Electron-transfer/higher-energy Collision Dissociation Research Articles

Enhancing De Novo Protein Sequencing through the C-Terminal Labeling Strategy: Resolving Isobaric Ambiguities by Electron-Transfer/Higher Energy Collision Dissociation (EThcD).

De novo protein sequencing via a bottom-up approach requires various proteases to produce overlapping peptides. However, peptides generated by proteases other than trypsin, LysC, and ArgC often yield C-terminal fragments with suboptimal ionization in positive mode mass spectrometry (MS). This study introduces a novel peptide labeling strategy that involves modifying peptides at the C-terminal and at the carboxyl groups of Aspartic and Glutamic acid with arginine methyl ester (R-met) to improve peptide fragmentation and resolve isobaric ambiguities encountered during sequencing. An amidation reaction is used with coupling reagents to conjugate R-met to the peptide's C-terminal end, introducing a functional group that enhances the detectability of C-terminal peptide fragment ions by mass spectrometry. Subsequently, selecting a charge state of +2 or higher can facilitate optimal fragmentation of the derivatized peptides using electron-transfer/higher energy collision dissociation (EThcD), thereby generating essential w-ions to resolve common isobaric ambiguities. Demonstrating this strategy across diverse protein types, including albumin and antibodies and using different proteases for digestion, highlights the unique characteristics of combining the proposed amidation reaction with the specific proteases tested.

Glycan-Tailored Glycoproteomic Analysis Reveals Serine is the Sole Residue Subjected to O-Linked Glycosylation in Acinetobacter baumannii.

Protein glycosylation is a ubiquitous process observed across all domains of life. Within the human pathogen Acinetobacter baumannii, O-linked glycosylation is required for virulence; however, the targets and conservation of glycosylation events remain poorly defined. In this work, we expand our understanding of the breadth and site specificity of glycosylation within A. baumannii by demonstrating the value of strain specific glycan electron-transfer/higher-energy collision dissociation (EThcD) triggering for bacterial glycoproteomics. By coupling tailored EThcD-triggering regimes to complementary glycopeptide enrichment approaches, we assessed the observable glycoproteome of three A. baumannii strains (ATCC19606, BAL062, and D1279779). Combining glycopeptide enrichment techniques including ion mobility (FAIMS), metal oxide affinity chromatography (titanium dioxide), and hydrophilic interaction liquid chromatography (ZIC-HILIC), as well as the use of multiple proteases (trypsin, GluC, pepsin, and thermolysis), we expand the known A. baumannii glycoproteome to 33 unique glycoproteins containing 42 glycosylation sites. We demonstrate that serine is the sole residue subjected to glycosylation with the substitution of serine for threonine abolishing glycosylation in model glycoproteins. An A. baumannii pan-genome built from 576 reference genomes identified that serine glycosylation sites are highly conserved. Combined this work expands our knowledge of the conservation and site specificity of A. baumannii O-linked glycosylation.

#2000 Analysis between site-specific N-glycosylation of monoclonal immunoglobulin and complement system activation

