Enzymatic Conversion of Cypridina Luciferyl Sulfate to Cypridina Luciferin with Coenzyme A as a Sulfate Acceptor in Cypridina (Vargula) hilgendorfii

In the luminous ostracod Cypridina (presently Vargula) hilgendorfii, Cypridina luciferyl sulfate (3‐enol sulfate of Cypridina luciferin) is converted to Cypridina luciferin by a sulfotransferase with 3′‐phosphoadenosine‐5′‐phosphate (PAP) as a sulfate acceptor. The resultant Cypridina luciferin is used for the luciferase–luciferin reaction of Cypridina to emit blue light. The luminescence stimulation with major organic cofactors was examined using the crude extracts of Cypridina specimens, and we found that the addition of coenzyme A (CoA) to the crude extracts significantly stimulated luminescence intensity. Further, the light‐emitting source in the crude extracts stimulated with CoA was identified as Cypridina luciferyl sulfate, and we demonstrated that CoA could act as a sulfate acceptor from Cypridina luciferyl sulfate. In addition, the sulfate group of Cypridina luciferyl sulfate was also transferred to adenosine 5′‐monophosphate (5′‐AMP) and adenosine 3′‐monophosphate (3′‐AMP) by a sulfotransferase. The sulfated products corresponding to CoA, 5′‐AMP and 3′‐AMP were identified using mass spectrometry. This is the first report that CoA can act as a sulfate acceptor in a sulfotransferase reaction.     

Recent advances in methods for the analysis of protein o‐glycosylation at proteome level

O‐Glycosylation, which refers to the glycosylation of the hydroxyl group of side chains of Serine/Threonine/Tyrosine residues, is one of the most common post‐translational modifications. Compared with N‐linked glycosylation, O‐glycosylation is less explored because of its complex structure and relatively low abundance. Recently, O‐glycosylation has drawn more and more attention for its various functions in many sophisticated biological processes. To obtain a deep understanding of O‐glycosylation, many efforts have been devoted to develop effective strategies to analyze the two most abundant types of O‐glycosylation, i.e. O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation. In this review, we summarize the proteomics workflows to analyze these two types of O‐glycosylation. For the large‐scale analysis of mucin‐type glycosylation, the glycan simplification strategies including the ‘‘SimpleCell’’ technology were introduced. A variety of enrichment methods including lectin affinity chromatography, hydrophilic interaction chromatography, hydrazide chemistry, and chemoenzymatic method were introduced for the proteomics analysis of O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation.     

Proteins from Erwinia asparaginase Erwinase® and E. coli asparaginase 2 MEDAC® for treatment of human leukemia,show a multitude of modifications for which the consequences are completely unclear

L ‐Asparaginase from Erwinia chrysanthemi (ASPG_ERWCH; UniProtKB accession number P06608 (Erwinase^®)) and L ‐asparaginase 2 from Escherichia coli (ASPG2_ECOLI; UniProtKB accession number P00805 (Medac^®)), both L ‐asparagine amidohydrolases, are widely used for the treatment of acute lymphoblastic leukemia. A series of serious side effects have been reported and this warrants studies into the protein chemistry of the medical products sold. Mass spectrometry (MS) data on ASPG_ERWCH and ASPG2_ECOLI have not been published so far and herein a gel‐based proteomics study was performed to provide information about sequence and modifications of the commercially available medical products. ASPG_ERWCH and ASPG2_ECOLI were applied onto two‐dimensional gel electrophoresis, spots were in‐gel digested with several proteases and resulting peptides and protein modifications were analysed by nano‐ESI‐LC‐MS/MS. Four spots were observed for ASPG_ERWCH, six spots were observed for ASPG2_ECOLI and the identified proteins showed high sequence coverage without sequence conflicts. Several protein modifications including technical and posttranslational modifications were demonstrated. Protein modifications are known to change physicochemical, immunochemical, biological and pharmacological properties and results from this work may challenge re‐designing of the product including possible removal of the modifications by the manufacturer because it is not known whether they are contributing to the serious adverse effects of the protein drug.     

Total Synthesis of Homogeneous Variants of Hirudin P6: A Post‐Translationally Modified Anti‐Thrombotic Leech‐Derived Protein

Hirudin P6 is a leech‐derived anti‐thrombotic protein which possesses two post‐translational modifications, O‐glycosylation and tyrosine sulfation. In this study we report the ligation‐based synthesis of a library of hirudin P6 proteins possessing homogeneous glycosylation and sulfation modifications. The nature of the modifications incorporated was shown to have a drastic effect on inhibition against both the fibrinogenolytic and amidolytic activities of thrombin and thus highlights a potential means for attenuating the biological activity of the protein.     

