Multiparameter screen optimizes immunoprecipitation.

Immunoprecipitation (IP) coupled with mass spectrometry effectively maps protein-protein interactions when genome-wide, affinity-tagged cell collections are used. Such studies have recorded significant portions of the compositions of physiological protein complexes, providing draft 'interactomes'; yet many constituents of protein complexes still remain uncharted. This gap exists partly because high-throughput approaches cannot optimize each IP. A key challenge for IP optimization is stabilizing in vivo interactions during the transfer from cells to test tubes; failure to do so leads to the loss of genuine interactions during the IP and subsequent failure to detect. Our high-content screening method explores the relationship between in vitro chemical conditions and IP outcomes, enabling rapid empirical optimization of conditions for capturing target macromolecular assemblies.

Improving impact of heparan sulfate on the endothelial glycocalyx abnormalities in atherosclerosis as revealed by glycan-protein interactome.

Endothelial dysfunction induced by oxidative stress is an early predictor of atherosclerosis, which can cause various cardiovascular diseases. The glycocalyx layer on the endothelial cell surface acts as a barrier to maintain endothelial biological function, and it can be impaired by oxidative stress. However, the mechanism of glycocalyx damage during the development of atherosclerosis remains largely unclear. Herein, we established a novel strategy to address these issues from the glycomic perspective that has long been neglected. Using countercharged fluorescence protein staining and quantitative mass spectrometry, we found that heparan sulfate, a major component of the glycocalyx, was structurally altered by oxidative stress. Comparative proteomics and protein microarray analysis revealed several new heparan sulfate-binding proteins, among which alpha-2-Heremans-Schmid glycoprotein (AHSG) was identified as a critical protein. The molecular mechanism of AHSG with heparin was characterized through several methods. A heparan analog could relieve atherosclerosis by protecting heparan sulfate from degradation during oxidative stress and by reducing the accumulation of AHSG at lesion sites. In the present study, the molecular mechanism of anti-atherosclerotic effect of heparin through interaction with AHSG was revealed. These findings provide new insights into understanding of glycocalyx damage in atherosclerosis and lead to the development of corresponding therapeutics.

MsPHep: An online application for low molecular weight heparin rapid characterization based on liquid chromatography-tandem mass spectrometry.

Low-molecular-weight heparins (LMWHs) are important anticoagulants widely used in clinic. Since they are comprised of complex and heterogenous glycan chains, liquid chromatography-tandem mass spectrometry (LC-MS) is commonly used for structural analysis and quality control of LMWHs to ensure their safety and efficacy. Yet, the structural complexity arising from the parent heparin macromolecules, as well as the different depolymerization methods used for preparing LMWHs, makes processing and assigning the LC-MS data of LWMHs very tedious and challenging. We therefore developed, and here report, an open-source and easy-to-use web application, MsPHep, to facilitate the LMWH analysis based on LC-MS data. MsPHep is compatible with various LMWHs and chromatographic separation methods. With the HepQual function, MsPHep is capable of annotating both the LMWH compound and its isotopic distribution from mass spectra. Moreover, the HepQuant function enables automatic quantification of LMWH compositions without prior knowledge or any database generation. To demonstrate the reliability and system stability of MsPHep, we tested various types of LMWHs that were analyzed with different chromatographic methods coupled to MS. The results show that MsPHep has its own advantages compared to another public tool GlycReSoft for LMWH analysis, and it is available online under an open-source license at https://ngrc-glycan.shinyapps.io/MsPHep.

Delayed Comparison and Apriori Algorithm (DCAA): A Tool for Discovering Protein-Protein Interactions From Time-Series Phosphoproteomic Data.

Analysis of high-throughput omics data is one of the most important approaches for obtaining information regarding interactions between proteins/genes. Time-series omics data are a series of omics data points indexed in time order and normally contain more abundant information about the interactions between biological macromolecules than static omics data. In addition, phosphorylation is a key posttranslational modification (PTM) that is indicative of possible protein function changes in cellular processes. Analysis of time-series phosphoproteomic data should provide more meaningful information about protein interactions. However, although many algorithms, databases, and websites have been developed to analyze omics data, the tools dedicated to discovering molecular interactions from time-series omics data, especially from time-series phosphoproteomic data, are still scarce. Moreover, most reported tools ignore the lag between functional alterations and the corresponding changes in protein synthesis/PTM and are highly dependent on previous knowledge, resulting in high false-positive rates and difficulties in finding newly discovered protein-protein interactions (PPIs). Therefore, in the present study, we developed a new method to discover protein-protein interactions with the delayed comparison and Apriori algorithm (DCAA) to address the aforementioned problems. DCAA is based on the idea that there is a lag between functional alterations and the corresponding changes in protein synthesis/PTM. The Apriori algorithm was used to mine association rules from the relationships between items in a dataset and find PPIs based on time-series phosphoproteomic data. The advantage of DCAA is that it does not rely on previous knowledge and the PPI database. The analysis of actual time-series phosphoproteomic data showed that more than 68% of the protein interactions/regulatory relationships predicted by DCAA were accurate. As an analytical tool for PPIs that does not rely on a priori knowledge, DCAA should be useful to predict PPIs from time-series omics data, and this approach is not limited to phosphoproteomic data.

Oligosaccharides mapping of nitrous acid degraded heparin through UHPLC-HILIC/WAX-MS.