Abstract Background and Aims Monoclonal immunoglobulin produced by clonal plasma cells is the main cause in multiple myeloma (MM) and monoclonal gammopathy of renal significance (MGRS). Hypocomplementemia and deposition of complement components in the kidney is observed in some patients with MM or MGRS. Previous studies have suggested that glycosylation of the immunoglobulin was associated with complement-mediated-effector activities. Whereas, site-specific N-glycosylation characterization for monoclonal immunoglobulin in MM or MGRS and its effects on complement system activation still remains unclear. Method We separated plasma monoclonal IgG1 and IgA immunoglobulin from patients with MM and human plasma immunoglobulin from healthy controls (HC). Purified IgG and IgA molecules were digested by trypsin. Tryptic peptides without enrichment of intact N-glycopeptides were analyzed using a combination of electron-transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (EThcD-sceHCD-MS/MS). N-glycosylation characterization was compared between monoclonal immunoglobulin from MM patients and healthy controls and its association with complement level was evaluated. Results A total of 46 IgG1 digestion samples (HC, n = 16; MM, n = 30) and 38 IgA1 digestion samples (HC, n = 16; MM, n = 22) were analyzed using the EThcDsceHCD-MS/MS. On average, 34 and 37 unique N-glycopeptides of IgG1, 19 unique N-glycans per IgG were identified from HC and MM. The relative abundance of fucosylated N-glycans of IgG1 in MM was significantly higher than HC (94.7% vs 88.8%, p = 0.002). Other types of N-glycans including sialylated, hybrid and complex were similar between the two groups. The types of N-glycans including fucosylated, sialylated, hybrid and complex were similar between patients with hypocomplementemia and normal complement levels. Regarding IgA1, on average, 63 and 52 unique N-glycopeptides of IgA1, 40 and 35 unique N-glycans per IgA were identified from HC and MM. The relative abundance of fucosylated (41.0% vs 26.4%, p < 0.001), sialylated (61.0% vs 45.6%, p = 0.003), mannose (2.44% vs 0.28%, p < 0.001) N-glycans of IgA1 in MM was significantly higher than HC. In MM, the relative abundance of mannose N-glycans of IgA1 in patients with hypocomplementemia was significantly lower than patients with normal complement levels (1.69% vs 7.52%, p < 0.001). The relative abundance of fucosylated and sialylated N-glycans were similar between patients with hypocomplementemia and normal complement levels. Conclusion Site-specific N-glycosylation characterization of monoclonal IgG1 and IgA1 in MM patients were differentially expressed from healthy controls. Certain specific N-glycan types were associated with complement levels. These findings laid a foundation for future investigations of their biological effects.

CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols

Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) of monoclonal antibodies (mAbs). The implemented reduction often leaves the intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on a neutral-coated capillary for the separation of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by mass spectrometry (MS), MS/MS identification, and trapped ion mobility mass spectrometry (timsTOF). Our CE approach enables the separation of (i) different subunit moieties, (ii) various reduction states, and (iii) positional isomers of these partly reduced subunit moieties. The location of the remaining disulfide bridges can be determined by middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All these CE-separated variants show differences in ion mobility in the timsTOF measurements. Applying the presented CE-MS/MS method, reduction parameters such as the use of chaotropic salts were studied. For the investigated antibodies, urea improved the subunit reduction significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals of the same subunit in the CE separation. The presented CE-MS method is a powerful tool for the disulfide-variant characterization of mAbs on the subunit level. It enables understanding disulfide bridge reduction processes in antibodies and potentially other proteins.

Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

Analysis of complex proteoglycans using serial proteolysis and EThcD provides deep N- and O-glycoproteomic coverage.

Proteoglycans are a small but diverse family of proteins that play a wide variety of roles at the cell surface and in the extracellular matrix. In addition to their glycosaminoglycan (GAG) chains, they are N- and O-glycosylated. All of these types of glycosylation are crucial to their function but present a considerable analytical challenge. We describe the combination of serial proteolysis followed by the application of higher-energy collisional dissociation (HCD) and electron transfer/higher-energy collisional dissociation (EThcD) to optimize protein sequence coverage and glycopeptide identification from proteoglycans. In many cases, the use of HCD alone allows the identification of more glycopeptides. However, the localization of glycoforms on multiply glycosylated peptides has remained elusive. We demonstrate the use of EThcD for the confident assignment of glycan compositions on multiply glycosylated peptides. Dense glycosylation on proteoglycans is key to their biological function; thus, developing tools to identify and quantify doubly glycosylated peptides is of interest. Additionally, glycoproteomics searches identify glycopeptides in otherwise poorly covered regions of proteoglycans. The development of these and other analytical tools may permit glycoproteomic similarity comparisons in biological samples.

Large-Scale and Site-Specific Mapping of the Murine Brain O-Glycoproteome with IMPa.

Altered protein glycosylation is typically associated with cognitive defects and other phenotypes, but there is a lack of knowledge about the brain glycoproteome. Here, we used the newly available O-glycoprotease IMPa from Pseudomonas aeruginosa for comprehensive O-glycoproteomic analyses of the mouse brain. In this approach, total tryptic glycopeptides were prepared, extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by electron-transfer/higher-energy collision dissociation (EThcD), which permits site-specific and global analysis of all types of O-glycans. We developed two complementary approaches for the analysis of the total O-glycoproteome using HEK293 cells and derivatives. The results demonstrated that IMPa and EThcD facilitate the confident localization of O-glycans on glycopeptides. We then applied these approaches to characterize the O-glycoproteome of the mouse brain, which revealed the high frequency of various sialylated O-glycans along with the unusual presence of the Tn antigen. Unexpectedly, the results demonstrated that glycoproteins in the brain O-glycoproteome only partly overlap with those reported for the brain N-glycoproteome. These approaches will aid in identifying the novel O-glycoproteomes of different cells and tissues and foster clinical and translational insights into the functions of protein O-glycosylation in the brain and other organs.

