-glycans in close proximity and the lack of efficient O-glycosidases. Even bovine fetuin, a frequently used test glycoprotein for glycosylation analysis, has hitherto not been fully characterized in terms of site occupancy. This gap shall hereby be closed by application of electron-transfer dissociation mass spectroscopy.

Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry

The rapid growth of approved biotherapeutics, e.g., monoclonal antibodies or immunoglobulins G (IgGs), demands improved techniques for their quality control. Traditionally, proteolysis-based bottom-up mass spectrometry (MS) has been employed. However, the long, multistep sample preparation protocols required for bottom-up MS are known to potentially introduce artifacts in the original sample. For this reason, a top-down MS approach would be preferable. The current performance of top-down MS of intact monoclonal IgGs, though, enables reaching only up to ∼30% sequence coverage, with incomplete sequencing of the complementarity determining regions which are fundamental for IgG's antigen binding. Here, we describe a middle-down MS protocol based on the use of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS), which is capable of digesting IgGs in only 30 min. After chemical reduction, the obtained ∼25 kDa proteolytic fragments were analyzed by reversed phase liquid chromatography (LC) coupled online with an electron transfer dissociation (ETD)-enabled hybrid Orbitrap Fourier transform mass spectrometer (Orbitrap Elite FTMS). Upon optimization of ETD and product ion transfer parameters, results show that up to ∼50% sequence coverage for selected IgG fragments is reached in a single LC run and up to ∼70% when data obtained by distinct LC-MS runs are averaged. Importantly, we demonstrate the potential of this middle-down approach in the identification of oxidized methionine residues. The described approach shows a particular potential for the analysis of IgG mixtures.

Practical Considerations for Improving the Productivity of Mass Spectrometry-based Proteomics

Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/ MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.