Building blocks characterization is a significant approach for understanding the molecular structure of heparin and its derivatives. Nitrous acid (HONO) depolymerization of heparin generates oligosaccharides that maintain the epimerization conformation on C5 of the uronic acids, reflecting the authentic structure of the parental chain. HONO treatment at pH 1.5 selectively cleaves the bond between N-sulfated glucosamine and hexuronic acid, resulting mainly disaccharides, as well as tetra-, tri-, and mono-saccharides. The tetrasaccharides are derived from the structure of N-acetylated domains while tri-, and mono-saccharides are derived from the reducing or the non-reducing end of the heparin chain. The resulted oligosaccharides were separated and analyzed using a UHPLC-HILIC/WAX-MS method. We succeeded in the identification of 19 tetrasaccharides, 19 trisaccharides and 4 monosaccharides species, majority of which is structurally characterized. By comparing the theoretical possibilities and actual occurrence of the well-characterized tetrasaccharides, we demonstrated that the biosynthesis of heparin is a systematic process.

Preparation of low molecular weight heparins from bovine and ovine heparins using nitrous acid degradation.

Low molecular weight heparins (LMWHs) are important anticoagulant drugs. Nitrous acid degradation is a major approach to produce LMWHs, such as dalteparin. Due to the foreseeable shortage of porcine intestinal mucosa heparin and other potential risks, expansion of other animal tissues for heparin preparation is necessary. Heparins from different tissues differ in structure and bioactivity potency, and these variations may be carried over to the LMWH products. Sophisticated analytical techniques have been applied to compare various versions of dalteparins produced from porcine intestinal, bovine lung and ovine intestinal heparins to elucidate the effects of different animal tissues starting materials and processing conditions on the properties of final dalteparin products. With adjusted depolymerization conditions, versions of dalteparins that qualify under the European Pharmacopeia (EP) specifications were manufactured using non-porcine heparins. Dissimilarities among the three interspecies animal tissue heparin-derived dalteparins regarding fine structures are also disclosed, and their origins are discussed.

Sequencing the oligosaccharide pool in the low molecular weight heparin dalteparin with offline HPLC and ESI-MS/MS.

Low molecular weight heparins (LMWHs) are widely used anticoagulant drugs. The composition and sequence of LMWH oligosaccharides determine their safety and efficacy. The short oligosaccharide pool in LMWHs undergoes more depolymerization reactions than the longer chains and is the most sensitive indicator of the manufacturing process. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has been demonstrated as a powerful tool to sequence synthetic heparin oligosaccharide but never been applied to analyze complicated mixture like LMWHs. We established an offline strong anion exchange (SAX)-high performance liquid chromatography (HPLC) and ESI-MS/MS approach to sequence the short oligosaccharides of dalteparin sodium. With the help of in-house developed MS/MS interpretation software, the sequences of 18 representative species ranging from tetrasaccharide to octasaccharide were obtained. Interestingly, we found a novel 2,3-disulfated hexauronic acid structure and reconfirmed it by complementary heparinase digestion and LC-MS/MS analysis. This approach provides straightforward and in-depth insight to the structure of LMWHs and the reaction mechanism of heparin depolymerization.

Synthesis of 3-O-Sulfated Oligosaccharides to Understand the Relationship between Structures and Functions of Heparan Sulfate.

The sulfation at the 3-OH position of glucosamine is an important modification in forming structural domains for heparan sulfate to enable its biological functions. Seven 3-O-sulfotransferase isoforms in the human genome are involved in the biosynthesis of 3-O-sulfated heparan sulfate. As a rare modification present in heparan sulfate, the availability of 3-O-sulfated oligosaccharides is very limited. Here, we report the use of a chemoenzymatic synthetic approach to synthesize six 3-O-sulfated oligosaccharides, including three hexasaccharides and three octasaccharides. The synthesis was achieved by rearranging the enzymatic modification sequence to accommodate the substrate specificity of 3-O-sulfotransferase 3. We studied the impact of 3-O-sulfation on the conformation of the pyranose ring of 2-O-sulfated iduronic acid using NMR, and on the correlation between ring conformation and anticoagulant activity. We identified a novel octasaccharide that interacts with antithrombin and displays anti factor Xa activity. Interestingly, the octasaccharide displays a faster clearance rate than fondaparinux, an FDA-approved pentasaccharide drug, in a rat model, making this octasaccharide a potential short-acting anticoagulant drug candidate that could reduce bleeding risk. Having access to a set of critically important 3-O-sulfated oligosaccharides offers the potential to develop new heparan sulfate-based therapeutics.

Comprehensive Identification and Quantitation of Basic Building Blocks for Low-Molecular Weight Heparin.

Low-molecular weight heparins (LMWHs) are widely used anticoagulant drugs. They inherit the heterogeneous backbone sequences of the parent heparin, while the chemical depolymerization process modifies the nonreducing end (NRE) and reducing end (RE) of their sugar chains. Some side reactions may also occur and increase the structural complexity of LMWHs. It is important to precisely characterize the structures of LMWHs, especially their chemical modifications, to ensure drug quality and safety. Compositional analysis provides a powerful approach to reveal the building blocks that make up the LMWHs, which are the mutual consequence of the heparin starting materials and the manufacturing process. Here, we introduce a comprehensive analytical method to recover the most basic building blocks of LMWHs. A strategy of combining both enzymatic digestion and oxidative degradation of LMWH was used to make the NRE, RE, and backbone structures differentiable from one another. Satisfactory separation, identification, and quantitation were achieved by coupling hydrophilic interaction chromatography with a triple quadrupole mass spectrometer operating under the multiple reaction monitoring mode. After enzymatic digestion, over 30 species were detected, with both natural and chemically modified heparin basic building blocks. Two novel structures, including a trisaccharide containing two glucosamine residues and a tetrasaccharide containing a 3-O-sulfated uronic acid residue, were discovered. Reduced and oxidatively degraded samples were analyzed to provide the complementary information on both termini of LMWHs. The reproducibility of this method was evaluated, and enoxaparin injections were analyzed to demonstrate the application of this method for evaluating the sameness of LMWH products.