Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 µg of HeLa protein. CSC also revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

Characterization and comparison site-specific N-glycosylation profiling of milk fat globule membrane proteome in donkey and human colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are highly glycosylated and involved in various biological processes within the body. However, information on site-specific N-glycosylation of MFGM glycoproteins in donkey and human milk remains limited. This study aimed to map the most comprehensive site-specific N-glycosylation fingerprinting of donkey and human MFGM glycoproteins using a site-specific glycoproteomics strategy. We identified 1,360, 457, 2,617, and 986 site-specific N-glycans from 296, 77, 214, and 196 N-glycoproteins in donkey colostrum (DC), donkey mature milk (DM), human colostrum (HC), and human mature milk (HM), respectively. Bioinformatics was used to describe the structure-activity relationships of DC, DM, HC, and HM MFGM N-glycoproteins. The results revealed differences in the molecular composition of donkey and human MFGM N-glycoproteins and the dynamic changes to site-specific N-glycosylation of donkey and human MFGM glycoproteins during lactation, deepening our understanding of the composition of donkey and human MFGM N-glycoproteins and their potential physiological roles.

Glycoproteomics analysis reveals differential site-specific N-glycosylation of donkey milk fat globule membrane protein during lactation.

N-glycosylation is a prevalent and complex post-translational modification of milk proteins with significant biological importance. However, the systematic characterisation of donkey milk fat globule membrane (MFGM) N-glycoproteins remains largely ill-defined. Here, 1443 intact N-glycopeptides from 336 MFGM glycoproteins in donkey colostrum (DC) and 489 intact N-glycopeptides from 86 MFGM glycoproteins in donkey mature milk (DM) were identified via label-free site-specific glycoproteomics. Mannosylation and fucosylation were predominant in DC MFGM N-glycoproteins compared to sialylation and mannosylation in DM. Among them, 22 site-specific N-glycans attached to 14 glycosites of eight glycoproteins were significantly increased, whereas 30 site-specific N-glycans attached to 19 glycosites of 16 glycoproteins were significantly decreased. Furthermore, the site-specific N-glycans with Neu5Gc moieties or simultaneous fucosylation and sialylation were not significantly increased, exhibiting significant site specificity. We provide new insights into the composition of donkey MFGM N-glycoproteins and their roles in donkey milk-related biological functions.

Glycan-selective in-situ growth of thermoresponsive polymers for thermoprecipitation and enrichment of N-glycoprotein/glycopeptides.

In view of the biological significance and micro-heterogeneity of protein glycosylation for human health, specific enrichment of N-glycosylated proteins/peptides from complex biological samples is a prerequisite for the discovery of disease biomarkers and clinical diagnosis. In this work, we propose a "grafting-from" N-glycoprotein enriching method based on the in-situ growth of thermoresponsive polymer brushes from the N-glycosylated site of proteins. The initiator was first attached to the pre-oxidized glycan moieties by hydrazide chemistry, from which the thermoresponsive polymers can be grown to form giant protein-polymer conjugates (PPC). The thermosensitive PPC can be precipitated and separated by raising the temperature to above its lower critical solubility temperature (LCST). Mass spectrometry verified 210 N-glycopeptides corresponding to 136 N-glycoproteins in the rabbit serum. These results demonstrate the capability of the tandem thermoprecipitation strategy to enrich and separate N-glycoprotein/glycopeptide. Due to its simplicity and efficiency specifically, this method holds the potential for identifying biomarkers from biological samples in N-glycoproteome analysis.

Progresses of mass spectrometry-based analysis of N-glycoproteins / 中国药科大学学报

@#N-linked glycosylation is a common post-translational modification on proteins, which exhibits the same macro-heterogeneity of modification site as other small molecule modifications (such as methylation, acetylation, phosphorylation), i.e., the amino acid sequence of a protein has multiple putative modification sites. However, compared to small molecule modifications with single structures, N-glycosylation modification have tens of thousands of structures from multiple structural dimensions such as different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformation.This results in additional micro-heterogeneity of modification site of N-glycosylation, i.e., the same N-glycosylation site can be modified with different glycans with a certain stoichiometric ratio.N-glycosylation modification regulates the structure and function of N-glycoproteins in a site- and structure-specific manner, and differential expression of N-glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis.This article mainly introduces the latest development of mass spectrometry-based site- and structure-specific quantitative N-glycoproteomics and its applications in biomedical fields.

