Unusual N-type glycosylation of salivary prolactin-inducible protein (PIP): multiple LewisY epitopes generate highly-fucosylated glycan structures.

Prolactin-inducible protein (PIP) is a glycoprotein found in body secretions from exocrine glands like saliva and seminal plasma. Important biological functions of PIP concentrations have been demonstrated, e.g. in tumor diagnosis and progression. PIP quantity has been also found useful to determine the success of chemotherapy of mammary carcinoma. Here, we present the analysis of the N-glycosylation of PIP isolated from different sources by LC-MS(/MS) and 1H-NMR. We found a very uncommon N-type glycosylation of PIP in healthy individuals from both, seminal fluid and saliva. PIP carries unusual highly fucosylated N-linked glycans with multiple Lewisy (Ley) epitopes on bi-, tri- and tetraantennary structures resulting in up to nine fucosyl residues on a tetraantennary glycan. In most organs, Ley epitopes are not present on N-glycans except in case of a tumor when it is highly up-regulated and important for prognosis. Here, for the first time on a specific glycoprotein Ley antigens are unambiguously characterized on an N-type glycan by NMR spectroscopy. So far, for specific glycoproteins Ley epitopes had only been reported on O-glycans. Furthermore, a correlation between a nonsynonymous single nucleotide polymorphism (SNP) and glycosylation pattern was detected: individuals heterozygous for the SNP causing the amino acid exchange 51Gln to 51His have glycan structures with a higher degree of sialylation compared to individuals lacking the SNP.

Insights into Microalga and Bacteria Interactions of Selected Phycosphere Biofilms Using Metagenomic, Transcriptomic, and Proteomic Approaches.

Microalga are of high relevance for the global carbon cycling and it is well-known that they are associated with a microbiota. However, it remains unclear, if the associated microbiota, often found in phycosphere biofilms, is specific for the microalga strains and which role individual bacterial taxa play. Here we provide experimental evidence that Chlorella saccharophila, Scenedesmus quadricauda, and Micrasterias crux-melitensis, maintained in strain collections, are associated with unique and specific microbial populations. Deep metagenome sequencing, binning approaches, secretome analyses in combination with RNA-Seq data implied fundamental differences in the gene expression profiles of the microbiota associated with the different microalga. Our metatranscriptome analyses indicates that the transcriptionally most active bacteria with respect to key genes commonly involved in plant-microbe interactions in the Chlorella (Trebouxiophyceae) and Scenedesmus (Chlorophyceae) strains belong to the phylum of the α-Proteobacteria. In contrast, in the Micrasterias (Zygnematophyceae) phycosphere biofilm bacteria affiliated with the phylum of the Bacteroidetes showed the highest gene expression rates. We furthermore show that effector molecules known from plant-microbe interactions as inducers for the innate immunity are already of relevance at this evolutionary early plant-microbiome level.

Reduction of GAPDH in lenses of Parkinson's disease patients: A possible new biomarker.

BACKGROUND: We previously showed that the protein pattern of lenses removed in cataract surgery differs between patients with Parkinson's disease and age-matched controls. In this study, we identified the protein reduced in abundance in the 34- to 37-kDa gel band. METHODS: During cataract surgery (phacoemulsification), we collected the rinsing fluid and lens particles from the eyes of PD patients and controls. Residual lens fragments in the supernatant of 3 PD patients and aged-matched controls were studied for protein profiles using liquid chromatography-mass spectrometry and Western blots. RESULTS: We identified glyceraldehyde-3-phosphate dehydrogenase by mass spectrometry as the protein reduced in abundance and verified this finding in Western blots. CONCLUSIONS: Glyceraldehyde-3-phosphate dehydrogenase has been implicated in PD development. The reduction of glyceraldehyde-3-phosphate dehydrogenase in the lenses of PD patients may be a new biomarker for PD and might also indicate an important role for this protein in PD development. © 2016 International Parkinson and Movement Disorder Society.

Unambiguous characterization of N-glycans of monoclonal antibody cetuximab by integration of LC-MS/MS and ¹H NMR spectroscopy.

Monoclonal antibodies are most rapidly emerging as therapeutic drugs for the treatment of cancer and of various other diseases such as autoimmunity or inflammation. Recently, it was found that nonhuman glycosylation of recombinant antibodies can cause tremendous problems for some patients. Therefore, unambiguous assignment of the glycosylation pattern of therapeutic antibodies is of high importance for assessment of human compatibility. Here we present results from a broad and detailed N-glycan analysis of the therapeutic antibody cetuximab by LC-MS/MS analyses tightly integrated with (1)H NMR to obtain unambiguous structures. Thirty-seven N-glycan compositions were identified by LC-MS(/MS). Subsequently, ten abundant structures were structurally characterized by applying the recently introduced method called three-dimensional cross correlation (3DCC). It was possible to extract NMR spectra of pure N-glycans that were heavily overlapping in a chromatographic separation by mathematically dissecting the NMR spectra obtained from chromatographic fractions. Even mass isobaric structures that differ only in the branching position of one monosaccharide unit were distinguished and characterized. We also developed an improvement of the 3DCC method by introducing singular value decomposition (SVD) for processing of the data. The smallest amount of the N-glycan characterized by 3DCC was approximately 400 pmol (836 ng). Among the ten unambiguously identified glycans, six N-glycans, representing 24% of all detected glycans, possess the immunogenic α-1,3-Gal epitope and/or N-glycolylneuraminic acid. These results illustrate the importance of integrated use of LC-MS(/MS) and (1)H NMR for the glycome analysis of biopharmaceuticals in research, development, and quality control.

Glycan analysis: scope and limitations of different techniques--a case for integrated use of LC-MS(/MS) and NMR techniques.

The structure of glycans from glycoproteins is highly relevant for their function. We tightly integrate liquid chromatography-mass spectrometry (LC-MS), MS/MS, and nuclear magnetic resonance (NMR) data to achieve a complete characterization of even isobaric glycans differing in only one linkage position or in the substitution in one branch. As example, we analyzed ten desialylated underivatized glycans from bovine fibrinogen. The molecules were separated on a PGC column, and LC-MS data allowed an assignment of the compositions of the glycans. MS/MS data of the same glycans allowed elucidation of sequence and to some extent of branching and linkage. All MS/MS fragmentation methods led to multiple dissociations, resulting in several cases in ambiguous data. The MS/MS data were interpreted both by scientists and automatically by software, and the differential results are compared. Additional data from a tight integration of LC-MS and NMR data resulted in a complete structural characterization of the glycans. The acquisition of simple 1D (1)H NMR data led--in combination with LC-MS and MS/MS data--to an unambiguous assignment of the isobaric glycans. Compounds that were not separated in the chromatography could easily be assigned structurally by applying the 3D cross-correlation (3DCC) technology to arrive at NMR spectra of the pure components-without actually separating them. By applying LC-MS, MS/MS, 1D (1)H NMR, and 3DCC together, one can assign glycan structures from glycoconjugates with high confidence affording only 200 pmol of glycan material.

Detection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.

Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using (14)N/(15)N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers.