Epitope Ligand Binding Sites of Blood Group Oligosaccharides in Lectins Revealed by Pressure-Assisted Proteolytic Excision Affinity Mass Spectrometry.

Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differentiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW) biosensor analysis and provided micromolar KD values for the intact lectins, in agreement with results of previous ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower, consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains. Graphical Abstract ᅟ.

Erratum: Growth of organic crystals via attachment and transformation of nanoscopic precursors.

This corrects the article DOI: 10.1038/ncomms15933.

Growth of organic crystals via attachment and transformation of nanoscopic precursors.

A key requirement for the understanding of crystal growth is to detect how new layers form and grow at the nanoscale. Multistage crystallization pathways involving liquid-like, amorphous or metastable crystalline precursors have been predicted by theoretical work and have been observed experimentally. Nevertheless, there is no clear evidence that any of these precursors can also be relevant for the growth of crystals of organic compounds. Herein, we present a new growth mode for crystals of DL-glutamic acid monohydrate that proceeds through the attachment of preformed nanoscopic species from solution, their subsequent decrease in height at the surface and final transformation into crystalline 2D nuclei that eventually build new molecular layers by further monomer incorporation. This alternative mechanism provides a direct proof for the existence of multistage pathways in the crystallization of molecular compounds and the relevance of precursor units larger than the monomeric constituents in the actual stage of growth.

Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry.

Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects. Graphical Abstract ᅟ.

Identification and affinity-quantification of ß-amyloid and α-synuclein polypeptides using on-line SAW-biosensor-mass spectrometry.

Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn for MS of the ligand eluate from the biosensor. Suitable MS-acquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF- MS systems. Applications are presented for mass spectrometric identifications and affinity (K(D)) determinations of the neurodegenerative polypeptides, ß-amyloid (Aß), and pathophysiological and physiological synucleins (α- and ß-synucleins), two key polypeptide systems for Alzheimer's disease and Parkinson's disease, respectively. Moreover, first in vivo applications of αSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions.

When is mass spectrometry combined with affinity approaches essential? A case study of tyrosine nitration in proteins.

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar K(D) values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

Surface-based and mass spectrometric approaches to deciphering sugar-protein interactions in a galactose-specific agglutinin.

Interest in powerful, nanosized tools to analyze in detail glycan-protein interactions has increased significantly over recent years. Here, we report two complementary approaches to characterize such interactions with high sensitivity, low sample consumption, and without the need for sample labeling, namely, surface plasmon resonance (SPR) and an approach that combines limited proteolysis and mass spectrometry. Combination of these two approaches to investigate glycan-protein interactions allows (1) to characterize interactions through kinetic and thermodynamic parameters, (2) to capture efficiently the carbohydrate-binding protein, and (3) to identify the interacted protein and its carbohydrate binding site by mass spectrometry. As a proof of principle, the interaction of the galactose-specific legume lectin Erythrina cristagalli agglutinin with several sugars has been characterized in-depth by means of these two approaches.

Toward bioinspired galectin mimetics: identification of ligand-contacting peptides by proteolytic-excision mass spectrometry.

Clinically relevant bioactivities of human galectins (adhesion/growth-regulatory galactoside-specific lectins) inspired the design of peptides as new tools to elicit favorable effects (e.g., in growth control) or block harmful binding (e.g., in tissue invasion). To obtain the bioinspired lead compounds, we combined a proteolytic fragmentation approach without/with ligand contact (excision) with mass spectrometric identification of affinity-bound protein fragments, using galectin-1 and -3 as models. Two peptides from the carbohydrate recognition domains were obtained in each case in experimental series rigorously controlled for specificity, and the [157-162] peptide of galectin-3 proved to be active in blocking lectin binding to a neoglycoprotein and to tumor cell surfaces. This approach affords peptide sequences for structural optimization and intrafamily/phylogenetic galectin comparison at the binding-site level with a minimal requirement of protein quantity, and it is even amenable to mixtures.

Epitope structure of the carbohydrate recognition domain of asialoglycoprotein receptor to a monoclonal antibody revealed by high-resolution proteolytic excision mass spectrometry.

Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with ß-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

"Unknown genome" proteomics: a new NADP-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR, and high resolution mass spectrometry.

We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences and high resolution peptide mass determination of proteolytic digests from two-dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two-dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular masses of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P) epimerase/dehydratase by specific assignment of peptide masses to a single ORF, excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations identified in the domains 23-47 and 91-118 of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two-dimensional gels by mass spectrometry provided a molecular mass of 35,400 Da, which is consistent with an average degree of three phosphorylations.

