Glycoscience aids in biomarker discovery

( Serenus Hua ),( Hyun Joo An ) 생화학분자생물학회 (구 한국생화학분자생물학회) 2012 BMB Reports Vol.45 No.6

The glycome consists of all glycans (or carbohydrates) within a biological system, and modulates a wide range of important biological activities, from protein folding to cellular communications. The mining of the glycome for disease markers represents a new paradigm for biomarker discovery; however, this effort is severely complicated by the vast complexity and structural diversity of glycans. This review summarizes recent developments in analytical technology and methodology as applied to the fields of glycomics and glycoproteomics. Mass spectrometric strategies for glycan compositional profiling are described, as are potential refinements which allow structure-specific profiling. Analytical methods that can discern protein glycosylation at a specific site of modification are also discussed in detail. Biomarker discovery applications are shown at each level of analysis, highlighting the key role that glycoscience can play in helping scientists understand disease biology. [BMB Reports 2012; 45(6): 323-330]

Development and Application of a Mouse Serum N-glycan Library for Automated Glycan Profiling and Biomarker Discovery

Serenus Hua,Inae Kang,Lauren M. Dimapasoc,Hyun Joo An 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

In vivo medical research relies primarily on the common laboratory mouse, who exhibits an inherent similarity to its fellow mammal, the human, yet is much more amenable to experimentation (for ethical, economic, and life cycle -related reasons). Numerous studies have elucidated the genome, proteome, and metabolome of the various mouse models available, but few if any have paid any attention to the mouse glycome. However, glycosylation is one of the most common eukaryotic post-translational modifications, and variations in glycosylation have been linked to cancer and other important human diseases by over fifty years of glycobiological research. Elucidation of the mouse glycome would aid glycomic and glycoproteomic biomarker studies based on the mouse model and advance our understanding of mice as models for humans. We have used retrosynthetic analysis to create a library of all possible mouse serum N-glycan compositions, based on our knowledge of the N-glycan biosynthesis pathway. Highly sensitive nano-LC/MS profiling of mouse serum N-glycans allowed us to annotate the theoretical library with experimental data, while isomer-specific nano-LC/MS/MS provided structural information. The annotated mouse serum N-glycan library was applied to automated data analysis of large sample sets for the purposes of biomarker discovery.

Glyco-Analytical Multispecific Proteolysis (Glyco-AMP): A Simple Method for Detailed and Quantitative Glycoproteomic Characterization

Hua, Serenus,Hu, Chloe Y.,Kim, Bum Jin,Totten, Sarah M.,Oh, Myung Jin,Yun, Nayoung,Nwosu, Charles C.,Yoo, Jong Shin,Lebrilla, Carlito B.,An, Hyun Joo American Chemical Society 2013 Journal of proteome research Vol.12 No.10

<P>Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site- and structure-specific information about protein glycosylation.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2013/jprobs.2013.12.issue-10/pr400442y/production/images/medium/pr-2013-00442y_0010.gif'></P>

Isomer-Specific LC/MS and LC/MS/MS Profiling of the Mouse Serum N-Glycome Revealing a Number of Novel Sialylated N-Glycans

Hua, Serenus,Jeong, Ha Neul,Dimapasoc, Lauren M.,Kang, Inae,Han, Chanyoung,Choi, Jong-Soon,Lebrilla, Carlito B.,An, Hyun Joo American Chemical Society 2013 ANALYTICAL CHEMISTRY - Vol.85 No.9

<P>Mice are the premier mammalian models for studies of human physiology and disease, bearing extensive biological similarity to humans with far fewer ethical, economic, or logistic complications. To facilitate glycomic studies based on the mouse model, we comprehensively profiled the mouse serum N-glycome using isomer-specific nano-LC/MS and -LC/MS/MS. N-Glycans were identified by accurate mass MS and structurally elucidated by MS/MS. Porous graphitized carbon nano-LC was able to separate out nearly 300 N-linked glycan compounds (including isomers) from just over 100 distinct N-linked glycan compositions. Additional MS/MS structural analysis was performed on a number of novel N-glycans, revealing the structural characteristics of modifications such as dehydration, O-acetylation, and lactylation. Experimental findings were combined with known glycobiology to generate a theoretical library of all biologically possible mouse serum N-glycan compositions. The library may be used for automated identification of complex mixtures of mouse N-glycans, with possible applications to a wide range of mouse-related research endeavors, including pharmaceutical drug development and biomarker discovery.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2013/ancham.2013.85.issue-9/ac400195h/production/images/medium/ac-2013-00195h_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac400195h'>ACS Electronic Supporting Info</A></P>

Multi-Level Characterization of Protein Glycosylation

( Serenus Hua ),( Myung Jin Oh ),( Hyun Joo An ) 한국질량분석학회 2013 Mass spectrometry letters Vol.4 No.1

Recent developments in MS-based glycomics and glycoproteomics have rapidly advanced the field and pushed the boundaries of glyco-analysis into new territories. This review will lay out current workflows and strategies for characterization of the glycoproteome, including (in order of increasing complexity and information content) preliminary site mapping, compositional glycan profiling, isomer-specific glycan profiling, glycosite-specific glycopeptide profiling, and finally, glycoproteomic profiling.