A combined mass spectrometry strategy for complete posttranslational modification mapping of Neisseria meningitidis major pilin

Herein, we report a new approach, based on the combination of mass profiling and tandem mass spectrometry, to address the issue of localising all post‐translational modifications (PTMs) on the major pilin protein PiIE expressed by the pathogenic Neisseria species. PilE is the main component of type IV pili; filamentous organelles expressed at the surface of many bacterial pathogens and important virulence factors. Previous reports have shown that PilE can harbour various combinations of PTMs and have established strong links between PTM and pathogenesis. Complete PTM mapping of proteins involved in bacterial infection is therefore highly desirable. The methodology we propose here allowed us to fully characterise the PilE proteoforms of Neisseria meningitidis strain 8013, definitively identifying all PTMs present on all proteoforms and localising their position on the protein backbone. These modifications include a processed and methylated N‐terminus, disulfide bridge, glycosylation and glycerophosphorylation at two different sites. A key element of our approach is high resolution, intact mass measurement of the proteoforms, a piece of information completely lacking in all classical bottom–up proteomics strategies used for PTM analysis and without which it is difficult to ensure complete PTM mapping. Copyright © 2013 John Wiley & Sons, Ltd.     

An online nano‐LC‐ESI‐FTICR‐MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid

Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low‐energy collision‐induced dissociation tandem mass spectrometry (MS/MS), mainly long b‐fragments are observed, limiting the possibility to determine variations such as amino acid variants or post‐translational modifications (PTMs) within the N‐terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann–Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O‐glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top‐down MS‐based method. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterizing protein glycosylation sites through higher‐energy C‐trap dissociation

Assigning glycosylation sites of glycoproteins and their microheterogeneity is still a very challenging analytical task despite the rapid advancements in mass spectrometry. It is shown here that glycopeptide ions can be fragmented efficiently using the higher‐energy C‐trap dissociation (HCD) feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap). An attractive aspect of this dissociation option is the generation of distinct Y1 ions (peptide+GlcNAc), thus allowing unequivocal assignment of N‐glycosylation sites of glycoproteins. The combination of the very informative collision‐induced dissociation spectra acquired in the linear ion trap with the distinct features of HCD offers very useful information aiding in the characterization of the glycosylation sites of glycoproteins. The HCD activation energy needed to obtain optimum Y1 ions was studied in terms of glycan structure and charge state, and size and structure of the peptide backbone. The latter appeared to be primarily dictating the needed HCD energy. The distinct Y1 ion formation in HCD facilitated an easy assignment of such an ion and its subsequent isolation and dissociation through multiple‐stage tandem mass spectrometry. The resulting MS³ spectrum of the Y1 ion facilitates database searching and de novo sequencing thus prompting the subsequent identification of the peptide backbone and associated glycosylation sites. Moreover, fragment ions formed by HCD are detected in the Orbitrap, thus overcoming the 1/3 cut‐off limitation that is commonly associated with ion trap mass spectrometers. As a result, in addition to the Y1 ion, the common glycan oxonium ions are also detected. The high mass accuracy offered by the LTQ Orbitrap mass spectrometer is also an attractive feature that allows a confident assignment of protein glycosylation sites and the microheterogeneity of such sites. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of and Specific Antibody Generation for Glycopeptides with Arginine N‐GlcNAcylation

As a unique and unappreciated protein posttranslational modification, arginine N‐glycosylation was recently discovered to play an important role in the process that bacteria counteract host defenses. To provide chemical tools for further proteomic and biochemical studies on arginine N‐glycosylation, we report the first general strategy for a rapid and cost‐effective synthesis of glycopeptides carrying single or multiple arginine N‐GlcNAcyl groups. These glycopeptides were successfully utilized to generate the first antibodies that can specifically recognize arginine N‐GlcNAcylated peptides or proteins in a sequence‐independent manner.     

Metabolic Labeling and Imaging of N‐Linked Glycans in Arabidopsis Thaliana

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N‐azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N‐acetylglucosamine (GlcNAc) and incorporated into N‐linked glycans, and possibly intracellular O‐GlcNAc. Click‐labeling with fluorescent probes enables visualization of newly synthesized N‐linked glycans. N‐glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N‐glycosylation is regulated in a developmental stage‐dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N‐linked glycosylation in plants.     

