Carol Stroble - Academia.edu (original) (raw)

Papers by Carol Stroble

Research paper thumbnail of Protein-specific differential Glycosylation of Immunoglobulins in serum of Ovarian Cancer Patients

Journal of proteome research, Jan 27, 2016

Earlier studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian ... more Earlier studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer. However, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight in biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, n=84) and matched healthy controls (n=84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed ...

Research paper thumbnail of Serum glycans as risk markers for non-small cell lung cancer

Cancer prevention research (Philadelphia, Pa.), Jan 26, 2016

Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung... more Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung cancer. Here, we aimed to determine the predictive value of serum glycans to distinguish non-small cell lung cancer (NSCLC) cases from controls in pre-diagnostic samples using a previously validated predictive protein marker pro-SFTPB, as anchor. Blinded pre-diagnostic serum samples were obtained from the Carotene and Retinol Efficacy Trial (CARET), and included a discovery set of 100 NSCLC cases and 199 healthy controls. A second test set consisted of 108 cases and 216 controls. Cases and controls were matched for age at baseline (5-yr groups), sex, smoking status (current vs. former), study enrollment cohort and date of blood draw. Serum glycan profiles were determined by mass spectrometry. Twelve glycan variables were identified to have significant discriminatory power between cases and controls in the discovery set (AUC>0.6). Of these, four were confirmed in the independent valid...

Research paper thumbnail of A method for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins

Journal of proteome research, Jan 29, 2015

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma usi... more A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and non-specific enzyme digestions followed by nano LC-Chip/QTOF mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20-min UPLC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantita...

Research paper thumbnail of Differential N-glycosylation patterns in lung adenocarcinoma tissue

Journal of Proteome Research, 2015

To decrease the mortality of lung cancer, better screening- and diagnostic tools as well as treat... more To decrease the mortality of lung cancer, better screening- and diagnostic tools as well as treatment options are needed. Protein glycosylation is one of the major post-translational modifications that is altered in cancer, but it is not exactly clear which glycan structures are affected. A better understanding of the glycan structures that are differentially regulated in lung tumor tissue is highly desirable and will allow us to gain greater insight into the underlying biological mechanisms of aberrant glycosylation in lung cancer. Here, we assess differential glycosylation patterns of lung tumor tissue and non-malignant tissue at the level of individual glycan structures using nLC Chip/TOF MS. Using tissue samples from 42 lung adenocarcinoma patients, 29 differentially expressed (FDR <0.05) glycan structures were identified. The levels of several oligomannose type glycans were upregulated in tumor tissue. Furthermore, levels of fully galactosylated glycans, some of which were of the hybrid type and mostly without fucose, were decreased in cancerous tissue, while levels of non- or lowly galactosylated glycans mostly with fucose were increased. To further assess the regulation of the altered glycosylation, the glycomics data was compared to publicly available gene expression data from lung adenocarcinoma tissue compared to non-malignant lung tissue. The results are consistent with the possibility that the observed N-glycan changes have their origin in differentially expressed glycosyltransferases. These results will be used as a starting point for the further development of clinical glycan applications in the fields of imaging, drug targeting and biomarkers for lung cancer.

Research paper thumbnail of Detection of milk oligosaccharides in plasma of infants

Analytical and Bioanalytical Chemistry, 2014

Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are nondiges... more Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are nondigestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here, we present a procedure for the profiling of milk oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction, and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3′SL, 6′SL, 3′SLN, and 6′SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast milk. Our results indicate that free MO similar in structure to those found in human milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant.

Research paper thumbnail of Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies

Analytical and Bioanalytical Chemistry, 2014

Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with ... more Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures.

Research paper thumbnail of The serum immunoglobulin G glycosylation signature of gastric cancer

EuPA Open Proteomics, 2015

Serum N-glycan Gastritis Duodenal ulcer Nano-LC-MS a b s t r a c t Biomarkers may facilitate dete... more Serum N-glycan Gastritis Duodenal ulcer Nano-LC-MS a b s t r a c t Biomarkers may facilitate detection of gastric cancer at an earlier stage and reduce mortality.