Conjugation site characterization of antibody–drug conjugates using electron-transfer/higher-energy collision dissociation (EThcD)

Antibody–drug conjugates (ADCs) are formed by binding of cytotoxic drugs to monoclonal antibodies (mAbs) through chemical linkers. A comprehensive evaluation of the critical quality attributes (CQAs) of ADCs is vital for drug development but remains challenging owing to ADC structural heterogeneity than mAbs. Drug conjugation sites can considerably affect ADC properties, such as stability and pharmacokinetics, however, few studies have focused on method development in this area owing to technical challenges. Hybrid electron-transfer/higher-energy collision dissociation (EThcD) produces more fragment ions than conventional higher-energy collision dissociation (HCD) fragmentation, which aids in identifying and localizing post-translational modifications. Herein, we systematically employ EThcD to assess the fragmentation mode impact on conjugation site characterization for randomly conjugated and site-specific ADCs. EThcD generates more fragment ions in tandem mass spectrometry (MS/MS) spectra compared with HCD. Additional ions aid in pinpointing the correct conjugation sites that bear complex linker payload structures. Our study may contribute to the quality control of various preclinical and clinical ADCs.

Novel Isobaric Tagging Reagent Enabled Multiplex Quantitative Glycoproteomics via Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry.

Protein glycosylation, covalent attachment of carbohydrates to polypeptide chains, is a highly important post-translational modification involved in many essential physiological processes. Comprehensive site-specific and quantitative analysis is crucial for revealing the diverse functions and dynamics of glycosylation. To characterize intact glycopeptides, mass spectrometry (MS)-based glycoproteomics employs versatile fragmentation methods, among which electron-transfer/higher-energy collision dissociation (EThcD) has gained great popularity. However, the inherent limitation of EThcD in fragmenting low-charge ions has prevented its widespread applications. Furthermore, there is a need to develop a high-throughput strategy for comparative glycoproteomics with a large cohort of samples. Herein, we developed isobaric N,N-dimethyl leucine-derivatized ethylenediamine (DiLeuEN) tags to increase the charge states of glycopeptides, thereby improving the fragmentation efficiency and allowing for in-depth intact glycopeptide analysis, especially for sialoglycopeptides. Moreover, the unique reporter ions of DiLeuEN-labeled glycopeptides generated in tandem MS spectra enable relative quantification of up to four samples in a single analysis, which represents a new high-throughput method for quantitative glycoproteomics.

The Key Role of Metal Adducts in the Differentiation of Phosphopeptide from Sulfopeptide Sequences by High-Resolution Mass Spectrometry.

Site localization of protein sulfation by high-throughput proteomics remains challenging despite the technological improvements. In this study, sequence analysis and site localization of sulfation in tryptic peptides were determined under a conventional nano-liquid chromatography-mass spectrometry configuration. Tryptic sulfopeptide standards were used to study different fragmentation strategies, including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), electron-transfer/higher-energy collision dissociation (EThcD), and electron-transfer/collision-induced dissociation (ETciD), in the positive ionization mode. Sulfopeptides displayed only neutral loss of SO3 under CID, while the sequence could be determined for all other tested fragmentation techniques. Results were compared to the same sequences with phosphotyrosine, indicating important differences, as the sequence and modification localization could be studied by all fragmentation strategies. However, the use of metal adducts, especially potassium, provided valuable information for sulfopeptide localization in ETD and ETD-hybrid strategies by stabilizing the modification and increasing the charge state of sulfopeptides. In these conditions, both the sequence and localization could be obtained. In-source neutral loss of SO3 under EThcD provided diagnostic peaks suitable to distinguish the sulfopeptides from the nearly isobaric phosphopeptides. Further confirmation on the modification type was found in the negative ionization mode, where phosphopeptides always had the typical phosphate product ion corresponding to PO3–.

