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2. Screening of glycosylation patterns in serum using natural glycoprotein microarrays and multi-lectin fluorescence detection

Author

Patwa, Tasneem H., Zhao, Jia, Anderson, Michelle A., Simeone, Diane M., and Lubman, David M.

Subjects
Serum -- Properties, Serum -- Research, Glycoproteins -- Structure, Glycoproteins -- Research, Glycosylation -- Analysis, Fluorescence -- Analysis, Chemistry
Abstract

Protein glycosylation has been implicated in key biological processes including immunological recognition, cellular adhesion, protein folding, and signaling as well as disease progression. Although several methods are available to assess glycosylation of protein structures, none of them is able to screen complex biological samples at a global as well as an individual scale. A novel strategy presented here uses an all-liquid phase enrichment and prefractionation methodology coupled to glycoprotein microarray technology using a multiple lectin-based, biotin--streptavidin detection scheme. Selective detection of glycan structures was made possible by employing multiple lectins to screen glycoprotein standards as well as serum samples from normal subjects or patients with chronic pancreatitis or pancreatic cancer. Interestingly, in some instances, a greater degree of glycosylation was seen in proteins that were underexpressed based on the reversed-phase chromatogram alone. Studies with standard proteins established the limits of detection to be in the 2.5-5-fmol range. Studies on serum samples showed differences in glycosylation patterns, particularly with respect to sialylation, mannosylation, and fucosylation, in normal, pancreatitis, and cancer sera. By coupling glycoprotein enrichment and fractionation with a microarray platform, we have shown that naturally occurring glycoproteins from human serum can be screened and characterized for different glycan structures, thereby allowing one to do comparative studies that monitor individual glycosylation changes within a glycoproteome representing different biological states. This approach may be useful to identify potential biomarkers in cancer.

Published
2006

6. The identification of phosphoglycerate kinase-1 and histone H4 autoantibodies in pancreatic cancer patient serum using a natural protein microarray

Author

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA, Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA, Department of Surgery, University of Michigan, Ann Arbor, MI, USA ; Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA, Department of Chemistry, University of Michigan, Ann Arbor, MI, USA ; Department of Surgery, University of Michigan, Ann Arbor, MI, USA ; Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, USA ; Department of Surgery, University of Michigan, 1150 West Medical Center Drive, Room A510B MSRB I, Ann Arbor, MI 48109-0656, USA Fax: +1-734-615-2088, Department of Statistics, Pennsylvania State University, University Park, PA, USA ; Department of Public Health Sciences, Penn State, University Park, PA, USA, Patwa, Tasneem H., Li, Chen, Poisson, Laila M., Kim, Hye-Yeung, Pal, Manoj, Ghosh, Debashis, Simeone, Diane M., Lubman, David M., Department of Chemistry, University of Michigan, Ann Arbor, MI, USA, Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA, Department of Surgery, University of Michigan, Ann Arbor, MI, USA ; Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA, Department of Chemistry, University of Michigan, Ann Arbor, MI, USA ; Department of Surgery, University of Michigan, Ann Arbor, MI, USA ; Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, USA ; Department of Surgery, University of Michigan, 1150 West Medical Center Drive, Room A510B MSRB I, Ann Arbor, MI 48109-0656, USA Fax: +1-734-615-2088, Department of Statistics, Pennsylvania State University, University Park, PA, USA ; Department of Public Health Sciences, Penn State, University Park, PA, USA, Patwa, Tasneem H., Li, Chen, Poisson, Laila M., Kim, Hye-Yeung, Pal, Manoj, Ghosh, Debashis, Simeone, Diane M., and Lubman, David M.

Abstract

Protein microarrays have been used to explore whether a humoral response to pancreatic cancer-specific tumor antigens has utility as a biomarker of pancreatic cancer. To determine if such arrays can be used to identify novel autoantibodies in the sera from pancreatic cancer patients, proteins from a pancreatic adenocarcinoma cell line (MIAPACA) were resolved by 2-D liquid-based separations, and then arrayed on nitrocellulose slides. The slides were probed with serum from a set of patients diagnosed with pancreatic cancer and compared with age- and sex-matched normal subjects. To account for patient-to-patient variability, we used a rank-based non-parametric statistical testing approach in which proteins eliciting significant differences in the humoral response in cancer compared with control samples were identified. The prediction analysis for microarrays classification algorithm was used to explore the classification power of the proteins found to be differentially expressed in cancer and control sera. The generalization error of the classification analysis was estimated using leave-one-out cross-validation. A serum diagnosis of pancreatic cancer in this set was predicted with 86.7% accuracy, with a sensitivity and specificity of 93.3 and 80%, respectively. Candidate autoantibody biomarkers identified using this approach were studied for their classification power by performing a humoral response experiment on recombinant proteins using an independent sample set of 238 serum samples. Phosphoglycerate kinase-1 and histone H4 were noted to elicit a significant differential humoral response in cancer sera compared with age- and sex-matched sera from normal patients and patients with chronic pancreatitis and diabetes. This work demonstrates the use of natural protein arrays to study the humoral response as a means to search for the potential markers of cancer in serum.