Research paper thumbnail of Enrichment strategies in glycomics-based lung cancer biomarker development

PROTEOMICS - Clinical Applications, 2013

There is a need to identify better glycan biomarkers for diagnosis, early detection, and treatmen... more There is a need to identify better glycan biomarkers for diagnosis, early detection, and treatment monitoring in lung cancer using biofluids such as blood. Biofluids are complex mixtures of proteins dominated by a few high abundance proteins that may not have specificity for lung cancer. Therefore, two methods for protein enrichment were evaluated; affinity capturing of IgG and enrichment of medium abundance proteins, thus allowing us to determine which method yields the best candidate glycan biomarkers for lung cancer. N-glycans isolated from plasma samples from 20 cases of lung adenocarcinoma and 20 matched controls were analyzed using nLC-PGC-chip-TOF-MS (where PGC is porous-graphitized carbon). N-glycan profiles were obtained for five different fractions: total plasma, isolated IgG, IgG-depleted plasma, and the bound and flow-through fractions of protein enrichment. Four glycans differed significantly (false discovery rate, FDR < 0.05) between cases and controls in whole unfractionated plasma, while four other glycans differed significantly by cancer status in the IgG fraction. No significant glycan differences were observed in the other fractions. These results confirm that the N-glycan profile in plasma of lung cancer patients is different from healthy controls and appears to be dominated by alterations in relatively abundant proteins.

Research paper thumbnail of Abstract 4804: Glycoproteomic analysis of lung cancer malignant pleural effusions identify glycosylated proteins being produced by metastatic tumor cells

Research paper thumbnail of Protein-specific differential Glycosylation of Immunoglobulins in serum of Ovarian Cancer Patients

Journal of proteome research, Jan 27, 2016

Earlier studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian ... more Earlier studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer. However, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight in biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, n=84) and matched healthy controls (n=84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed ...

Research paper thumbnail of Serum glycans as risk markers for non-small cell lung cancer

Cancer prevention research (Philadelphia, Pa.), Jan 26, 2016

Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung... more Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung cancer. Here, we aimed to determine the predictive value of serum glycans to distinguish non-small cell lung cancer (NSCLC) cases from controls in pre-diagnostic samples using a previously validated predictive protein marker pro-SFTPB, as anchor. Blinded pre-diagnostic serum samples were obtained from the Carotene and Retinol Efficacy Trial (CARET), and included a discovery set of 100 NSCLC cases and 199 healthy controls. A second test set consisted of 108 cases and 216 controls. Cases and controls were matched for age at baseline (5-yr groups), sex, smoking status (current vs. former), study enrollment cohort and date of blood draw. Serum glycan profiles were determined by mass spectrometry. Twelve glycan variables were identified to have significant discriminatory power between cases and controls in the discovery set (AUC>0.6). Of these, four were confirmed in the independent valid...

Research paper thumbnail of A method for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins

Journal of proteome research, Jan 29, 2015

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma usi... more A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and non-specific enzyme digestions followed by nano LC-Chip/QTOF mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20-min UPLC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantita...

Research paper thumbnail of Differential N-glycosylation patterns in lung adenocarcinoma tissue

