Cross-identification of N-Glycans by CE-LIF using two capillary coatings and three labeling dyes.

Recombinant protein biopharmaceuticals comprise a significant portion of the current drug development landscape. The glycosylation profile of these proteins is a key quality parameter as it can affect their safety, efficacy, and stability. However, glycan analysis is challenging because of the complexity of their structures. To overcome this challenge in achieving accurate glycan identification, cross-identification of N-Glycans by CE-LIF method using two capillary coatings and three labeling dyes was developed in this work. This work explored whether complementary separation capabilities can be achieved using homemade polyvinyl alcohol (PVA) coating and commercial Guarant™ (Guarant) coating in the analysis of N-glycans. Similar separation profiles were observed using the two capillary coatings, and hence the N-glycan GU databases generated by these coatings were comparable and complementary. The performance of cross-validation by labeling with three fluorescent dyes indicated that low covariance of APTS and Turquoise™ labeling can be obtained, and hence these two labeling mechanisms provided better accuracy for the identification of glycans. Superior reproducibility with RSDs less than 1% for all target glycan standards was achieved by the internal standards (IS) method using maltodextrin ladders as additives in the separation buffer. The developed CE-LIF analysis method was applied to the identification of N-glycans in IgG samples.

N-glycan profiles of acute myocardial infarction patients reveal potential biomarkers for diagnosis, severity assessment and treatment monitoring.

Acute myocardial infarction (AMI) is a leading cause of mortality and morbidity worldwide. Diagnostic challenges remain in this highly time-sensitive condition. Using capillary electrophoresis-laser-induced fluorescence, we analyzed the blood plasma N-glycan profile in a cohort study comprising 103 patients with AMI and 69 controls. Subsequently, the data generated was subjected to classification modeling to identify potential AMI biomarkers. An area under the Receiving Operating Characteristic curve (AUCROC) of 0.81 was obtained when discriminating AMI vs. non-MI patients. We postulate that the glycan profile involves a switch from a pro- to an anti-inflammatory state in the AMI pathophysiology. This was supported by significantly decreased levels in galactosylation, alongside increased levels in sialylation, afucosylation and GlcNAc bisection levels in the blood plasma of AMI patients. By substantiating the glycomics analysis with immunoglobulin G (IgG) protein measurements, robustness of the glycan-based classifiers was demonstrated. Changes in AMI-related IgG activities were also confirmed to be associated with alterations at the glycosylation level. Additionally, a glycan-biomarker panel derived from glycan features and current clinical biomarkers performed remarkably (AUCROC = 0.90, sensitivity = 0.579 at 5% false positive rate) when discriminating between patients with ST-segment elevation MI (n = 84) and non-ST-segment elevation MI (n = 19). Moreover, by applying the model trained using glycomics information, AMI and controls can still be discriminated at 1 and 6 months after baseline. Thus, glycomics biomarkers could potentially serve as a valuable complementary test to current diagnostic biomarkers. Additional research on their utility and associated biomechanisms via a large-scale study is recommended.

High throughput human plasma N-glycan analysis using DNA analyzer and multivariate analysis for biomarker discovery.

Carbohydrates form the majority of organic compounds found in nature and their presence on proteins influences many important bioactivities. Therefore, glycan profiling shows potential in clinical applications. This work demonstrates the use of a high-throughput GlycanAssure™ sample preparation technology and multi-capillary DNA analyzer for the analysis of the major N-linked glycans (N-glycans) found in human plasma. The application involves two biomarker studies: (1) in profiling patients with chronic kidney disease and (2) in differentiating heart disease patients with normal controls in response to an antiplatelet drug from hypo-responders. Due to complexity of the study data, bio-statistical methods were applied to data processing. 37 N-glycan peaks were observed from separation results, with confirmed structure for most glycans. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to build models to differentiate the patient groups. The percentages of correct classification of the models reached 95.45% for the chronic kidney disease dataset and 85.42% for the anti-platelet drug response dataset. Given that blood N-glycan profiles had been shown to reflect certain disease states, this high-throughput platform could potentially be used for the simultaneous screening of multiple glycan biomarkers, with as little as one drop of blood sample.

Multiplexing N-glycan analysis by DNA analyzer.

Analysis of N-glycan structures has been gaining attentions over the years due to their critical importance to biopharma-based applications and growing roles in biological research. Glycan profiling is also critical to the development of biosimilar drugs. The detailed characterization of N-glycosylation is mandatory because it is a nontemplate driven process and that significantly influences critical properties such as bio-safety and bio-activity. The ability to comprehensively characterize highly complex mixtures of N-glycans has been analytically challenging and stimulating because of the difficulties in both the structure complexity and time-consuming sample pretreatment procedures. CE-LIF is one of the typical techniques for N-glycan analysis due to its high separation efficiency. In this paper, a 16-capillary DNA analyzer was coupled with a magnetic bead glycan purification method to accelerate the sample preparation procedure and therefore increase N-glycan assay throughput. Routinely, the labeling dye used for CE-LIF is 8-aminopyrene-1,3,6-trisulfonic acid, while the typical identification method involves matching migration times with database entries. Two new fluorescent dyes were used to either cross-validate and increase the glycan identification precision or simplify sample preparation steps. Exoglycosidase studies were carried out using neuramididase, galactosidase, and fucosidase to confirm the results of three dye cross-validation. The optimized method combines the parallel separation capacity of multiple-capillary separation with three labeling dyes, magnetic bead assisted preparation, and exoglycosidase treatment to allow rapid and accurate analysis of N-glycans. These new methods provided enough useful structural information to permit N-glycan structure elucidation with only one sample injection.