A new sight to explore site-specific N-glycosylation in donkey colostrum milk fat globule membrane proteins with glycoproteomics analysis.

Donkey colostrum milk fat globule membrane (DCMFGM) proteins are involved in multiple biological functions. However, the effect of N-glycosylation on their physiological properties are unknown. The aim of this study was to map the DCMFGM protein site-specific N-glycosylation landscape using a label-free glycoproteomic approach. A total of 1,443 unique intact N-glycopeptides mapping to 453 unique N-glycosites on 336 N-glycoproteins were identified. The macro- and microheterogeneity of DCMFGM glycoproteins were explored at the N-glycosite level and the site-specific N-glycan level, respectively, and it was found that the N-glycosylation profiles of the DCMFGM proteins varied based on subcellular localisation and protein domain types. Our findings reveal the heterogeneity and functional diversity of N-glycosylation of DCMFGM proteins and provide theoretical support for the promotion of DCMFGM proteins as a functional food ingredient.

Upregulation of protein N-glycosylation plays crucial roles in the response of Camellia sinensis leaves to fluoride.

The tea plant (Camellia sinensis) is one of the three major beverage crops in the world with its leaves consumption as tea. However, it can hyperaccumulate fluoride with about 98% fluoride deposition in the leaves. Our previously studies found that cell wall proteins (CWPs) might play a central role in fluoride accumulation/detoxification in C. sinensis. CWP is known to be glycosylated, however the response of CWP N-glycosylation to fluoride remains unknown in C. sinensis. In this study, a comparative N-glycoproteomic analysis was performed through HILIC enrichment coupled with UPLC-MS/MS based on TMT-labeling approach in C. sinensis leaves. Totally, 237 N-glycoproteins containing 326 unique N-glycosites were identified. 73.4%, 18.6%, 6.3% and 1.7% of these proteins possess 1, 2, 3, and ≥4 modification site, respectively. 93.2% of these proteins were predicted to be localized in the secretory pathway and 78.9% of them were targeted to the cell wall and the plasma membrane. 133 differentially accumulated N-glycosites (DNGSs) on 100 N-glycoproteins (DNGPs) were detected and 85.0% of them exhibited upregulated expression after fluoride treatment. 78.0% DNGPs were extracellular DNGPs, which belonged to CWPs, and 53.0% of them were grouped into protein acting on cell wall polysaccharides, proteases and oxido-reductases, whereas the majority of the remaining DNGPs were mainly related to N-glycoprotein biosynthesis, trafficking and quality control. Our study shed new light on the N-glycoproteome study, and revealed that increased N-glycosylation abundance of CWPs might contribute to fluoride accumulation/detoxification in C. sinensis leave.

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum.

N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

Cryo-EM structure of fission yeast tetrameric α-mannosidase Ams1.

Fungal α-mannosidase Ams1 and its mammalian homolog MAN2C1 hydrolyze terminal α-linked mannoses in free oligosaccharides released from misfolded glycoproteins or lipid-linked oligosaccharide donors. Ams1 is transported by selective autophagy into vacuoles. Here, we determine the tetrameric structure of Ams1 from the fission yeast Schizosaccharomyces pombe at 3.2 Å resolution by cryo-electron microscopy. Distinct from a low resolution structure of S. cerevisiae Ams1, S. pombe Ams1 has a prominent N-terminal tail that mediates tetramerization and an extra ß-sheet domain. Ams1 shares a conserved active site with other enzymes in glycoside hydrolase family 38, to which Ams1 belongs, but contains extra N-terminal domains involved in tetramerization. The atomic structure of Ams1 reported here will aid understanding of its enzymatic activity and transport mechanism.

