Electron Transfer Dissociation Mass Spectrometry Research Articles

O-GlcNAcylation of YTHDF2 antagonizes ERK-dependent phosphorylation and inhibits lung carcinoma

The intracellular O-linked N-acetylglucosamine (O-GlcNAc) glycosylation mediates many signal transduction events and regulates tumorigenesis. Previously the RNA N6-methyladenosine (m6A) reader, YTH (YT521-B homology) domain 2 (YTHDF2), has been shown to be O-GlcNAcylated on Ser-263 during Hepatitis B virus (HBV) infection and promote HBV-related hepatocellular carcinoma. Herein we mapped YTHDF2 O-GlcNAcylation at Thr-49 via electron-transfer dissociation mass spectrometry under unperturbed conditions. We show that YTHDF2 Thr-49 O-GlcNAcylation antagonizes Extracellular-signal regulated kinase (ERK)-dependent phosphorylation at Ser-39 and promotes YTHDF2 degradation. The downstream signaling pathway of YTHDF2 in lung carcinoma is thus upregulated, which leads to the downregulation of c-Myc. We further used mouse xenograft models to show that YTHDF2-T49A mutants increased lung cancer mass and size. Our work reveals a key role of YTHDF2 O-GlcNAcylation in tumorigenesis and suggests that O-GlcNAcylation exerts distinct functions under different biological stress.

Deciphering the Roles of the Key Oligomerization Domains in Amyloid‐β to Design Amyloid Aggregation Inhibitors

AbstractBackgroundProtein folding issues are significant difficulties since they are linked to various diseases, including dementia. Fibrillar amyloid aggregates composed of misfolded proteins are pathological features of several neurodegenerative diseases. Numerous attempts have been made to treat neurodegenerative diseases by focusing on amyloid aggregates of pathologically disordered proteins. The first Alzheimer’s disease (AD) curative drug was introduced as a consequence of attempts to combat the most frequent kind of dementia. Even though antibody strategies reduce clinical deterioration in mild cognitive disease, alternate techniques must be developed over a single approach (i.e., antibody treatment). Recent studies have attempted to rationally design candidate drugs to improve existing therapeutic approaches or suppress the fibrillar amyloid aggregation process altogether. Prompted by previous findings, we rationally designed amyloid‐β (1‐42) point mutants to inhibit amyloid aggregation.MethodWe investigated the structural ensembles of the monomers using small‐angle X‐ray scattering. In addition, we performed hydrogen‐deuterium exchange mass spectrometry (MS) and ion mobility MS to elucidate the molecular principles governing Aβ42‐inhibitor interactions. We also used microsecond‐scale molecular dynamics simulations to investigate the structures and interactions, and combined them with electron transfer dissociation MS analysis to tackle challenges in identifying the multiple binding sites of intrinsically disordered protein complexes.ResultSpecifically designed mutations disrupt not only amyloid aggregation but reduce its cytotoxicity. Furthermore, we proposed minimal mutation sites to control amyloid‐β (1‐42) self‐assembly behavior. We are now determining the key domains of amyloid‐β (1‐42) that facilitate its self‐assembly to design aggregation inhibitors as potential therapeutic agents. We identified three critical hydrophobic domains (17LVF19, 32IGL34, and 41IA42) on amyloid‐β (1‐42) and investigated their role in the protein’s self‐assembly process. Our results suggest that interchain interactions in the central hydrophobic region (17LVF19) of amyloid‐β (1‐42) are essential for fibrillar aggregation and that their interaction with other domains is related to the accessibility of the central hydrophobic region for initiating the pathological cascade. Our findings shed light on the mechanisms underlying amyloid‐β (1‐42) self‐assembly and reveal key structural domains that aid in this process.ConclusionOur findings can be used to improve the rational design of amyloid‐β (1‐42) aggregation inhibitors in the future.

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.