Normal and functional TP53 in genetically stable myxoid/round cell liposarcoma.

Myxoid/round-cell liposarcoma (MLS/RCLS) is characterized by either the fusion gene FUS-DDIT3 or the less commonly occurring EWSR1-DDIT3 and most cases carry few or no additional cytogenetic changes. There are conflicting reports concerning the status and role of TP53 in MLS/RCLS. Here we analysed four MLS/RCLS derived cell lines for TP53 mutations, expression and function. Three SV40 transformed cell lines expressed normal TP53 proteins. Irradiation caused normal posttranslational modifications of TP53 and induced P21 expression in two of these cell lines. Transfection experiments showed that the FUS-DDIT3 fusion protein had no effects on irradiation induced TP53 responses. Ion Torrent AmpliSeq screening, using the Cancer Hotspot panel, showed no dysfunctional or disease associated alleles/mutations. In conclusion, our results suggest that most MLS/RCLS cases carry functional TP53 genes and this is consistent with the low numbers of secondary mutations observed in this tumor entity.

Comparison of cancer-associated genetic abnormalities in columnar-lined esophagus tissues with and without goblet cells.

OBJECTIVE: To determine and compare the frequency of cancer-associated genetic abnormalities in esophageal metaplasia biopsies with and without goblet cells. BACKGROUND: Barrett's esophagus is associated with increased risk of esophageal adenocarcinoma (EAC), but the appropriate histologic definition of Barrett's esophagus is debated. Intestinal metaplasia (IM) is defined by the presence of goblet cells whereas nongoblet cell metaplasia (NGM) lacks goblet cells. Both have been implicated in EAC risk but this is controversial. Although IM is known to harbor genetic changes associated with EAC, little is known about NGM. We hypothesized that if NGM and IM infer similar EAC risk, then they would harbor similar genetic aberrations in genes associated with EAC. METHODS: Ninety frozen NGM, IM, and normal tissues from 45 subjects were studied. DNA copy number abnormalities were identified using microarrays and fluorescence in situ hybridization. Targeted sequencing of all exons from 20 EAC-associated genes was performed on metaplasia biopsies using Ion AmpliSeq DNA sequencing. RESULTS: Frequent copy number abnormalities targeting cancer-associated genes were found in IM whereas no such changes were observed in NGM. In 1 subject, fluorescence in situ hybridization confirmed loss of CDKN2A and amplification of chromosome 8 in IM but not in a nearby NGM biopsy. Targeted sequencing revealed 11 nonsynonymous mutations in 16 IM samples and 2 mutations in 19 NGM samples. CONCLUSIONS: This study reports the largest and most comprehensive comparison of DNA aberrations in IM and NGM genomes. Our results show that IM has a much higher frequency of cancer-associated mutations than NGM.

Applications of quantitative polymerase chain reaction protein assays during reprogramming.

The capability to reprogram human somatic cells to induced pluripotent stem cells (iPSCs) has opened a new area of biology and provides unprecedented access to patient-specific iPSCs for drug screening, disease models, and transplantation therapies. Although the process of obtaining iPSC lines is technically simple, reprogramming is a slow and inefficient process consisting of a largely uncharacterized chain of molecular events. To date, researchers have reported a wide range of reprogramming efficiencies, from <0.01% to >1%, depending on the specific reprogramming factors used, the mode of delivery of the reprogramming factors, properties of the starting cells, and culture conditions. We have applied a quantitative polymerase chain reaction methodology, TaqMan Protein Assays to directly quantify the kinetics, and cellular levels of crucial transcription factors during the reprogramming process. Further, we have used the assays to ascertain the threshold levels of reprogramming protein factors required to generate iPSC colonies, to characterize the protein expression signatures of different iPSC lines, and to rapidly identify iPS versus non-iPSC colonies based on expression of pluripotency markers. These data demonstrate that TaqMan Protein Assays can be used as tools to dissect and gain greater understanding of the mechanisms guiding reprogramming and to further characterize individual established iPSC lines.

Expanding applications of protein analysis using proximity ligation and qPCR.

The correlation of gene and protein expression changes in biological systems has been hampered by the need for separate sample handling and analysis platforms for nucleic acids and proteins. In contrast to the simple, rapid, and flexible workflow of quantitative PCR (qPCR) methods, which enable characterization of several classes of nucleic acid biomarkers (i.e. DNA, mRNA, and microRNAs), protein analysis methods such as Western blotting are cumbersome, laborious, and much less quantitative. However, TaqMan(R) Protein Assays, which use the proximity ligation assay (PLA) technology, now expand the range of qPCR applications to include the direct detection of proteins through the amplification of a surrogate DNA template after antibody binding. Here we describe an integrated qPCR approach for measuring relative changes in gene and protein expression from the same starting sample and on a single analytical platform that pairs TaqMan Gene Expression (GEx) Assays with TaqMan Protein Assays. We have monitored the changes in mRNA, microRNA, and protein expression of relevant biomarkers in the pluripotent human embryonal carcinoma cell line, NTERA2, upon differentiation to neuronal cells. In addition, TaqMan Protein Assays have been used to monitor protein expression in induced pluripotent stem cells (iPSC) that have been reprogrammed from human somatic cells. The data presented establishes a general paradigm utilizing real-time PCR instruments and reagents for studying the relationship between the stem cell transcriptome and proteome.