Proteomic Analysis of the Air-Way Fluid in Lung Cancer. Detection of Periostin in Bronchoalveolar Lavage (BAL).

Background: Bronchoalveolar lavage (BAL) is a specific type of air-way fluid. It is a commonly used clinical specimen for the diagnosis of benign diseases and cancers of the lung. Although previous studies have identified several disease-associated proteins in the BAL, the potential utility of BAL in lung cancer is still not well-studied. Based upon the fact that the majority of secreted proteins are glycoproteins, we have profiled N-glycoproteins in BAL collected from lung cancers, and investigated the expression of glycoproteins such as the matrix N-glycoprotein, periostin, in lung cancers. Methods: BAL specimens (n = 16) were collected from lung cancer patients, and analyzed using mass spectrometry-based quantitative N-glycoproteomic technique. Additional BAL specimens (n = 39) were independently collected to further evaluate the expression of periostin by using an enzyme-linked immunosorbent assay (ELISA). Results: A total of 462 glycoproteins were identified in BAL samples using N-glycoproteomic technique, including 290 in lung adenocarcinoma (ADC, n = 5), 376 in squamous cell carcinoma (SQCC, n = 4), 309 in small cell lung carcinoma (SCLC, n = 4), and 316 in benign lung disease (n = 3). The expressions of several glycoproteins were elevated, including 8 in ADC, 12 in SQCC, and 17 in SCLC, compared to benign BALs. The expression of periostin was detected in all subtypes of lung cancers. To further investigate the expression of periostin, an ELISA assay was performed using additional independently collected BALs (n = 39) The normalized levels of periostin in benign disease, ADC, SQCC, and SCLC were 255 ± 104 (mean ± SE) and 4,002 ± 2,181, 3,496 ± 1,765, and 1,772 ± 1,119 ng/mg of total BAL proteins. Conclusion: Our findings demonstrate that proteomic analysis of BAL can be used for the study of cancer-associated extracellular proteins in air-way fluid from lung cancer patients.

Integrated proteomic, phosphoproteomic and N-glycoproteomic analyses of chicken eggshell matrix.

Eggshell matrix (EM) proteins play an important biological role in eggshell mineralization and embryo development. Many studies have demonstrated that some matrix proteins undergo posttranslational modifications, including phosphorylation and glycosylation, which have important regulatory effects on the functional properties of the proteins. Systematic analysis of the proteome, the phosphorylated modified proteome and the glycosylated modified proteome of the chicken EM was performed using a proteomics strategy. A total of 112 phosphorylation sites from 69 phosphoproteins and 297 N-glycosylation sites from 182 N-glycoproteins were identified in the chicken EM. Among all these identified modified proteins, 129 were not identified in the proteome (547 proteins). Therefore, a total of 676 EM proteins were identified in this study. Gene ontology (GO) enrichment analysis indicated that EM proteins and phosphoproteins were mainly enriched in regulation of enzyme activity, while EM N-glycoproteins were enriched in immune response regulation.

An Engineered Pathway for Production of Terminally Sialylated N-glycoproteins in the Periplasm of Escherichia coli.

Terminally sialylated N-glycoproteins are of great interest in therapeutic applications. Due to the inability of prokaryotes to carry out this post-translational modification, they are currently predominantly produced in eukaryotic host cells. In this study, we report a synthetic pathway to produce a terminally sialylated N-glycoprotein in the periplasm of Escherichia coli, mimicking the sialylated moiety (Neu5Ac-α-2,6-Gal-ß-1,4-GlcNAc-) of human glycans. A sialylated pentasaccharide, Neu5Ac-α-2,6-Gal-ß-1,4-GlcNAc-ß-1,3-Gal-ß-1,3-GlcNAc-, was synthesized through the activity of co-expressed glycosyltransferases LsgCDEF from Haemophilus influenzae, Campylobacter jejuni NeuBCA enzymes, and Photobacterium leiognathi α-2,6-sialyltransferase in an engineered E. coli strain which produces CMP-Neu5Ac. C. jejuni oligosaccharyltransferase PglB was used to transfer the terminally sialylated glycan onto a glyco-recognition sequence in the tenth type III cell adhesion module of human fibronectin. Sialylation of the target protein was confirmed by lectin blotting and mass spectrometry. This proof-of-concept study demonstrates the successful production of terminally sialylated, homogeneous N-glycoproteins with α-2,6-linkages in the periplasm of E. coli and will facilitate the construction of E. coli strains capable of producing terminally sialylated N-glycoproteins in high yield.

