Pia Hønnerup Jensen

Structural analysis of N - and O -glycans released from glycoproteins

This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.

Mucin-type O-glycosylation--putting the pieces together.

The O‐glycosylation of Ser and Thr by N‐acetylgalactosamine‐linked (mucin‐type) oligosaccharides is often overlooked in protein analysis. Three characteristics make O‐linked glycosylation more difficult to analyse than N‐linked glycosylation, namely: (a) no amino acid consensus sequence is known; (b) there is no universal enzyme for the release of O‐glycans from the protein backbone; and (c) the density and number of occupied sites may be very high. For significant biological conclusions to be drawn, the complete picture of O‐linked glycosylation on a protein needs to be determined. This review specifically addresses the analytical approaches that have been used, and the challenges remaining, in the characterization of both the composition and structure of mucin‐type O‐glycans, and the determination of the occupancy and heterogeneity at each amino acid attachment site.

Determination of site-specific glycan heterogeneity on glycoproteins

The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3–5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.

GlycoSpectrumScan : fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA

With the emergence of glycoproteomics, there is a need to develop bioinformatic tools to identify glycopeptides in protease digests of glycoproteins. GlycoSpectrumScan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass spectrometric data. Two experimental data sets are required as inputs: (1) oligosaccharide compositions of the N- and/or O-linked glycans present in the sample and (2) in silico derived peptide masses of proteolytically digested proteins with a potential number of N- and/or O-glycosylation sites. GlycoSpectrumScan uses MS data, rather than MS/MS data, to identify glycopeptides and determine the relative distribution of N- and O-glycoforms at each site. It is functional for assigning monosaccharide compositions on glycopeptides with single and multiple sites of glycosylation. The algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both ESI and MALDI data from all mass spectrometer platforms. Manual analysis time for identifying glycosylation heterogeneity at each site on glycoprotein(s) is substantially decreased. The application of this tool to characterize the N- and O-linked glycopeptides from human secretory IgA (sIgA), consisting of secretory component (7 N-linked sites), IgA1 (2 N-linked, <or=5 O-linked sites), IgA2 (4 N-linked sites) and J-chain (1 N-linked site) is described. GlycoSpectrumScan is freely available at www.glycospectrumscan.org .

Heteromeric Complexes of Native Collectin Kidney 1 and Collectin Liver 1 Are Found in the Circulation with MASPs and Activate the Complement System

The complement system is an important part of the innate immune system. The complement cascade may be initiated downstream of the lectin activation pathway upon binding of mannan-binding lectin, ficolins, or collectin kidney 1 (CL-K1, alias CL-11) to suitable microbial patterns consisting of carbohydrates or acetylated molecules. During purification and characterization of native CL-K1 from plasma, we observed that collectin liver 1 (CL-L1) was copurified. Based on deglycosylation and nonreduced/reduced two-dimensional SDS-PAGE, we detected CL-K1 and CL-L1 in disulfide bridge-stabilized complexes. Heteromeric complex formation in plasma was further shown by ELISA and transient coexpression. Judging from the migration pattern on two-dimensional SDS-PAGE, the majority of plasma CL-K1 was found in complex with CL-L1. The ratio of this complex was in favor of CL-K1, suggesting that a heteromeric subunit is composed of one CL-L1 and two CL-K1 polypeptide chains. We found that the complex bound to mannan-binding lectin–associated serine proteases (MASPs) with affinities in the nM range in vitro and was associated with both MASP-1/-3 and MASP-2 in plasma. Upon binding to mannan or DNA in the presence of MASP-2, the CL-L1–CL-K1 complex mediated deposition of C4b. In favor of large oligomers, the activity of the complex was partly determined by the oligomeric size, which may be influenced by an alternatively spliced variant of CL-K1. The activity of the native heteromeric complexes was superior to that of recombinant CL-K1. We conclude that CL-K1 exists in circulation in the form of heteromeric complexes with CL-L1 that interact with MASPs and can mediate complement activation.

Rat liver membrane glycoproteome : enrichment by phase partitioning and glycoprotein capture

Past proteomic studies of membrane proteins have often been hampered by the low abundance and relatively high hydrophobicity of these proteins. Proteins are often glycosylated, particularly on the extracellular surface of the plasma membrane, and this characteristic was targeted as an enrichment strategy for identifying membrane proteins. Here, we report a strategy for identifying the tissue membrane glycoproteome, which involves (1) Triton X-114 phase partitioning, (2) isolation of glycosylphosphatidylinositol (GPI)-anchored proteins, and (3) glycoprotein capture using lectin affinity or hydrazine chemistry. Surprisingly, the capture of membrane proteins by lectin affinity and hydrazine chemistry resulted in mostly different populations of enriched glycoproteins. Lectins enriched high molecular weight functional membrane proteins with more potential glycosylation such as those involved in signal transduction and cell adhesion. Conversely, hydrazine chemistry isolated a higher proportion of smaller, enzymatic and peripheral membrane proteins such as solute carrier transporters and cytochrome p450s. We have applied our strategy to characterize the rat liver membrane glycoproteome and identified four new predicted GPI-anchored proteins and two that have not previously been seen in the liver. We also identified 424 nonredundant membrane proteins, of which 335 had potential N-linked glycosylation sites.