Journal of Proteome Research, 2015

To decrease the mortality of lung cancer, better screening- and diagnostic tools as well as treat... more To decrease the mortality of lung cancer, better screening- and diagnostic tools as well as treatment options are needed. Protein glycosylation is one of the major post-translational modifications that is altered in cancer, but it is not exactly clear which glycan structures are affected. A better understanding of the glycan structures that are differentially regulated in lung tumor tissue is highly desirable and will allow us to gain greater insight into the underlying biological mechanisms of aberrant glycosylation in lung cancer. Here, we assess differential glycosylation patterns of lung tumor tissue and non-malignant tissue at the level of individual glycan structures using nLC Chip/TOF MS. Using tissue samples from 42 lung adenocarcinoma patients, 29 differentially expressed (FDR <0.05) glycan structures were identified. The levels of several oligomannose type glycans were upregulated in tumor tissue. Furthermore, levels of fully galactosylated glycans, some of which were of the hybrid type and mostly without fucose, were decreased in cancerous tissue, while levels of non- or lowly galactosylated glycans mostly with fucose were increased. To further assess the regulation of the altered glycosylation, the glycomics data was compared to publicly available gene expression data from lung adenocarcinoma tissue compared to non-malignant lung tissue. The results are consistent with the possibility that the observed N-glycan changes have their origin in differentially expressed glycosyltransferases. These results will be used as a starting point for the further development of clinical glycan applications in the fields of imaging, drug targeting and biomarkers for lung cancer.

Research paper thumbnail of Abstract A41: Dasatinib regulation of N-glycans induce anticancer activity in hematological and solid tumor malignancies

Cancer Epidemiology Biomarkers & Prevention, 2012

Research paper thumbnail of Detection of milk oligosaccharides in plasma of infants

Analytical and Bioanalytical Chemistry, 2014

Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are nondiges... more Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are nondigestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here, we present a procedure for the profiling of milk oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction, and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3′SL, 6′SL, 3′SLN, and 6′SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast milk. Our results indicate that free MO similar in structure to those found in human milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant.

Research paper thumbnail of Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies

Analytical and Bioanalytical Chemistry, 2014

Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with ... more Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures.

Research paper thumbnail of The serum immunoglobulin G glycosylation signature of gastric cancer

EuPA Open Proteomics, 2015

Serum N-glycan Gastritis Duodenal ulcer Nano-LC-MS a b s t r a c t Biomarkers may facilitate dete... more Serum N-glycan Gastritis Duodenal ulcer Nano-LC-MS a b s t r a c t Biomarkers may facilitate detection of gastric cancer at an earlier stage and reduce mortality.

Research paper thumbnail of Enrichment strategies in glycomics-based lung cancer biomarker development

PROTEOMICS - Clinical Applications, 2013

There is a need to identify better glycan biomarkers for diagnosis, early detection, and treatmen... more There is a need to identify better glycan biomarkers for diagnosis, early detection, and treatment monitoring in lung cancer using biofluids such as blood. Biofluids are complex mixtures of proteins dominated by a few high abundance proteins that may not have specificity for lung cancer. Therefore, two methods for protein enrichment were evaluated; affinity capturing of IgG and enrichment of medium abundance proteins, thus allowing us to determine which method yields the best candidate glycan biomarkers for lung cancer. N-glycans isolated from plasma samples from 20 cases of lung adenocarcinoma and 20 matched controls were analyzed using nLC-PGC-chip-TOF-MS (where PGC is porous-graphitized carbon). N-glycan profiles were obtained for five different fractions: total plasma, isolated IgG, IgG-depleted plasma, and the bound and flow-through fractions of protein enrichment. Four glycans differed significantly (false discovery rate, FDR < 0.05) between cases and controls in whole unfractionated plasma, while four other glycans differed significantly by cancer status in the IgG fraction. No significant glycan differences were observed in the other fractions. These results confirm that the N-glycan profile in plasma of lung cancer patients is different from healthy controls and appears to be dominated by alterations in relatively abundant proteins.

Research paper thumbnail of Abstract 4804: Glycoproteomic analysis of lung cancer malignant pleural effusions identify glycosylated proteins being produced by metastatic tumor cells

Research paper thumbnail of Evaluation of Glycomic Profiling as a Diagnostic Biomarker for Epithelial Ovarian Cancer

Cancer Epidemiology Biomarkers & Prevention, 2014

Background: Prior studies suggested that glycans were differentially expressed in patients with o... more Background: Prior studies suggested that glycans were differentially expressed in patients with ovarian cancer and controls. We hypothesized that glycan-based biomarkers might serve as a diagnostic test for ovarian cancer and evaluated the ability of glycans to distinguish ovarian cancer cases from matched controls.