A pain-causing and paralytic ant venom glycopeptide

SummaryAnts (Hymenoptera: Formicidae) are familiar inhabitants of most terrestrial environments. Although we are aware of the ability of many species to sting, knowledge of ant venom chemistry remains limited. Herein, we describe the discovery and characterization of an O-linked glycopeptide (Mg7a) as a major component of the venom of the ant Myrmecia gulosa. Electron transfer dissociation and higher-energy collisional dissociation tandem mass spectrometry were used to localize three α-N-acetylgalactosaminyl residues (α-GalNAc) present on the 63-residue peptide. To allow for functional studies, we synthesized the full-length glycosylated peptide via solid-phase peptide synthesis, combined with diselenide–selenoester ligation-deselenization chemistry. We show that Mg7a is paralytic and lethal to insects, and triggers pain behavior and inflammation in mammals, which it achieves through a membrane-targeting mode of action. Deglycosylation of Mg7a renders it insoluble in aqueous solution, suggesting a key solubilizing role of the O-glycans.

Ribonucleic Acid Sequence Characterization by Negative Electron Transfer Dissociation Mass Spectrometry.

Modified oligonucleotides represent a promising avenue for drug development, with small interfering RNAs (siRNA) and microRNAs gaining traction in the therapeutic market. Mass spectrometry (MS)-based analysis offers many benefits for characterizing modified nucleic acids. Negative electron transfer dissociation (NETD) has proven valuable in sequencing oligonucleotide anions, particularly because it can retain modifications while generating sequence-informative fragments. We show that NETD can be successfully implemented on a widely available quadrupole-Orbitrap-linear ion trap mass spectrometer that uses a front-end glow discharge source to generate radical fluoranthene reagent cations. We characterize both unmodified and modified ribonucleic acids and present the first application of activated-ion negative electron transfer dissociation (AI-NETD) to nucleic acids. AI-NETD achieved 100% sequence coverage for both a 6-mer (5'-rGmUrArCmUrG-3') with 2'-O-methyl modifications and a 21-mer (5'-rCrArUrCrCrUrCrUrArGrArGrGrArUrArGrArArUrG-3'), the luciferase antisense siRNA. Both NETD and AI-NETD afforded complete sequence coverage of these molecules while maintaining a relatively low degree of undesired base-loss products and internal products relative to collision-based methods.

Mapping binding epitopes of monoclonal antibodies targeting major histocompatibility complex class I chain-related A (MICA) with hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry.

Major histocompatibility complex class I chain-related A and B (MICA/B) are cell-surface proteins that act as ligands to natural killer cell receptors, NKG2D, expressed on immune cells. Prevention of proteolytic shedding of MICA/B to retain their integrity on the cell surface has become a therapeutic strategy in immuno-oncology. Given the unique mechanism of MICA/B shedding, structural characterization of MICA/B and therapeutic agent interaction is important in the drug discovery process. In this study, we describe the practical utility of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in epitope mapping studies of a cohort of four monoclonal antibodies targeting MICA in a rapid manner. HDX-MS followed by electron-transfer dissociation allows high-resolution refinement of binding epitopes. This integrated strategy offers, for the first time, molecular-level understanding of MICA's conformational dynamics in solution as well as the unique mechanism of actions of these antibodies in targeting MICA. Graphical abstract.

Clinical method evaluation of hemoglobin S and C identification by top-down selected reaction monitoring and electron transfer dissociation

BackgroundBiological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Currently, hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Co-elution and co-migration may represent major issues for precise identification of Hb variants, even for the most common ones such as Hb S and C.MethodsWe adapted a top-down selected reaction monitoring (SRM) electron transfer dissociation (ETD) mass spectrometry (MS) method to fit with a clinical laboratory environment. An automated analytical process with semi-automated data analysis compatible with a clinical practice was developed. A comparative study between a reference HPLC method and the MS assay was performed on 152 patient samples.ResultsThe developed workflow allowed to identify with high specificity and selectivity the most common Hb variants (Hb S and Hb C). Concordance of the MS-based approach with HPLC was 71/71 (100%) for Hb S and 11/11 (100%) for Hb C.ConclusionsThis top-down SRM ETD method can be used in a clinical environment to detect Hb S and Hb C.

O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry.

As a dynamic post-translational modification, O-linked β- N-acetylglucosamine ( O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-β-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.

Investigation of the position of the radical in z3-ions resulting from electron transfer dissociation using infrared ion spectroscopy.