Determination of primary structure and microheterogeneity of a beta-amyloid plaque-specific antibody using high-performance LC-tandem mass spectrometry.

Using the bottom-up approach and liquid chromatography (LC) in combination with mass spectrometry, the primary structure and sequence microheterogeneity of a plaque-specific anti-beta-amyloid (1-17) monoclonal antibody (clone 6E10) was characterized. This study describes the extent of structural information directly attainable by a high-performance LC-tandem mass spectrometric method in combination with both protein database searching and de novo sequence determination. Using trypsin and chymotrypsin for enzymatic digestion, 95% sequence coverage of the light chain and 82% sequence coverage of the heavy chain of the 6E10 antibody were obtained. The primary structure determination of a large number of peptides from the antibody variable regions was obtained through de novo interpretation of the data. In addition, N-terminal truncations of the heavy chain were identified as well as low levels of pyroglutamic acid formation. Surprisingly, pronounced sequence microheterogeneities were determined for the CDR 2 region of the light chain, indicating that changes at the protein level derived from somatic hypermutation of the Ig V(L) genes in mature B-cells might contribute to unexpected structural diversity. Furthermore, the major glycoforms at the conserved heavy chain N-glycosylation site, Asn-292, were determined to be core-fucosylated, biantennary, complex-type structures containing zero to two galactose residues. [figure: see text]

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry.

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.

Designing better radiology workstations: impact of two user interfaces on interpretation errors and user satisfaction.

This paper presents our solution for supporting radiologists' interpretation of digital images by automating image presentation during sequential interpretation steps. We extended current hanging protocols with support for "stages" which reflect the presentation of digital information required to complete a single step within a complex task. We demonstrated the benefits of staging in a user experiment with 20 lay subjects involved in a comparative visual search for targets, similar to a radiology task of identifying anatomical abnormalities. We designed a task and a set of stimuli that allowed us to simulate the interpretation workflow from a typical radiology scenario-reading a chest radiography exam when a prior study is also available. The simulation was enabled by abstracting both the radiologist's task and the basic workstation navigation functionality. The staged interface was significantly faster than the traditional user interface, provided a 37% reduction in the interpretation errors, and improved user satisfaction.

Design requirements for radiology workstations.

This article stresses the importance of capturing feedback from representative users in the early stages of product development. We present our solution to producing quality requirement specifications for radiology workstations, specifications that remain valid over time because we successfully anticipated the industry trends and the user's needs. We present the results from a user study performed in December 1999 in a radiology clinic equipped with state-of-the-art Picture Archiving and Communications Systems (PACS) and imaging scanners. The study involved eight radiologists who answered questions and provided comments on three complementary research topics. First, we asked our subjects to enumerate the advantages and the disadvantages for both softcopy and hardcopy reading. We identified the two major factors for productivity improvement through the use of PACS workstations: workflow re-engineering and process automation. Second, we collected radiologist feedback on the use of hanging protocols (HPs). The results indicated the high importance of automatic image organization through HPs, with the potential effect of reducing the interpretation time by 10-20%. Our subjects estimated that 10-15 HPs would cover about 85%-95% of the regular radiological examinations. Third, we investigated the impact of the display devices on the radiologist's workflow. Our results indicated that the number and the properties of the monitors is a modality-specific requirement. The main results from this study on key functional requirements for softcopy interpretation only recently were incorporated in most of the current, successful PACS workstations.

Radiology workstation design for the medical intensive care unit.

The "one-size-fits-all" approach for radiology workstation design is not good enough anymore. While most of the picture archiving and communication system (PACS) vendors are racing to add more features to the radiology workstation, there is little interest in addressing the specific needs of other hospital departments. Significant delays in the availability of radiology reports are often caused by the fact there is not enough Intensive Care Unit (ICU) volume to justify a full time radiologist. Consequently, the radiologist assigned to cover the ICU exams, most likely working from a different building, will read the ICU exams only at certain times, depending on the limitations for remote image availability. This paper addresses the main objectives in designing a digital radiology workstation for use in the medical ICU (MICU), requiring enhancements to current PACS systems. Our suggestions for PACS improvement follow the ICU digital workflow starting with the transfer of the images from the modality, continuing with the presentation of the radiology examination to different types of users (radiologists or ICU staff), up to the creation and distribution of the reports.