The Preparation and Solution NMR Spectroscopy of Human Glycoproteins Is Accessible and Rewarding.

The majority of proteins excreted by human cells and borne at the cell surface are modified with carbohydrates. Glycoproteins mediate a wide range of processes and adopt fundamental roles in many diseases. The carbohydrates covalently attached to proteins during maturation in the cell directly impact protein structure and function as integral and indispensable components. However, the ability to study the structure of glycoproteins to high resolution was historically limited by technical barriers including a limited availability of appropriate recombinant protein expression platforms, limited methods to generate compositional homogeneity, and difficulties analyzing glycoprotein composition. Furthermore, glycoproteins and in particular the glycan moieties themselves often exhibit a high degree of conformational heterogeneity. Solution NMR spectroscopy is a powerful tool to study biological macromolecules that is capable of characterizing mobile elements of molecules with atomic-level resolution. Methods to express glycoproteins, incorporate stable isotope labels, and analyze glycoproteins have recently opened new avenues to prepare and investigate glycoproteins. These methods are accessible to many laboratories with experience expressing and purifying proteins from prokaryotic expression hosts.

Hydrazide chemistry based enrichment method for N-glycoprotein analysis from human plasma exosomes / 中国药理学与毒理学杂志

OBJECTIVE To analyze the composition and function of N-glycoproteins in human plasma exosomes. METHODS Exosomes from human plasma were extracted by ultracentrifugation. The proteins from plasma exosomes were released by ultrasound and the obtained proteins were enzymatically degraded by trypsin to peptides. The N-glycopeptides in the mixture of the digested peptides was enriched by the hydrazide resin. The enriched N-glycoproteins were identified by mass spectrometry and subjected to gene ontology (GO) annotation analysis. RESULTS The plasma exosomes isolated by ultracentrifugation had a typical cup-shaped structure and the particle sizes were approximately 100 nm. Totally, 252 N-glycosylation sites corresponding to 131 N-glycoproteins were enriched and identified from exosomes of 1 mL human plasma digestion, which participated in many important biological processes, such as serine-type endopeptidase activity, serine-type endopeptidedase inhibitor acitivity and calcium ion binding. Sixty-seven of the N-glycoproteins had close relations with the occurrence of diseases. CONCLUSION Totally, 131 N-glycoproteins were identified in the research from the plasma exosome protein. These N-glycoproteins precipitate in many important biological processes and have close correlations with the occurrence and progression of various diseases.

Development a hydrazide-functionalized thermosensitive polymer based homogeneous system for highly efficient N-glycoprotein/glycopeptide enrichment from human plasma exosome.

As one of the most common post-translational modifications, protein N-glycosylation precipitates in many important biological processes and has closely correlations with the occurrence and progression of multiple diseases. Plasma exosomes secreted by cells contain various bioactive N-glycoproteins which may serve as potential biomarkers for early disease diagnosis and treatment. However, the protein N-glycosylation profile in human plasma exosome is largely unknown, due to the technical challenges in glycoprotein identification. Signals of the rare N-glycoproteins/N-glycopeptides are severely suppressed by the abundant coexisting non-glycosylated counterparts in mass spectrometry analysis. Therefore, specific enrichment of N-glycoprotein/glycopeptide is a prerequisite for large scale N-glycosylation profiling. In this work, we developed a hydrazide functionalized thermosensitive polymer for efficient enrichment and in-depth identification of protein N-glycosylation in human plasma exosome by mass spectrometry. The polymer chains completely dissolve in the enrichment system to form a homogeneous solution. Therefore, efficient covalent coupling between the N-glycoprotein/glycopeptide and the polymer chain is achieved, due to the reduced interfacial mass transfer resistance and the densely packed accessible functional groups on the polymer chains. Furthermore, the thermosensitive polymer can be easily precipitated and recovered by simply rising the system temperature to above 34 °C. As a result, 329 N-glycosylation sites corresponding to 180 N-glycoproteins were enriched and identified from plasma exosomes of glioma patients and healthy subjects using the thermosensitive polymer. By quantitative comparison, we found 26 N-glycoproteins significantly changed between the glioma patients and the healthy subjects, demonstrating the potential of this new strategy for N-glycoproteome research of plasma exosome and biomarker discovery.