The molecular structures of six open-shell z3-ions resulting from electron transfer dissociation mass spectrometry (ETD MS) were investigated using infrared ion spectroscopy in the 800-1850 and 3200-3700 cm-1 spectral ranges in combination with density functional theory and molecular mechanics/molecular dynamics calculations. We assess in particular the question of whether the radical remains at the Cα-site of the backbone cleavage, or whether it migrates by H-atom transfer to another, energetically more favorable position. Calculations performed herein as well as by others show that radical migration to an amino acid side chain or to an α-carbon along the peptide backbone can lead to structures that are more stable, by up to 33 kJ mol-1 for the systems investigated here, by virtue of resonance stabilization of the radical in these alternative positions. Nonetheless, for four out of the six z3-ions considered here, our results quite clearly indicate that radical migration does not occur, suggesting that the radical is kinetically trapped at the site of ETD cleavage. For the two remaining systems, a structural assignment is less secure and we suggest that a mixture of migrated and unmigrated structures may be formed.

Electron transfer dissociation mass spectrometry of acidic phosphorylated peptides cationized with trivalent praseodymium.

The lanthanide ion praseodymium, Pr(III), was employed to study metallated ion formation and electron transfer dissociation (ETD) of 27 biological and model highly acidic phosphopeptides. All phosphopeptides investigated form metallated ions by electrospray ionization (ESI) that can be studied by ETD to yield abundant sequence information. The ions formed are [M+Pr-H]2+ , [M+Pr]3+ , and [M+Pr+H]4+ . All biological phosphopeptides with a chain length of seven or more residues generate [M+Pr]3+ . For biological phosphopeptides, [M+Pr]3+ undergoes more backbone cleavage by ETD than [M+Pr - H]2+ and, in some cases, full sequence coverage occurs. Acidic model phosphorylated hexa-peptides and octa-peptides, composed of alanine residues and one phosphorylated residue, form exclusively [M+Pr - H]2+ by ESI. Limited sequence information is obtained by ETD of [M+Pr - H]2+ with only metallated product ions being generated. For two biological phosphopeptides, [M+Pr+H]4+ is observed and may be due to the presence of at least one residue with a highly basic side chain that facilitates the addition of an extra proton. For the model phosphopeptides, more sequence coverage occurs when the phosphorylated residue is in the middle of the sequence than at either the N- or C-terminus. ETD of the metallated precursor ions formed by ESI generates exclusively metallated and nonmetallated c- and z-ions for the biological phosphopeptides, while metallated c-ions, z-ions, and a few y-ions form for the model phosphopeptides. Most of the product ions contain the phosphorylated residue indicating that the metal ion binds predominantly at the deprotonated phosphate group. The results of this study indicate that ETD is a promising tool for sequencing highly acidic phosphorylated peptides by metal adduction with Pr (III) and, by extension, all nonradioactive lanthanide metal ions.

A Targeted, Differential Top-Down Proteomic Methodology for Comparison of ApoA-I Proteoforms in Individuals with High and Low HDL Efflux Capacity.

Top-down proteomics (TDP) allows precise determination/characterization of the different proteoforms derived from the expression of a single gene. In this study, we targeted apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein cholesterol efflux (HDL-E), which is inversely associated with coronary heart disease risk. Absolute ApoA-I concentration and allelic variation only partially explain interindividual HDL-E variation. Therefore, we hypothesize that differences in HDL-E are associated with the abundances of different ApoA-I proteoforms. Here, we present a targeted TDP methodology to characterize ApoA-I proteoforms in serum samples and compare their abundances between individuals. We characterized 18 ApoA-I proteoforms using selected-ion monitoring coupled to electron-transfer dissociation mass spectrometry. We then compared the abundances of these proteoforms between two groups of four participants, representing the individuals with highest and lowest HDL-E values within the Chicago Healthy Aging Study ( n = 420). Six proteoforms showed significantly ( p < 0.0005) higher intensity in high HDL-E individuals: canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated (fd = 2.31) and one bearing two modifications: palmitoylation and truncation (fd = 2.13). These results demonstrate translational potential for targeted TDP in revealing, with high sensitivity, associations between interindividual proteoform variation and physiological differences underlying disease risk.

Effects of acidic peptide size and sequence on trivalent praseodymium adduction and electron transfer dissociation mass spectrometry.