Published
2009

7. A novel phosphoprotein analysis scheme for assessing changes in premalignant and malignant breast cell lines using 2-D liquid separations, protein microarrays, and tandem mass spectrometry

Author

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA, Department of Internal Medicine, Division of Nephrology, University of Michigan Medical Center, Ann Arbor, MI, USA, Department of Chemistry, University of Michigan, Ann Arbor, MI, USA ; Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA ; Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, MI, USA ; Department of Surgery, The University of Michigan Medical Center, MSRB1, Rm A510B, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0656, USA Fax: +1-734-615-2088, Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA, Department of Surgery, University of Florida, Jacksonville, FL, USA, Eprogen Inc., Darien, IL, USA, Patwa, Tasneem H., Wang, Yanfei, Miller, Fred R., Goodison, Steve, Pennathur, Subramaniam, Barder, Timothy J., Lubman, David M., Department of Chemistry, University of Michigan, Ann Arbor, MI, USA, Department of Internal Medicine, Division of Nephrology, University of Michigan Medical Center, Ann Arbor, MI, USA, Department of Chemistry, University of Michigan, Ann Arbor, MI, USA ; Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA ; Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, MI, USA ; Department of Surgery, The University of Michigan Medical Center, MSRB1, Rm A510B, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0656, USA Fax: +1-734-615-2088, Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA, Department of Surgery, University of Florida, Jacksonville, FL, USA, Eprogen Inc., Darien, IL, USA, Patwa, Tasneem H., Wang, Yanfei, Miller, Fred R., Goodison, Steve, Pennathur, Subramaniam, Barder, Timothy J., and Lubman, David M.

Abstract

An analysis of phosphorylation changes that occur during cancer progression would provide insights into the molecular pathways responsible for a malignant phenotype. In this study we employed a novel coupling of 2-D liquid separations and protein microarray technology to reveal changes in phosphoprotein status between premalignant (AT1) and malignant (CA1a) cell lines derived from the human MCF10A breast cell lines. Intact proteins were first separated according to their p I and hydrophobicities, then arrayed on SuperAmine glass slides. Phosphoproteins were detected using the universal, inorganic phospho-sensor dye, Pro-Q Diamond. Using this dye, out of 140 spots that were positive for phosphorylation, a total of 85 differentially expressed spots were detected over a pH range of 7.2–4.0. Proteins were identified and their peptides sequenced by MS. The strategy enabled the identification of 75 differentially expressed phosphoproteins, from which 51 phosphorylation sites in 27 unique proteins were confirmed. Interestingly, the majority of differentially expressed phosphorylated proteins observed were nuclear proteins. Three regulators of apoptosis, Bad, Bax, and Acinus, were also differentially phosphorylated in the two cell lines. Further development of this strategy will facilitate an understanding of the mechanisms involved in malignancy progression and other disease-related phenotypes.

Published
2009

8. Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

Author

Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA ; Amgen Inc., Chemistry Research and Discovery, Analytical Chemistry, One Amgen Center Drive, Mail Stop 25-2-A, Thousand Oaks, CA 91 320., Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA, Department of Pathology, The University of Michigan Medical Center, Ann Arbor, MI 48109, USA, Department of Surgery, The University of Michigan Medical Center, Ann Arbor, MI 48109, USA ; Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA ; Department of Pathology, The University of Michigan Medical Center, Ann Arbor, MI 48109, USA ; Comprehensive Cancer Center, The University of Michigan Medical Center, Ann Arbor, MI 48109, USA ; University of Michigan Medical Center, Department of Surgery, MSRBI, A510B, Box 0658, Ann Arbor, MI 48109, USA., Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA, Department of Chemistry, Instrumental Analysis and Bioanalysis, Saarland University, 66123 Saarbrucken, Germany, Yoo, Chul, Patwa, Tasneem H., Kreunin, Paweena, Miller, Fred R., Huber, Christian G., Nesvizhskii, Alexey I., Lubman, David M., Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA ; Amgen Inc., Chemistry Research and Discovery, Analytical Chemistry, One Amgen Center Drive, Mail Stop 25-2-A, Thousand Oaks, CA 91 320., Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA, Department of Pathology, The University of Michigan Medical Center, Ann Arbor, MI 48109, USA, Department of Surgery, The University of Michigan Medical Center, Ann Arbor, MI 48109, USA ; Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA ; Department of Pathology, The University of Michigan Medical Center, Ann Arbor, MI 48109, USA ; Comprehensive Cancer Center, The University of Michigan Medical Center, Ann Arbor, MI 48109, USA ; University of Michigan Medical Center, Department of Surgery, MSRBI, A510B, Box 0658, Ann Arbor, MI 48109, USA., Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA, Department of Chemistry, Instrumental Analysis and Bioanalysis, Saarland University, 66123 Saarbrucken, Germany, Yoo, Chul, Patwa, Tasneem H., Kreunin, Paweena, Miller, Fred R., Huber, Christian G., Nesvizhskii, Alexey I., and Lubman, David M.