Multi-Level Characterization of Protein Glycosylation

Hua, Serenus,Oh, Myung Jin,An, Hyun Joo Korean Society for Mass Spectrometry 2013 Mass spectrometry letters Vol.4 No.1

Recent developments in MS-based glycomics and glycoproteomics have rapidly advanced the field and pushed the boundaries of glyco-analysis into new territories. This review will lay out current workflows and strategies for characterization of the glycoproteome, including (in order of increasing complexity and information content) preliminary site mapping, compositional glycan profiling, isomer-specific glycan profiling, glycosite-specific glycopeptide profiling, and finally, glycoproteomic profiling.

Designation of fingerprint glycopeptides for targeted glycoproteomic analysis of serum haptoglobin : insights into gastric cancer biomarker discovery

Jua Lee,Serenus Hua,Sung Hyeon Lee,Myung Jin Oh,JaekyungYun,Jin Young Kim,Jae-Han Kim,Jung Hoe Kim,Hyun Joo An 한국당과학회 2018 한국당과학회 학술대회 Vol.2018 No.01

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. In spite of accumulating evidence linking aberrant glycosylation and GC, site-specific localization of the glycosylation for improving clinical specificity and sensitivity is still in analytical challenge. Here, we constructed an analytical platform with targeted glycoproteomic approach for GC biomarker discovery. Unlike conventional glycomic approach characterized by untargeted MS profiling of released glycan, developed platform can be characterized by three key features: target protein-specific, glycosylation site-specific, and structure-specific platform with one-shot non-specific enzyme digestion. Serum haptoglobin (Hp) was enriched by immunoaffinity purification and subjected to pronase to generate site-specific glycopeptides. Glycopeptides were identified and quantified by nano LC/MS and -LC/MS/MS, resulting in full characterization of glycan macro- and micro-heterogeneity. A total of 96 glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Significant alterations in site-specific glycosylation have been observed in Hp in GC patients.

Comprehensive Glycan Profiling of Exosomes Derived from Several Cancer Cell Lines for Biomarker Discovery

Seunghyup Jeong,Serenus Hua,Do-Young Choi,Pyong-Gon Moon,Rudolf Grimm,Kwang Pyo Kim,Moon Chang Baek,Hyun Joo An 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1

Exosomes (microvesicles, or micropaticles) are small membrane-enclosed vesicles and these are secreted by various cell types, including tumor cells. These vesicles play an important role as mediators in extracellular communication. They are composed of membrane, cellular proteins, DNA, and RNA derived from their origin cells. It is also known that exosomes are involved in tumor metastasis, angiogenesis, and antitumor immunity. These biological functions are probably due to the glycosylation on their membrane proteins. Thus, the study of glycosylation of exosomes will be another potential source of new biomarker. However, there is a little study about the glycosylation of exosomes. Here, we targeted and analyzed N-glycans of exosomes derived from five cancer cell lines (A549, PC9, PC9/ZD, MCF-7, and MDA-MB231) using nano-LC/MS. We also have compared glycans of exosomes with glycans on their origin cell membrane to examine glycosylation correlation between origin cells and exosomes on three lung cancer cell lines. Additionally, we have compared anticancer drug resistant cell line PC9/ZD (Gefitinib resistant) and untreated PC9 in terms of origin cells and exosomes glycan profiling via isomer separation. Exosomes and cell membranes were prepared from three lung cancer cell lines: A549, PC9, and PC9/ZD. And two breast cancer exosomes were prepared: MCF-7 and MDA-MB231. Glycans were released by PNGase F, then enriched by graphitized carbon solid phase extraction. Nano-LC/chip Q-TOF MS was used for overall glycan profiling and quantitation. We successfully release and profile N-glycans from exosomes. Origin cells and exosomes of lung cancer contain high-mannose glycans in abundance. Although, exosomes have less high-mannose glycans compared with origin cells. Both origin cells and exosomes, isomer separation of sialylated glycans are different between PC9 and PC9/ZD. On breast cancer exosomes, two different matastatic samples represent quite different profiling. This is the first study of comprehensive glycan profiling of exosomes using mass spectrometry.

Designation of fingerprint glycopeptides for targeted glycoproteomic analysis of serum haptoglobin: insights into gastric cancer biomarker discovery

Lee, Jua,Hua, Serenus,Lee, Sung Hyeon,Oh, Myung Jin,Yun, Jaekyung,Kim, Jin Young,Kim, Jae-Han,Kim, Jung Hoe,An, Hyun Joo Springer-Verlag 2018 Analytical and bioanalytical chemistry Vol.410 No.6

Analytical Platform for Glycomic Characterization of Recombinant Erythropoietin Biotherapeutics and Biosimilars by Mass Spectrometry

Myung Jin Oh,Serenus Hua,Chan Young Han,Jong Shin Yoo,Hyun Joo An 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1

Erythropoietin is a therapeutic glycoprotein that stimulates red blood cell production. The quality, safety, and potency of recombinant erythropoietins are determined largely by their glycosylation. Small variations in cell culture conditions can significantly affect the glycosylation, and therefore the efficacy, of recombinant erythropoietins. Thus, detailed glycomic analyses are necessary to assess biotherapeutic quality. We have developed a platform for qualitative and quantitative glycomic analysis of recombinant erythropoietins. The platform was used to profile native N-glycans from three different type erythropoietin a common form of recombinant erythropoietin. Darbepoetin alfa was found to contain an abundance of large, multi-antennary N-glycans with high levels of sialylation, O-acetylation, and dehydration. Results were verified by independent orthogonal analysis with both MALDI-TOF and nano-LC/Q-TOF MS. This platform may be applied to quality control and batch analysis of not only recombinant erythropoietin but also other complex, glycosylated biotherapeutics and biosimilars.