Using the lanthanide ion praseodymium, Pr(III), metallated ion formation and electron transfer dissociation (ETD) were studied for 25 biological and model acidic peptides. For chain lengths of seven or more residues, even highly acidic peptides that can be difficult to protonate by electrospray ionization will metallate and undergo abundant ETD fragmentation. Peptides composed of predominantly acidic residues form only the deprotonated ion, [M+Pr - H]2+ ; this ion yields near complete ETD sequence coverage for larger peptides. Peptides with a mixture of acidic and neutral residues generate [M+Pr]3+ , which cleaves between every residue for many peptides. Acidic peptides that contain at least one residue with a basic side chain also produce the protonated ion, [M+Pr+H]4+ ; this ion undergoes the most extensive sequence coverage by ETD. Primarily metallated and non-metallated c- and z-ions form for all peptides investigated. Metal adducted product ions are only present when at least half of the peptide sequence can be incorporated into the ion; this suggests that the metal ion simultaneously attaches to more than one acidic site. The only site consistently lacking dissociation is at the N-terminal side of a proline residue. Increasing peptide chain length generates more backbone cleavage for metal-peptide complexes with the same charge state. For acidic peptides with the same length, increasing the precursor ion charge state from 2+ to 3+ also leads to more cleavage. The results of this study indicate that highly acidic peptides can be sequenced by ETD of complexes formed with Pr(III). Copyright © 2017 John Wiley & Sons, Ltd.

Analysis of Protein O-GlcNAcylation by Mass Spectrometry.

O-linked β-D-N-acetyl glucosamine (O-GlcNAc) addition (O-GlcNAcylation), a post-translational modification of serine/threonine residues of proteins, is involved in diverse cellular metabolic and signaling pathways. Aberrant O-GlcNAcylation underlies the initiation and progression of multiple chronic diseases including diabetes, cancer, and neurodegenerative diseases. Numerous methods have been developed for the analysis of protein O-GlcNAcylation, but instead of discussing the classical biochemical techniques, this unit covers O-GlcNAc characterization by combining several enrichment methods and mass spectrometry detection techniques [including collision-induced dissociation (CID), higher energy collision dissociation (HCD), and electron transfer dissociation (ETD) mass spectrometry]. © 2017 by John Wiley & Sons, Inc.

High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex.

Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS2) with electron transfer dissociation (ETD) has proven to be a promising method for phosphoproteomic applications owing to its ability to determine phosphorylation sites on proteins. However, low precursor charge states hinder the ability to obtain useful information through peptide sequencing by ETD, and the presence of acidic phosphate groups contributes to a low charge state of peptide ions. In the present report, we used a dinuclear zinc complex, (Zn2L)3+ (L = alkoxide form of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol) for electrospray ionization (ESI), followed by ETD-MS2 analysis. Since (Zn2L)3+ selectively bound to phosphopeptide with addition of a positive charge per phosphate group, the use of (Zn2L)3+ for ESI improved the ionization yield of phosphopeptides in phosphoprotein digest. Additionally, an increase in the charge state of phosphopeptides were observed by addition of (Zn2L)3+, facilitating phosphopeptide sequencing by ETD-MS2. Since the binding between (Zn2L)3+ and the phosphate group was retained during the ETD process, a comparison between the ETD mass spectra obtained using two dinuclear zinc complex derivatives containing different zinc isotopes, namely (64Zn2L)3+ and (68Zn2L)3+, provided information about the number of phosphate groups in each fragment ion, allowing the phosphorylation site to be unambiguously determined. The details of the fragmentation processes of the (Zn2L)3+-phosphopeptide complex were investigated using a density functional theory calculation. As in the case of protonated peptides, ETD induced peptide backbone dissociation in the (Zn2L)3+-phosphopeptide complex proceeded through an aminoketyl radical intermediate.

Gas-Phase Fragmentation Behavior of Oxidized Prenyl Peptides by CID and ETD Tandem Mass Spectrometry.

Farnesylation and geranylgeranylation are the two types of prenyl modification of proteins. Prenylated peptides are highly hydrophobic and their abundances in biological samples are low. In this report, we studied the oxidized prenylated peptides by electrospray ionization mass spectrometry and identified them by collision-induced dissociation (CID) and electron-transfer dissociation (ETD) tandem mass spectrometry. Modified prenyl peptides were generated utilizing strong and low strength oxidizing agents to selectively oxidize and epoxidize cysteine sulfur and prenyl side chain. We selected three peptides with prenyl motifs and synthesized their prenylated versions. The detailed characteristic fragmentations of oxidized and epoxidized farnesylated and geranylgeranylated peptides were studied side by side with two popular fragmentation techniques. CID and ETD mass spectrometry clearly distinguished the modified version of these peptides. ETD mass spectrometry provided sequence information of the highly labile modified prenyl peptides and showed different characteristic fragmentations compared with CID. A detailed fragmentation of modified geranylgeranylated peptides was compared by CID and ETD mass spectrometry for the first time. Graphical Abstract ᅟ.