Challenges of determining o-glycopeptide heterogeneity: A fungal glucanase model system

O-Linked glycosylation often occurs in mucin-type domains that are heavily and heterogeneously glycosylated and are challenging to analyze. The analysis of these domains is often overlooked because of these difficulties, but changes in mucinlike domain glycosylation are implicated in many diseases. Here we have explored several strategies to determine the heterogeneity of mucinlike O-glycosylated domains. Four glucanases secreted in large quantities from Trichoderma reesei, all containing heavily O-glycosylated mucinlike linker regions, were used as a model system. The strategies involved monosaccharide compositional analysis and identification of the released glycans by HPAEC-PAD and carbon-LC ESI-MS/MS. Glycosylated peptides were generated by different protease digestions (trypsin, papain, Asp-N, PreTAQ) and enriched by HILIC microcolumns, to determine the glycopeptide heterogeneity and glycosylation sites. The complex O-glycan heterogeneity on the intact glycoproteins and the enriched mucin-type domains was determined by MALDI-MS and ESI-MS, but the dense O-glycosylation in the mucin-type domains conferred high resistance to protease cleavage. ETD-MS/MS of the glycopeptide-enriched protease digests was unsuccessful for the de novo assignment of O-glycosylation at individual sites within the mucin-type domains but allowed several previously unknown O-linked sites outside the defined linker region to be found on two of the four glucanases. The protease digests produced many glycopeptides as determined by CID-MS/MS, but ETD fragmentation of these resulted in only a few interpretable spectra, suggesting that the use of ETD for determining the heterogeneous O-glycosylation at specific sites in regions of multiple occupancy is still in its infancy.

Glycopeptide Enrichment for MALDI-TOF Mass Spectrometry Analysis by Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction (HILIC SPE)

Glycoproteins, and in particular glycopeptides, are highly hydrophilic and are often not retained by reversed phase (RP) chromatography. The separation principle of normal phase (NP) is based on hydrophilic interactions, which in many aspects is complementary to RP separations. Hydrophilic interaction liquid chromatography (HILIC) is a fairly new variation of the NP separations used in the 1970s, the major difference being the use of aqueous solvents. HILIC provides a versatile tool for enrichment of glycopeptides before mass spectrometric (MS) analysis, particularly when used for solid phase extraction (SPE), or in combination with other chromatographic resins or ion-pairing reagents. HILIC SPE can be used for glyco-profiling, i.e., for determining the glycan heterogeneity at one specific glycosylation site, for enrichment of glycopeptides from a complex mixture of peptides, as well as for pre-fractionation of complex samples at the protein or peptide level. In this chapter we present a straightforward HILIC SPE enrichment technique and then combine C18 RP and HILIC enrichment for analysis of glycopeptides. Finally, we demonstrate HILIC enrichment using trifluoroacetic acid as an ion-pairing reagent for the enrichment of glycopeptides prior to mass spectrometry analysis.

Selective renal vasoconstriction, exaggerated natriuresis and excretion rates of exosomic proteins in essential hypertension.

In essential hypertension (EH), the regulation of renal sodium excretion is aberrant. We hypothesized that in mild EH, (i) abnormal dynamics of plasma renin concentration (PRC) and atrial natriuretic peptide (ANP) are responsible for the exaggerated natriuresis, and (ii) exosomic protein patterns reflect the renal tubular abnormality involved in the dysregulation of sodium excretion.

Recycling for all: preliminary criteria for the design of disability-friendly receptacles

The design of current waste bins, paper and bottle banks presents barriers for disabled and elderly persons, as pointed out by several Danish studies. In drawing up design criteria for the development of receptacles to be usable by persons with disabilities and senior citizens it is essential to be aware of the problems caused by different types of disability as well as old age. On the basis of general user requirements, preliminary criteria for the design of disability-friendly receptacles are proposed. In collaboration with a collecting agent, current receptacles modified in accordance to the preliminary design criteria will be tested in pilot studies in three selected housing estates in the municipalities of Frederiksberg and Copenhagen. It is the authors’ hope that the testing of the modified receptacles will result in achieving more efficient source separation not only for disabled and elderly persons, but for all user groups.