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).

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.

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.

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.

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.

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.

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.

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.

"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.

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.

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).

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.

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.

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.