Knocking out Lpp gene of E.coli CLM37 strain and extracellular production of N -glycosylated recombinant proteins / 上海交通大学学报（医学版）

Objective • To construct the E.coli CLM37 strain with Lpp gene deletion and to study the production of N-glycosylated recombinant proteins in this E.coli strain. Methods • Firstly, Red homologous recombination system was used to knock out the Lpp gene from the genome of E.coli CLM37. And then, the growth curve was detected to study the effects of deleted Lpp gene on the growth states of E.coli strain. Finally, the vector pIG6-rFn3-Gly which expresses receptor protein and the vector pACYCpgl, which carries N-glycosylation gene cluster that derives from Campylobacter jejuni, were co-transformed into E.coli CLM37ΔLpp to investigate the extracellular production of N-glycosylated recombinant proteins. Results • The E. coli CLM37ΔLpp with Lpp gene deletion was obtained, and the extracellular production of N-glycosylated rFn3-Gly was successfully achieved in this strain. Compared with E. coli CLM37, the total amount of rFn3-Gly produced via extracellular production of E. coli CLM37ΔLpp increased about 4 times, and the glycosylation efficiency increased about 6 times. Conclusion • N-glycosylated rFn3-Gly was successfully produced via extracellular production in E. coli CLM37ΔLpp, and the production of interest glycoprotein and the glycosylation efficiency were improved.

N-glycoproteomic analysis of human follicular fluid during natural and stimulated cycles in patients undergoing in vitro fertilization.

OBJECTIVE: Hyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry. METHODS: For profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples. RESULTS: We detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9. CONCLUSION: We identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS.

N-glycoproteomic analysis of human follicular fluid during natural and stimulated cycles in patients undergoing in vitro fertilization / 대한생식의학회지

OBJECTIVE: Hyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry. METHODS: For profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples. RESULTS: We detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9. CONCLUSION: We identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS.

Htm1p-Pdi1p is a folding-sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation.

Our understanding of how the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the complex of the mannosidase Htm1p and the protein disulfide isomerase Pdi1p (Htm1p-Pdi1p) acts as a folding-sensitive mannosidase for catalyzing this first committed step in Saccharomyces cerevisiae We reconstitute this step in vitro with Htm1p-Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p-Pdi1p is a glycoprotein-specific mannosidase that preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p-Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

Comparative secretomic and N-glycoproteomic profiling in human MCF-7 breast cancer and HMEpC normal epithelial cell lines using a gel-based strategy.

BACKGROUND: Concurrent study of secretomic and glycoproteomic profiles in cancer cell lines represents an excellent approach for investigating cancer progression and identifying novel biomarker candidates. In this study, we performed a comparative secretomic and N-glycoprotein profiling from the secretions of normal human mammary epithelial cells (HMEpC) and the MCF-7 human breast cancer cell line. METHOD: We analyzed these cell lines using a combined methodology involving glycan-binding lectins and two-dimensional electrophoresis and identified several differentially secreted factors, including osteonectin and haptoglobin. RESULT: Notably, comparative analyses also revealed that MCF-7 cells produced differentially N-glycosylated forms of haptoglobin. CONCLUSION: The present data suggested that osteonectin and haptoglobin might have potential to be served as potential biomarkers for breast cancer. However, further investigation is needed to validate the findings.