Template-Assisted De Novo Sequencing of SARS-CoV-2 and Influenza Monoclonal Antibodies by Mass Spectrometry

In this study, we used multiple enzyme digestions, coupled with higher-energy collisional dissociation (HCD) and electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to develop a mass-spectrometric (MS) method for determining the complete protein sequence of monoclonal antibodies (mAbs). The method was refined on an mAb of a known sequence, a SARS-CoV-1 antireceptor binding domain (RBD) spike monoclonal antibody. The data were searched using Supernovo to generate a complete template-assisted de novo sequence for this and two SARS-CoV-2 mAbs of known sequences resulting in correct sequences for the variable regions and correct distinction of Ile and Leu residues. We then used the method on a set of 25 antihemagglutinin (HA) influenza antibodies of unknown sequences and determined high confidence sequences for >99% of the complementarity determining regions (CDRs). The heavy-chain and light-chain genes were cloned and transfected into cells for recombinant expression followed by affinity purification. The recombinant mAbs displayed binding curves matching the original mAbs with specificity to the HA influenza antigen. Our findings indicate that this methodology results in almost complete antibody sequence coverage with high confidence results for CDR regions on diverse mAb sequences.

Precise O-Glycosylation Site Localization of CD24Fc by LC-MS Workflows.

CD24Fc is a homodimeric recombinant Fc-fusion protein comprised of human CD24 connected to immunoglobulin G1 (IgG1) Fc fragment. CD24 is heavily glycosylated, and its biological function is considered mainly mediated by its glycosylation. Identification of the O-glycosylation sites would facilitate an in-depth understanding of the functional role of O-glycans in CD24. However, the presence of clustered mucin-type O-glycans together with N-glycans makes the determination of O-glycosylation sites and abundance very challenging. In this study, two sets of liquid chromatography-mass spectrometry (LC-MS) workflows were developed for the comprehensive characterization of O-glycosylation in CD24: (1) Fractionation and collision-induced dissociation (CID) workflow involving multienzyme digestion, fractionation, OpeRATOR/SialEXO digestion, and CID analysis; (2) Direct OpeRATOR/SialEXO digestion followed by electron-transfer/higher-energy collision dissociation (EThcD) analysis. The precise O-glycosylation sites were identified in CD24 for the first time, and the site occupancy was assessed. A total of 12 O-glycosylation sites were identified. Seven glycosylation sites were identified by both workflows, and five additional sites were identified only by the EThcD workflow. The predominant O-glycosylation site in CD24 was Thr25 followed by Thr15. The CID workflow provided an overall relative quantitation of O-glycoforms at the CD24 level and site localization for singly O-glycosylated peptides. The EThcD workflow directly identified glycosylation sites by tandem mass spectrometry (MS/MS) for singly, doubly, and triply O-glycosylated peptides. Together, both workflows validated each other's results and can be applied to a complex mucin-type O-glycosylation site analysis of other glycoproteins and Fc-fusion therapeutics.

Comparative N-Glycoproteomics Analysis of Clinical Samples Via Different Mass Spectrometry Dissociation Methods.

Site-specific N-glycosylation characterization requires intact N-glycopeptide analysis based on suitable tandem mass spectrometry (MS/MS) method. Electron-transfer/higher-energy collisional dissociation (EThcD), stepped collision energy/higher-energy collisional dissociation (sceHCD), higher-energy collisional dissociation-product-dependent electron-transfer dissociation (HCD-pd-ETD), and a hybrid mass spectrometry fragmentation method EThcD-sceHCD have emerged as valuable approaches for glycoprotein analysis. However, each of them incurs some compromise, necessitating the systematic performance comparisons when applied to the analysis of complex clinical samples (e.g., plasma, urine, cells, and tissues). Herein, we compared the performance of EThcD-sceHCD with those previous approaches (EThcD, sceHCD, HCD-pd-ETD, and sceHCD-pd-ETD) in the intact N-glycopeptide analysis, and determined its applicability for clinical N-glycoproteomic study. The intact N-glycopeptides of distinct samples, namely, plasma from prostate cancer (PCa) patients, urine from immunoglobulin A nephropathy (IgAN) patients, human hepatocarcinoma cell line (HepG2), and thyroid tissues from thyroid cancer (TC) patients were analyzed by these methods. We found that EThcD-sceHCD outperformed other methods in the balance of depth and accuracy of intact N-glycopeptide identification, and sceHCD and EThcD-sceHCD have good complementarity. EThcD-sceHCD holds great potential for biomarker discovery from clinical samples.