Research paper thumbnail of Protein-specific differential Glycosylation of Immunoglobulins in serum of Ovarian Cancer Patients

Journal of proteome research, Jan 27, 2016

Earlier studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian ... more Earlier studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer. However, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight in biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, n=84) and matched healthy controls (n=84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed ...

Research paper thumbnail of Serum glycans as risk markers for non-small cell lung cancer

Cancer prevention research (Philadelphia, Pa.), Jan 26, 2016

Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung... more Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung cancer. Here, we aimed to determine the predictive value of serum glycans to distinguish non-small cell lung cancer (NSCLC) cases from controls in pre-diagnostic samples using a previously validated predictive protein marker pro-SFTPB, as anchor. Blinded pre-diagnostic serum samples were obtained from the Carotene and Retinol Efficacy Trial (CARET), and included a discovery set of 100 NSCLC cases and 199 healthy controls. A second test set consisted of 108 cases and 216 controls. Cases and controls were matched for age at baseline (5-yr groups), sex, smoking status (current vs. former), study enrollment cohort and date of blood draw. Serum glycan profiles were determined by mass spectrometry. Twelve glycan variables were identified to have significant discriminatory power between cases and controls in the discovery set (AUC>0.6). Of these, four were confirmed in the independent valid...

Research paper thumbnail of A method for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins

Journal of proteome research, Jan 29, 2015

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma usi... more A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and non-specific enzyme digestions followed by nano LC-Chip/QTOF mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20-min UPLC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantita...

Research paper thumbnail of Differential N-glycosylation patterns in lung adenocarcinoma tissue

Journal of Proteome Research, 2015

To decrease the mortality of lung cancer, better screening- and diagnostic tools as well as treat... more To decrease the mortality of lung cancer, better screening- and diagnostic tools as well as treatment options are needed. Protein glycosylation is one of the major post-translational modifications that is altered in cancer, but it is not exactly clear which glycan structures are affected. A better understanding of the glycan structures that are differentially regulated in lung tumor tissue is highly desirable and will allow us to gain greater insight into the underlying biological mechanisms of aberrant glycosylation in lung cancer. Here, we assess differential glycosylation patterns of lung tumor tissue and non-malignant tissue at the level of individual glycan structures using nLC Chip/TOF MS. Using tissue samples from 42 lung adenocarcinoma patients, 29 differentially expressed (FDR <0.05) glycan structures were identified. The levels of several oligomannose type glycans were upregulated in tumor tissue. Furthermore, levels of fully galactosylated glycans, some of which were of the hybrid type and mostly without fucose, were decreased in cancerous tissue, while levels of non- or lowly galactosylated glycans mostly with fucose were increased. To further assess the regulation of the altered glycosylation, the glycomics data was compared to publicly available gene expression data from lung adenocarcinoma tissue compared to non-malignant lung tissue. The results are consistent with the possibility that the observed N-glycan changes have their origin in differentially expressed glycosyltransferases. These results will be used as a starting point for the further development of clinical glycan applications in the fields of imaging, drug targeting and biomarkers for lung cancer.

Research paper thumbnail of Detection of milk oligosaccharides in plasma of infants

Analytical and Bioanalytical Chemistry, 2014

Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are nondiges... more Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are nondigestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here, we present a procedure for the profiling of milk oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction, and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3′SL, 6′SL, 3′SLN, and 6′SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast milk. Our results indicate that free MO similar in structure to those found in human milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant.

Research paper thumbnail of Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies

Analytical and Bioanalytical Chemistry, 2014

Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with ... more Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures.

Research paper thumbnail of The serum immunoglobulin G glycosylation signature of gastric cancer

EuPA Open Proteomics, 2015

Serum N-glycan Gastritis Duodenal ulcer Nano-LC-MS a b s t r a c t Biomarkers may facilitate dete... more Serum N-glycan Gastritis Duodenal ulcer Nano-LC-MS a b s t r a c t Biomarkers may facilitate detection of gastric cancer at an earlier stage and reduce mortality.