Difference of Electron Capture and Transfer Dissociation Mass Spectrometry on Ni(2+)-, Cu(2+)-, and Zn(2+)-Polyhistidine Complexes in the Absence of Remote Protons.

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) in metal-peptide complexes are dependent on the metal cation in the complex. The divalent transition metals Ni(2+), Cu(2+), and Zn(2+) were used as charge carriers to produce metal-polyhistidine complexes in the absence of remote protons, since these metal cations strongly bind to neutral histidine residues in peptides. In the case of the ECD and ETD of Cu(2+)-polyhistidine complexes, the metal cation in the complex was reduced and the recombination energy was redistributed throughout the peptide to lead a zwitterionic peptide form having a protonated histidine residue and a deprotonated amide nitrogen. The zwitterion then underwent peptide bond cleavage, producing a and b fragment ions. In contrast, ECD and ETD induced different fragmentation processes in Zn(2+)-polyhistidine complexes. Although the N-Cα bond in the Zn(2+)-polyhistidine complex was cleaved by ETD, ECD of Zn(2+)-polyhistidine induced peptide bond cleavage accompanied with hydrogen atom release. The different fragmentation modes by ECD and ETD originated from the different electronic states of the charge-reduced complexes resulting from these processes. The details of the fragmentation processes were investigated by density functional theory. Graphical Abstract ᅟ.

The active site of O-GlcNAc transferase imposes constraints on substrate sequence.

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoans. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay to a library of peptides. We mapped sites of O-GlcNAc modification by electron transfer dissociation MS and found that they correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with Homo sapiens OGT suggest that a combination of size and conformational restriction defines sequence specificity in the -3 to +2 subsites. This work reveals that although the N-terminal TPR repeats of OGT may have roles in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a substantial contribution to O-GlcNAc site specificity.

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.

Detailed characterization of the O-linked glycosylation of the neuropilin-1 c/MAM-domain.

Neuropilins are involved in angiogenesis and neuronal development. The membrane proximal domain of neuropilin-1, called c or MAM domain based on its sequence conservation, has been implicated in neuropilin oligomerization required for its function. The c/MAM domain of human neuropilin-1 has been recombinantly expressed to allow for investigation of its propensity to engage in molecular interactions with other protein or carbohydrate components on a cell surface. We found that the c/MAM domain was heavily O-glycosylated with up to 24 monosaccharide units in the form of disialylated core 1 and core 2 O-glycans. Attachment sites were identified on the chymotryptic c/MAM peptide ETGATEKPTVIDSTIQSEFPTY by electron-transfer dissociation mass spectrometry (ETD-MS/MS). For highly glycosylated species consisting of carbohydrate to about 50%, useful results could only be obtained upon partial desialylation. ETD-MS/MS revealed a hierarchical order of the initial O-GalNAc addition to the four different glycosylation sites. These findings enable future functional studies about the contribution of the described glycosylations in neuropilin-1 oligomerization and the binding to partner proteins as VEGF or galectin-1.As a spin-off result the sialidase from Clostridium perfringens turned out to discriminate between galactose- and N-acetylgalactosamine-linked sialic acid.

Identification of hemoglobin variants by top-down mass spectrometry using selected diagnostic product ions.

Hemoglobin disorder diagnosis is a complex procedure combining several analytical steps. Due to the lack of specificity of the currently used protein analysis methods, the identification of uncommon hemoglobin variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissociation mass spectrometry method was developed for hemoglobin β chain analysis. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the hemoglobin β chain sequence. The method was applied to the analysis of rare hemoglobin β chain variants and an (A)γ-β fusion protein. The results showed that the developed data analysis process allows fast and reliable interpretation of top-down electron transfer dissociation mass spectra by nonexpert users in the clinical area.