Sequential Analysis of the N/O-Glycosylation of Heavily Glycosylated HIV-1 gp120 Using EThcD-sceHCD-MS/MS.

Deciphering the glycosylation of the viral envelope (Env) glycoprotein is critical for evaluating viral escape from the host’s immune response and developing vaccines and antiviral drugs. However, it is still challenging to precisely decode the site-specific glycosylation characteristics of the highly glycosylated Env proteins, although glycoproteomics have made significant advances in mass spectrometry techniques and data analysis tools. Here, we present a hybrid dissociation technique, EThcD-sceHCD, by combining electron transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) into a sequential glycoproteomic workflow. Following this scheme, we characterized site-specific N/O-glycosylation of the human immunodeficiency virus type 1 (HIV-1) Env protein gp120. The EThcD-sceHCD method increased the number of identified glycopeptides when compared with EThcD, while producing more comprehensive fragment ions than sceHCD for site-specific glycosylation analysis, especially for accurate O-glycosite assignment. Finally, eighteen N-glycosites and five O-glycosites with attached glycans were assigned unambiguously from heavily glycosylated gp120. These results indicate that our workflow can achieve improved performance for analysis of the N/O-glycosylation of a highly glycosylated protein containing numerous potential glycosites in one process. Knowledge of the glycosylation landscape of the Env glycoprotein will be useful for understanding of HIV-1 infection and development of vaccines and drugs.

Suppression of O-Linked Glycosylation of the SARS-CoV-2 Spike by Quaternary Structural Restraints.

Understanding the glycosylation of the envelope spike (S) protein of SARS-CoV-2 is important in defining the antigenic surface of this key viral target. However, the underlying protein architecture may significantly influence glycan occupancy and processing. There is, therefore, potential for different recombinant fragments of S protein to display divergent glycosylation. Here, we show that the receptor binding domain (RBD), when expressed as a monomer, exhibits O-linked glycosylation, which is not recapitulated in the native-like soluble trimeric protein. We unambiguously assign O-linked glycosylation by homogenizing N-linked glycosylation using the enzymatic inhibitor, kifunensine, and then analyzing the resulting structures by electron-transfer higher-energy collision dissociation (EThcD) in an Orbitrap Eclipse Tribrid instrument. In the native-like trimer, we observe a single unambiguous O-linked glycan at T323, which displays very low occupancy. In contrast, several sites of O-linked glycosylation can be identified when RBD is expressed as a monomer, with T323 being almost completely occupied. We ascribe this effect to the relaxation of steric restraints arising from quaternary protein architecture. Our analytical approach has also highlighted that fragmentation ions arising from trace levels of truncated N-linked glycans can be misassigned as proximal putative O-linked glycan structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we show that in matched expression systems the quaternary protein architecture limits O-linked glycosylation of the spike protein.

A Linkage-specific Sialic Acid Labeling Strategy Reveals Different Site-specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes the extensively glycosylated spike (S) protein protruding from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the main recombinant S protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, a recombinant S protein truncated at the transmembrane domain and engineered to express a C-terminal trimerization motif was transiently produced in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on S protein, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on the glycans and glycopeptides from the S protein. The process also adds a charge to Asp and Glu which aids in ionization. We used MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to determine global and site-specific N-linked glycosylation patterns. We identified 21 and 19 out of the 22 predicted N-glycosites of the SARS-CoV-2 S proteins produced in CHO and HEK, respectively. It was found that the N-glycosite at 1,158 position (N1158) and at 122, 282 and 1,158 positions (N122, N282 and N1158) were absent on S from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human S proteins reveals that CHO-Spike exhibits more complex and higher sialylation (α2,3-linked) content while HEK-Spike exhibits more high-mannose and a small amount of α2,3- and α2,6-linked sialic acids. The N74 site represents the most abundant glycosite on both spike proteins. The relatively higher amount of high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801, and N1134) on HEK-Spike suggests that glycan-shielding may differ among the two constructs. HEK-Spike can also provide different host immune system interaction profiles based on known immune system active lectins. Collectively, these data underscore the importance of characterizing the site-specific glycosylation of recombinant human spike proteins from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in research, diagnostics and vaccines.