Research paper thumbnail of Enrichment strategies in glycomics-based lung cancer biomarker development

PROTEOMICS - Clinical Applications, 2013

There is a need to identify better glycan biomarkers for diagnosis, early detection, and treatmen... more There is a need to identify better glycan biomarkers for diagnosis, early detection, and treatment monitoring in lung cancer using biofluids such as blood. Biofluids are complex mixtures of proteins dominated by a few high abundance proteins that may not have specificity for lung cancer. Therefore, two methods for protein enrichment were evaluated; affinity capturing of IgG and enrichment of medium abundance proteins, thus allowing us to determine which method yields the best candidate glycan biomarkers for lung cancer. N-glycans isolated from plasma samples from 20 cases of lung adenocarcinoma and 20 matched controls were analyzed using nLC-PGC-chip-TOF-MS (where PGC is porous-graphitized carbon). N-glycan profiles were obtained for five different fractions: total plasma, isolated IgG, IgG-depleted plasma, and the bound and flow-through fractions of protein enrichment. Four glycans differed significantly (false discovery rate, FDR < 0.05) between cases and controls in whole unfractionated plasma, while four other glycans differed significantly by cancer status in the IgG fraction. No significant glycan differences were observed in the other fractions. These results confirm that the N-glycan profile in plasma of lung cancer patients is different from healthy controls and appears to be dominated by alterations in relatively abundant proteins.

Research paper thumbnail of Abstract 4804: Glycoproteomic analysis of lung cancer malignant pleural effusions identify glycosylated proteins being produced by metastatic tumor cells

Research paper thumbnail of Protein-specific differential Glycosylation of Immunoglobulins in serum of Ovarian Cancer Patients

Journal of proteome research, Jan 27, 2016

Earlier studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian ... more Earlier studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer. However, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight in biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, n=84) and matched healthy controls (n=84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed ...

Research paper thumbnail of Serum glycans as risk markers for non-small cell lung cancer

Cancer prevention research (Philadelphia, Pa.), Jan 26, 2016

Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung... more Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung cancer. Here, we aimed to determine the predictive value of serum glycans to distinguish non-small cell lung cancer (NSCLC) cases from controls in pre-diagnostic samples using a previously validated predictive protein marker pro-SFTPB, as anchor. Blinded pre-diagnostic serum samples were obtained from the Carotene and Retinol Efficacy Trial (CARET), and included a discovery set of 100 NSCLC cases and 199 healthy controls. A second test set consisted of 108 cases and 216 controls. Cases and controls were matched for age at baseline (5-yr groups), sex, smoking status (current vs. former), study enrollment cohort and date of blood draw. Serum glycan profiles were determined by mass spectrometry. Twelve glycan variables were identified to have significant discriminatory power between cases and controls in the discovery set (AUC>0.6). Of these, four were confirmed in the independent valid...

Research paper thumbnail of A method for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins

Journal of proteome research, Jan 29, 2015

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma usi... more A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and non-specific enzyme digestions followed by nano LC-Chip/QTOF mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20-min UPLC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantita...

Research paper thumbnail of Differential N-glycosylation patterns in lung adenocarcinoma tissue