Identification of<Emphasis Type="Italic"> N</Emphasis>-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform–ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.Abbreviations amu atomic mass units - AspN endoproteinase AspN - CAM cell adhesion molecule - ESI electrospray ionization - FTICR Fourier transform–ion cyclotron resonance - IgSF immunoglobulin superfamily - MALDI-TOF matrix-assisted laser desorption ionization–time of flight - MS mass spectrometry - NCAM neural cell adhesion molecule - PNGase F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase - PSA polysialic acid - TFA trifluoroacetic acid     

Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow region

A high‐performance liquid chromatography nano‐electrospray ionization Fourier transform mass spectrometry (HPLC/nanoESI‐FTMS) approach involving recording of collision‐activated dissociation (CAD) and electron‐capture dissociation (ECD) spectra of an intact sample and two its modifications after performic oxidation and reduction followed by carboxamidomethylation helps to establish peptide profiles in the crude secretion of frog species at mid‐throughput level, including de novo sequencing. The proposed derivatization procedures allow increasing of the general sequence coverage in the backbone, providing complementary information and, what is more important, reveal the amino acid sequence in the cystine ring (‘rana box’). Thus purely mass spectrometric efficient sequencing becomes possible for longer than usual proteolytic peptides. Seventeen peptides belonging to four known families were identified in the secretion of the European brown frog Rana arvalis inhabiting the Moscow region in Russia. Ranatuerins, considered previously a unique feature of the North American species, as well as a new melittin‐related peptide, are worth special mention. The developed approach was previously successfully used for the identification of peptides in the skin secretion of the Caucasian green frog Rana ridibunda. Copyright © 2009 John Wiley & Sons, Ltd.     

Glycosylation Enhances Peptide Hydrophobic Collapse by Impairing Solvation

Post‐translational N‐glycosylation of proteins is ubiquitous in eukaryotic cells, and has been shown to influence the thermodynamics of protein collapse and folding. However, the mechanism for this influence is not well understood. All‐atom molecular dynamics simulations are carried out to study the collapse of a peptide linked to a single N‐glycan. The glycan is shown to perturb the local water hydrogen‐bonding network, rendering it less able to solvate the peptide and thus enhancing the hydrophobic contribution to the free energy of collapse. The enhancement of the hydrophobic collapse compensates for the weakened entropic coiling due to the bulky glycan chain and leads to a stronger burial of hydrophobic surface, presumably enhancing folding. This conclusion is reinforced by comparison with coarse‐grained simulations, which contain no explicit solvent and correspondingly exhibit no significant thermodynamic changes on glycosylation.     

Chemical Synthesis of Asparagine‐Linked Archaeal N‐Glycan from Methanothermus fervidus

Several N‐linked glycoproteins have been identified in archaea and there is growing evidence that the N‐glycan is involved in survival and functioning of archaea in extreme conditions. Chemical synthesis of the archaeal N‐glycans represents a crucial step towards understanding the putative function of protein glycosylation in archaea. Herein the first total synthesis of the archaeal L ‐asparagine linked hexasaccharide from Methanothermus fervidus is reported using a highly convergent [3+3] glycosylation approach in high overall yields. The synthesis relies on efficient preparation of regioselectively protected thioglycoside building blocks for orthogonal glycosylations and late stage N‐aspartylation.     

Cis‐trans Isomerism in Copper(II) Complexes with N‐acyl Thiourea Ligands

The N‐acyl thiourea complexes bis[N,N‐diethyl‐N′‐(p‐nitrobenzoyl)‐thioureato]copper(II) ( 1a,1b ) and bis(N,N‐diphenyl‐N′‐benzoylthioureato)copper(II) ( 2a,2b ) crystallize in each case in two modifications. X‐ray structural analysis shows that 1a and 1b are cis‐trans isomers. This is very unusual for N‐acyl thioureato complexes because with exception of one platinum(II) complex up to now only cis complexes have been found. In contrast X‐ray structural analysis of both forms 2a and 2b of the other complex shows no cis‐trans pair. Both modifications are cis complexes. In solution both isomers of the copper(II) complexes are observable by EPR spectroscopy.     

N‐Methylation of Peptides and Proteins: An Important Element for Modulating Biological Functions

N‐Methylation is one of the simplest chemical modifications often occurring in peptides and proteins of prokaryotes and higher eukaryotes. Over years of evolution, nature has employed N‐methylation of peptides as an ingenious technique to modulate biological function, often as a mode of survival through the production of antibiotics. This small structural change can not only mobilize large protein complexes (as in the histone methylation), but also inhibits the action of enzymes by selective recognition of protein–protein interaction surfaces. In recent years through the advancement in synthetic approaches, the potential of N‐methylation has begun to be revealed, not only in modulating biological activity and selectivity as well as pharmacokinetic properties of peptides, but also in delivering novel drugs. Herein, we summarize the current knowledge of the versatility of N‐methylation in modulating biological, structural, and pharmacokinetic properties of peptides.     