Abstract

A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 µg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis. Copyright © 2007 John Wiley & Sons, Ltd.

Published
2007

12. Plasma Glycoprotein Profiling for Colorectal Cancer Biomarker Identification by Lectin Glycoarray and Lectin Blot

Author

Qiu, Yinghua, primary, Patwa, Tasneem H., additional, Xu, Li, additional, Shedden, Kerby, additional, Misek, David E., additional, Tuck, Missy, additional, Jin, Gracie, additional, Ruffin, Mack T., additional, Turgeon, Danielle K., additional, Synal, Sapna, additional, Bresalier, Robert, additional, Marcon, Norman, additional, Brenner, Dean E., additional, and Lubman, David M., additional

Published
2008
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19. An Integrated Approach Utilizing Liquid Separations, Protein Microarrays and Tandem Mass Spectrometry Towards Understanding Phosphorylation, Glycosylation and Humoral Response Changes in Cancer.

Author

Patwa, Tasneem H.

Subjects
Protein Microarrays, Protein Phosphorylation and Glycosylation, Humoral Response
Abstract

The development and application of an integrated approach that utilizes liquid separations, protein microarrays and mass spectrometry with the goal of understanding breast, pancreatic and colon cancer progression is described. In order to understand breast cancer progression, two liquid separation techniques for the generation of protein microarrays from pre-malignant and malignant breast cancer cell lines which were subsequently probed for phosphoproteins was pursued. Out of 140 positively phosphorylated array spots, 85 were differentially phosphorylated. This corresponded to 75 unique proteins. 51 phosphorylation sites were identified by tandem MS/MS in 27 of these proteins. Phosphorylation changes were dominant in proteins involved in transcriptional and translational control as well as apoptosis and cytoskeletal regulation. A strategy for global glycoprotein profiling in serum and plasma was also developed. Glycoprotein microarrays of enriched and separated N-linked glycoproteins from serum and plasma from normal controls and patients with pancreatic cancer and colorectal cancer were generated. The specific sugar moieties on glycans were assessed using biotinylated lectins coupled with Alexa-flor conjugated streptavidin. In both cancers sialylation and fucosylation changes were dominant. Principal component analysis and hierarchical clustering analysis showed that this strategy successfully classified serum/plasma groups based on overall glycan expression profiles. The utility of protein microarrays for identifying potential markers of pancreatic cancer was also explored. Proteins from MIAPACA, a pancreatic cancer cell line, were fractionated in 2 dimensions and arrayed on nitrocellulose slides. The slides were hybridized with multiple serum samples from normal controls and patients diagnosed with pancreatic cancer in order to find specific cancer proteins that elicited an antigen-antibody response to the serum samples. Wilcoxon rank sum tests followed by the PAM classification algorithm highlighted 9 fractions that together classified serum samples with 87% accuracy and sensitivity and specificity of 93% and 80% respectively. Further validation experiments with recombinant proteins showed good correlation for two of the identified proteins, phosphoglycerate kinase and histone H4. A modified arraying technique utilizing CNBr digestion was also successful, suggesting that exposure of binding sites to serum during hybridization is critical in maximizing the sensitivity of the microarray.

Published
2008

20. All-liquid separations, protein microarrays, and mass spectrometry to interrogate serum proteomes: an application to serum glycoproteomics.

Author

Patwa TH, Qiu Y, Zhao J, Simeone DM, and Lubman DM

Subjects
Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Glycosylation, Humans, Lectins isolation & purification, Membranes, Artificial, Peptide Mapping, Porosity, Trypsin metabolism, Blood Proteins analysis, Glycoproteins blood, Mass Spectrometry, Protein Array Analysis methods, Proteome analysis, Proteomics methods
Abstract

Disease-related changes in serum proteins are reasonable targets for early detection particularly due to the noninvasive approach in obtaining samples. Glycoproteins specifically have been implicated in a variety of disease types ranging from immune diseases to cancers. High-throughput screening methods that can assess glycosylation states of all serum proteins in normal and diseased sample groups can facilitate early detection as well as shed light on disease progression mechanisms. Outlined here is a combination of liquid separation, protein microarray, and mass spectrometry approach to highlight candidate proteins involved in diseases through glycosylation mechanisms.