Journal of Proteome Research, 2015

To decrease the mortality of lung cancer, better screening- and diagnostic tools as well as treat... more To decrease the mortality of lung cancer, better screening- and diagnostic tools as well as treatment options are needed. Protein glycosylation is one of the major post-translational modifications that is altered in cancer, but it is not exactly clear which glycan structures are affected. A better understanding of the glycan structures that are differentially regulated in lung tumor tissue is highly desirable and will allow us to gain greater insight into the underlying biological mechanisms of aberrant glycosylation in lung cancer. Here, we assess differential glycosylation patterns of lung tumor tissue and non-malignant tissue at the level of individual glycan structures using nLC Chip/TOF MS. Using tissue samples from 42 lung adenocarcinoma patients, 29 differentially expressed (FDR <0.05) glycan structures were identified. The levels of several oligomannose type glycans were upregulated in tumor tissue. Furthermore, levels of fully galactosylated glycans, some of which were of the hybrid type and mostly without fucose, were decreased in cancerous tissue, while levels of non- or lowly galactosylated glycans mostly with fucose were increased. To further assess the regulation of the altered glycosylation, the glycomics data was compared to publicly available gene expression data from lung adenocarcinoma tissue compared to non-malignant lung tissue. The results are consistent with the possibility that the observed N-glycan changes have their origin in differentially expressed glycosyltransferases. These results will be used as a starting point for the further development of clinical glycan applications in the fields of imaging, drug targeting and biomarkers for lung cancer.

Research paper thumbnail of Abstract A41: Dasatinib regulation of N-glycans induce anticancer activity in hematological and solid tumor malignancies

Cancer Epidemiology Biomarkers & Prevention, 2012

Research paper thumbnail of Detection of milk oligosaccharides in plasma of infants

Analytical and Bioanalytical Chemistry, 2014

Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are nondiges... more Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are nondigestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here, we present a procedure for the profiling of milk oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction, and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3′SL, 6′SL, 3′SLN, and 6′SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast milk. Our results indicate that free MO similar in structure to those found in human milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant.

Research paper thumbnail of Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies

Analytical and Bioanalytical Chemistry, 2014

Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with ... more Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures.

Research paper thumbnail of The serum immunoglobulin G glycosylation signature of gastric cancer

EuPA Open Proteomics, 2015

Serum N-glycan Gastritis Duodenal ulcer Nano-LC-MS a b s t r a c t Biomarkers may facilitate dete... more Serum N-glycan Gastritis Duodenal ulcer Nano-LC-MS a b s t r a c t Biomarkers may facilitate detection of gastric cancer at an earlier stage and reduce mortality.

Research paper thumbnail of Enrichment strategies in glycomics-based lung cancer biomarker development

PROTEOMICS - Clinical Applications, 2013

There is a need to identify better glycan biomarkers for diagnosis, early detection, and treatmen... more There is a need to identify better glycan biomarkers for diagnosis, early detection, and treatment monitoring in lung cancer using biofluids such as blood. Biofluids are complex mixtures of proteins dominated by a few high abundance proteins that may not have specificity for lung cancer. Therefore, two methods for protein enrichment were evaluated; affinity capturing of IgG and enrichment of medium abundance proteins, thus allowing us to determine which method yields the best candidate glycan biomarkers for lung cancer. N-glycans isolated from plasma samples from 20 cases of lung adenocarcinoma and 20 matched controls were analyzed using nLC-PGC-chip-TOF-MS (where PGC is porous-graphitized carbon). N-glycan profiles were obtained for five different fractions: total plasma, isolated IgG, IgG-depleted plasma, and the bound and flow-through fractions of protein enrichment. Four glycans differed significantly (false discovery rate, FDR < 0.05) between cases and controls in whole unfractionated plasma, while four other glycans differed significantly by cancer status in the IgG fraction. No significant glycan differences were observed in the other fractions. These results confirm that the N-glycan profile in plasma of lung cancer patients is different from healthy controls and appears to be dominated by alterations in relatively abundant proteins.

Research paper thumbnail of Abstract 4804: Glycoproteomic analysis of lung cancer malignant pleural effusions identify glycosylated proteins being produced by metastatic tumor cells

Research paper thumbnail of Evaluation of Glycomic Profiling as a Diagnostic Biomarker for Epithelial Ovarian Cancer

Cancer Epidemiology Biomarkers & Prevention, 2014

Background: Prior studies suggested that glycans were differentially expressed in patients with o... more Background: Prior studies suggested that glycans were differentially expressed in patients with ovarian cancer and controls. We hypothesized that glycan-based biomarkers might serve as a diagnostic test for ovarian cancer and evaluated the ability of glycans to distinguish ovarian cancer cases from matched controls.