Systematic Evaluation of Fragmentation Methods for Unlabeled and Isobaric Mass Tag-Labeled O-Glycopeptides.

Dissecting site-specific functions of O-glycosylation requires simultaneous identification and quantification of differentially expressed O-glycopeptides by mass spectrometry. However, different dissociation methods have not been systematically compared in their performance in terms of identification, glycosite localization, and quantification with isobaric labeling. Here, we conducted this comparison on highly enriched unlabeled O-glycopeptides with higher-energy collision dissociation (HCD), electron-transfer/collision-induced dissociation (ETciD), and electron transfer/higher-energy collisional dissociation (EThcD), concluding that ETciD and EThcD with optimal supplemental activation resulted in superior identification of glycopeptides and unambiguous site localizations than HCD in a database search by Sequest HT. We later described a pseudo-EThcD strategy that in silico concatenates the electron transfer dissociation spectrum with the paired HCD spectrum acquired sequentially for the same precursor ions, which combines the identification advantage of ETciD/EThcD with the superior reporter ion quality of HCD. We demonstrated its improvements in identification and quantification of isobaric mass tag-labeled O-glycopeptides and showcased the discovery of the specific glycosites of GalNAc transferase 11 (GALNT11) in HepG2 cells.

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.

Site-Specific O-Glycosylation Analysis by Liquid Chromatography-Mass Spectrometry with Electron-Transfer/Higher-Energy Collisional Dissociation.

O-glycosylation is a major post-translational modification of proteins. Accurate and detailed analysis to reveal O-glycosylation patterns at each site (site-specific O-glycosylation analysis) is essential to deeply understand glycoprotein function. Recent reports also demonstrated that unintended O-glycosylation occurs on therapeutic fusion glycoproteins; therefore, it is increasingly important to perform detailed and exhaustive O-glycosylation analysis during the development of therapeutic glycoproteins. Here, we describe a method of in-depth site-specific O-glycosylation analysis by liquid chromatography-mass spectrometry using electron-transfer/higher-energy collisional dissociation (EThcD) and database analysis.

Systematic examination of protein extraction, proteolytic glycopeptide enrichment and MS/MS fragmentation techniques for site-specific profiling of human milk N-glycoproteins

Human milk contains numerous N-glycoproteins with functions that provide protection to the infant. Increasing understanding of the functional role of human milk glycoproteins within the infant requires toolsets to comprehensively profile their site-specific glycosylation patterns. However, optimized methods for site-specific glycosylation analysis across the entire human milk proteome are not available. Therefore, we performed a systematic analysis of techniques for profiling the sites and compositions of N-glycans in human milk using liquid chromatography/mass spectrometry. To decrease interference from non-target molecules, we compared techniques for protein extraction, including ethanol (EtOH) precipitation, trichloroacetic acid precipitation, molecular weight cut-off filtration and techniques for tryptic glycopeptide enrichment, including C18-, porous graphitized carbon and hydrophilic interaction liquid chromatography (HILIC)-solid phase extraction (SPE) and acetone precipitation. We compared the capacity of higher-energy collision dissociation, electron-transfer dissociation and electron-transfer/higher-energy collision dissociation (EThcD) to produce fragment ions that would enable effective identification of the glycan composition, peptide sequence and glycosylation site. Of these methods, a combination of EtOH precipitation, HILIC-SPE and EThcD-fragmentation was the most effective for human milk N-glycopeptide profiling. This optimized approach significantly increased the number of N-glycopeptides and precursor N-glycoproteins (246 N-glycopeptides from 29 glycoproteins) compared with a more common extraction approach with no protein extraction and C18 clean-up (62 N-glycopeptides from 11 glycoproteins). The advancement in methods for human milk N-glycoproteins provided by this study represents a key step for better understanding the function of glycoproteins within the breast milk-fed infant.