Research Interests

Overview

We are best known for our technology development in the area of proteomics - the genome-scale study of changes in proteins. As part of our biomedical engineering effort, we have successfully applied the proteomics approach to develop important new diagnostic tests for prion disease as well as Alzheimer's disease. In partnership with collaborators, we extend the use of our proteomic approach to study relevant molecular changes in response to various treatments that are being developed and are developing techniques to better understand how medicines cross the blood-brain barrier. We have also used our approach to develop nanoscale devices for the improved separation and analysis of proteins.

More recently, we have been interested in developing technologies to improve the manufacturing of biopharmaceuticals by enhancing the production and secretion of recombinant proteins using CHO cells (the most commonly used mammalian cell line in biomanufacturing) via biomolecular & bioprocess engineering. Our work in the genomics space as part of an international consortium of CHO researchers has resulted in major accomplishments including the sequencing and assembly of Chinese hamster and CHO reference genomes, which serve as invaluable tools in these efforts. Our systems biology efforts rely on proteomic and genomic data, as well as studies of gene expression and mathematical frameworks, to develop a better understanding of these processes.

Mass Spectrometry Results: Protein Identification

Proteomics

We have always been interested in the development of next generation technologies for studying living systems. Beyond the novel bioinformatics tools we work on and the (sobaric tagging chemistries for shotgun proteomics we help report, we are also active in the application of state of the art methods to better understand the molecules that are found in living systems.

Mass spectrometry is an analytical tool for measuring the mass to charge ratio of molecules and we are fortunate to have been one of the first laboratories to employ tandem time of flight mass spectrometry (aka MALDI TOF-TOF). Our laboratory is equipped with a suite of state of the art mass spectrometers to collect information about protein mixtures. Moreover, we are fortunate to have published important papers demonstrating the use of 4plex and 8plex isobaric tagging methods for the quantitation of proteins.

While the application of existing technology is critical to our work, we are also interested in the development of next generation tools. One way in which we do this is to work in the field of nanobiotechnology. By employing the methods and tools of the semiconductor industry, we have developed micro and nanoscale devices for the separation of proteins based on photolithography. These tools have shown the ability to separate mixtures of proteins based on charge, size, affinity, and hydrophobicity and include the ability to inject separated peptides and proteins directly in the electrospray ionization source of a mass spectrometer.

Mass Spectrometers

Back to Top

Genomics

As part of an international consortium, we helped sequence the CHO genome and now are working to broadly share information related to the CHO genome. The www.CHOgenome.org international community seeks to share data freely and make tools available to support the CHO biotechnology community. More recently, we were also part of an international effort to resequence and assemble a high quality Chinese hamster genome that can serve as a stable reference point when studying genomic instability/plasticity in CHO cells. We are now using this reference genome and its substantially improved annotation to study changes in the epigenome and transcriptome that occur in response to various bioprocess-related stresses.

Genome Assembly Continuity Metrics
PICR = Newest Chinese Hamster Genome

Back to Top

Biomolecular & Bioprocess Engineering

One of the key questions that biochemical engineers face is how to control and optimize the amount and quality of a biopharmaceutical product. These products are typically recombinant proteins that are expressed either in E. coli or Chinese hamster ovary (CHO) cells. By studying changes in protein and gene expression, we previously found that one could make very subtle changes to a gene sequence and observe a substantial (nearly 10-fold) increase in the active product secreted by E. coli cells. We also have observed that making changes to the cytoskeleton of CHO cells can increase productivity by 50%. The ability to make quantitative measurements on gene expression and genome mutations has significantly accelerated metabolic and cellular engineering efforts.

More recently, we have been studying engineering methods to increase productivity in monoclonal antibody (mAb) and viral vector production processes. We aim to develop model processes and cell lines, improve site-specific integration methods, and alleviate bottlenecks caused by difficult to express antibody constructs. Some of our projects are carried out in collaboration with industrial and academic partners in the AMBIC (Advanced Mammalian Biomanufacturing Innovation Center) and CHOg2p (CHO genotype to phenotype) consortia.

Production CHO Cell Line Development

Back to Top

Biomedical Engineering

We are well known for our work on studying changes in the proteome of cerebrospinal fluid (CSF). Going back a number of years, we were involved in the development of the first antemortem diagnostic test for Creutzfeldt-Jakob disease (CJD). CJD is one of a group of prion diseases (which includes "Mad Cow disease") and in humans, this disease was previously only definitively diagnosed upon autopsy. We helped identify a protein marker in cerebrospinal fluid called 14-3-3 that is used to help evaluate patients suspected of having prion disease. More recently, we identified a panel of 23 protein spots that are indicative of the presence of Alzheimer's disease (another disease that is diagnosed definitely only upon autopsy). By studying a large group of known Alzheimer cases and other cases, we found a panel of markers that seems effective in separating these two groups. The hope is that this information can be used to better identify Alzheimer patients when they are alive. We are working to develop technology that will allow this test to be standardized and easy to use by a variety of laboratories.

More recently, in collaboration with our clinical partners at the Weill Cornell Medical Center, we have been studying CSF in subjects undergoing a new immunotherapy for Alzheimer's disease. The concept is to help the body treat Alzheimer's disease by providing them with antibodies that will reverse the cause of the disease.

One of the important limitations in the development of medicines is to have a good understanding of how the medicine crosses the blood-brain barrier. We have also been working to develop and improve on available blood-brain barrier models by studying material and cell characteristics.

2D Gel

Back to Top

Systems Biology

We are active both in bioinformatics as well as in computational systems biology. Bioinformatics efforts intersect computer science with life science and computational systems biology efforts intersect applied mathematics with life science. In the proteomics space, we have been involved in the development of bioinformatic tools that facilitate the analysis of shotgun proteomics datasets such as those obtained using isobaric tagging reagents. We developed a package called iTRAQPak which facilitates visualization of such datasets and addresses important issues related to protein versus peptide measurements during large scale shotgun proteomics studies.

We also have an interest in developing mathematical frameworks to describe molecular processes inside cells. More specifically, we are interested in the relationship between DNA sequence, mRNA expression, and protein expression in cells on a genome-wide scale. We developed a framework that predicts protein expression changes genome-wide when changes in mRNA expression are measured and when DNA sequence is known. The balance between modeling and experiment is key to our approach.

iTraq Labeling
Image Source: www.creative-proteomics.com

Back to Top