Density Functional Study on Adsorption of NO on AuSe (010) Surface

NO molecule adsorption on (010) surface of gold selenide (AuSe) has been studied with a periodic slab model by means of the GGA‐PW91 exchange‐correlation functional within the framework of density functional theory (DFT). Four different on‐top adsorption sites Au(1), Au(2), Se(1) and Se(2) were considered for α‐AuSe and three on‐top adsorption sites Au(1), Au(2) and Se(1) for β‐AuSe. N‐end and O‐end adsorptions of NO were investigated for the above sites. The results show that N‐end adsorptions are preferred for α‐ and β‐AuSe and O‐end adsorptions are not feasible and thought as physisorption with the weak adsorption energies from 6.0 to 10.8 kJ/mol. For the N‐end adsorptions on α‐ and β‐AuSe (010) surfaces, Au(2) sites are most favorable with the adsorption energies 89.0 and 78.0 kJ/mol for α‐ and β‐AuSe, respectively. However, the adsorptions at Au1 sites are very weak with the adsorption energies of 27.8 and 7.5 kJ/mol, respectively. In case of the adsorption of N‐down orientations of NO at Se sites for α‐ and β‐AuSe (010) surfaces, the adsorption activities of Se(1) and Se(2) sites on the α‐AuSe (010) surface and Se(1) site on the β‐AuSe (010) surface are almost the same with the adsorption energies 51.2, 52.7 and 49.2 kJ/mol. The geometric optimizations for adsorption configurations were calculated along with accounting for stretching frequency and density of states in our work.     

Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry

Glycosylation plays a critical role in the in vivo efficacy of both endogenous and recombinant erythropoietin (EPO). Using mass spectrometry, we characterized the N‐/O‐linked glycosylation of recombinant human EPO (rhEPO) produced in glycoengineered Pichia pastoris and compared with the glycosylation of Chinese hamster ovary (CHO) cell‐derived rhEPO. While the three predicted N‐linked glycosylation sites (Asn24, Asn38 and Asn83) showed complete site occupancy, Pichia‐ and CHO‐derived rhEPO showed distinct differences in the glycan structures with the former containing sialylated bi‐antennary glycoforms and the latter containing a mixture of sialylated bi‐, tri‐ and tetra‐antennary structures. Additionally, the N‐linked glycans from Pichia‐produced rhEPO were similar across all three sites. A low level of O‐linked mannosylation was detected on Pichia‐produced rhEPO at position Ser126, which is also the O‐linked glycosylation site for endogenous human EPO and CHO‐derived rhEPO. In summary, the mass spectrometric analyses revealed that rhEPO derived from glycoengineered Pichia has a highly uniform bi‐antennary N‐linked glycan composition and preserves the orthogonal O‐linked glycosylation site present on endogenous human EPO and CHO‐derived rhEPO. Copyright © 2013 John Wiley & Sons, Ltd.     

Total Chemical Synthesis and Biological Activities of Glycosylated and Non‐Glycosylated Forms of the Chemokines CCL1 and Ser‐CCL1

CCL1 is a naturally glycosylated chemokine protein that is secreted by activated T‐cells and acts as a chemoattractant for monocytes. 1 Originally, CCL1 was identified as a 73 amino acid protein having one N‐glycosylation site, 1 and a variant 74 residue non‐glycosylated form, Ser‐CCL1, has also been described. 2 There are no systematic studies of the effect of glycosylation on the biological activities of either CCL1 or Ser‐CCL1. Here we report the total chemical syntheses of both N‐glycosylated and non‐glycosylated forms of (Ser‐)CCL1, by convergent native chemical ligation. We used an N‐glycan isolated from hen egg yolk together with the Nbz linker for Fmoc chemistry solid phase synthesis of the glycopeptide‐^αthioester building block. 3 Chemotaxis assays of these glycoproteins and the corresponding non‐glycosylated proteins were carried out. The results were correlated with the chemical structures of the (glyco)protein molecules. To the best of our knowledge, these are the first investigations of the effect of glycosylation on the chemotactic activity of the chemokine (Ser‐)CCL1 using homogeneous N‐glycosylated protein molecules of defined covalent structure.     

Global quantification of phosphoproteins combining metabolic labeling and gel‐based proteomics in B. pumilus

Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post‐translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two‐dimensional gels after differential phosphoprotein staining or gel‐free by stable isotope labeling, sequential phosphopeptide enrichment and following LC‐MS analysis. In the current work, we combined in a proof‐of‐principle experiment these techniques using ¹⁴N/¹⁵N metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC‐MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide‐based gel‐free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.