Published
2009
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21. Protein biomarkers in cancer: natural glycoprotein microarray approaches.

Author

Zhao J, Patwa TH, Lubman DM, and Simeone DM

Subjects
Animals, Biomarkers, Tumor metabolism, Glycoproteins chemistry, Glycoproteins metabolism, Glycosylation, Humans, Lectins analysis, Models, Biological, Neoplasms diagnosis, Biomarkers, Tumor analysis, Glycoproteins analysis, Neoplasms metabolism, Protein Array Analysis methods
Abstract

Protein glycosylation is the most versatile and common protein modification and plays important roles in various biological processes and disease progression. In this review, the development of microarray technology for protein glycosylation analysis is described. Three types are discussed: carbohydrate, lectin and natural glycoprotein microarrays. The advantages of microarray technology to study protein glycosylation are high-volume throughput coupled with a highly miniaturized platform. These techniques show great promise for detecting interactions that involve carbohydrates and as a screening tool to detect glycan patterns important for the early diagnosis of disease.

Published
2008

22. A novel phosphoprotein analysis scheme for assessing changes in premalignant and malignant breast cell lines using 2D liquid separations, protein microarrays and tandem mass spectrometry.

Author

Patwa TH, Wang Y, Miller FR, Goodison S, Pennathur S, Barder TJ, and Lubman DM

Abstract

An analysis of phosphorylation changes that occur during cancer progression would provide insights into the molecular pathways responsible for a malignant phenotype. In this study we employed a novel coupling of 2D-liquid separations and protein microarray technology to reveal changes in phosphoprotein status between premalignant (AT1) and malignant (CA1a) cell lines derived from the human MCF10A breast cell lines. Intact proteins were first separated according to their isoelectric point and hydrophobicities, then arrayed on SuperAmine glass slides. Phosphoproteins were detected using the universal, inorganic phospho-sensor dye, ProQ Diamond. Using this dye, out of 140 spots that were positive for phosphorylation, a total of 85 differentially expressed spots were detected over a pH range of 7.2 to 4.0. Proteins were identified and their peptides sequenced by mass spectrometry. The strategy enabled the identification of 75 differentially expressed phosphoproteins, from which 51 phosphorylation sites in 27 unique proteins were confirmed. Interestingly, the majority of differentially expressed phosphorylated proteins observed were nuclear proteins. Three regulators of apoptosis, Bad, Bax and Acinus, were also differentially phosphorylated in the two cell lines. Further development of this strategy will facilitate an understanding of the mechanisms involved in malignancy progression and other disease-related phenotypes.

Published
2008
Full Text
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23. Differential expression of acidic proteins with progression in the MCF10 model of human breast disease.

Author

Buchanan NS, Zhao J, Zhu K, Patwa TH, Miller FR, and Lubman DM

Subjects
Chromatography, High Pressure Liquid, Disease Progression, Electrophoresis, Gel, Two-Dimensional, Humans, Isoelectric Focusing, Peptide Mapping, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Biomarkers, Tumor metabolism, Breast Neoplasms metabolism, Neoplasm Proteins metabolism, Proteome analysis
Abstract

A proteomic characterization of one premalignant (MCF10AT1) and two malignant (MCF10CA1a and MCF10 CA1d) human breast cancer cell lines has been performed using a combination of two-dimensional liquid separations and mass spectrometry. Chromatofocusing (CF) and NPS-RP-HPLC are combined with ESI-TOF-MS to resolve and detect intact proteins. Simultaneously, fractions are collected and digested for protein identification using MALDI-MS peptide mass fingerprinting. Following protein identification a small database was compiled for use in comparison between IDs and measured masses taking into account variables such as pI, hydrophobicity and potential modifications. Out of 239 mass bands detected between pH 4.6 and 6.0, 133 have been definitively associated with identified proteins and 67 show consistent up/down regulation in two malignant breast cancer cell lines relative to the precursor premalignant cell line. Of these, 8 are also altered in the premalignant MCF10AT1 cell line by treatment with estradiol. Differentially expressed proteins indicate significant changes to the cytoskeleton, cellular metabolism, and adaptation to environmental stressors in malignant cell lines.

Published
2007

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