Proteomics and bio analytical mass spectometry research

  • Global and targeted discovery of protein-protein/protein ligand interactions by chemical cross-linking and mass spectrometry
  • Identification and quantitative characterization of protein posttranslational modifications(PTMs)
  • Elucidation of protien structures by mass spectrometry
  • Quantitative Proteomics(bio-markers discovery)

Protein-protein interactions and protein posttranslational modifications (PTMs) are the major events which regulate signaling cascades in the cells. In disease states, these interaction networks get altered, disrupting normal cellular processes. Understanding the overall network “interactome” or a particular receptor’s interaction network, in diseased and normal states, will provide insight into the physiological and pathological processes of those diseases at a molecular level. Mass spectrometer is a major instrument for identifying and quantifying proteins, but deciphering these large-scale interaction networks in vivo is a major challenge in functional proteomics research. Due to current remarkable advancements in mass spectrometry instrumentations, there is a great opportunity to develop mass spectrometry-based quantitative and chemical proteomics strategies that can give us precise information about signaling complexes in vivo or in vitro. The ‘proteomics and bio-analytical mass spectrometry’ group at UT Arlington focuses on the development of cutting-edge mass spectrometry-supportive novel quantitative and chemical proteomics methods and tools, as well as the application of these methods for proteome-wide identification of protein expressions, protein-protein interactions and PTMs. My laboratory focuses on mass spectrometry-based method development in the following specific areas of proteome research in order to study environmental diseases impacted by innate immunity.

1. Global and targeted discovery of protein-protein/protein-ligand interactions by antibody/affinity based enrichment methods in combination with novel mass spectrometry-supportive chemical cross-linking approaches.

2. Identification and quantitative characterization of protein posttranslational modifications (PTMs) by mass spectrometry- supportive chemical probes.

3. Elucidation of protein structures by mass spectrometry.

4. Quantitative proteomics studies for the discovery and validation of cellular protein targets (bio-signatures or bio-markers) The focused biological application of my research is to understand the role of lipid rafts and Toll-like Receptors (TLRs) in inflammatory signaling pathways. We focus on studying several environmental diseases impacted by innate immunity, such as atherosclerosis, sepsis, asthma, etc. by mass spectrometry-based novel proteomics tools. In addition, the approaches we develop can be generally applied to any biological systems; hence we also welcome researchers to initiate collaborative research efforts with us.

Current biochemical methods are not very efficient to analyze system-level or large-scale protein interaction networks. Most of the studies utilize a technique called “co-immunoprecipitation,” where a protein is isolated along with its interacting partners (protein complexes) by using an antibody or by incorporating an affinity group in the protein which can be used as a hook to selectively purify that protein. This method is applicable for very strong and stable interactions, but most of the cellular interactions are very transient and weak, and during the purification process these interactions get lost completely. Besides, this identification is very qualitative and does not put emphasis on the protein-to-protein interaction domain. To solve this very important problem, one chemistry-based fixation method combined with mass spectrometry technology has come into limelight wherein a reactive compound, called cross-linker, is utilized to stabilize proteins with its interaction partners (protein complexes) by using certain side chain of proteins (proteins are made of 20 amino acids) before performing cell lysis. Cross-linker can fix the nearby proteins or protein complexes by chemical reactions and hold them tightly so they will not detach after cell lysis and will not be affected by the subsequent strict purification conditions. In addition, cross-linker reacts within a limited distance; hence, protein reactive sites can be measured by calculating the distances of the reactive sites.
This method has two advantages:
1) it can identify large-scale protein interactions.
2) it can identify protein structures in their native biological conditions. The universal use of this technology is hindered due to several bottlenecks. Traditional cross-linking strategies generate enormous amount of mass spectrometry data which is extremely difficult to analyze by routine software tools. Finding these interactions in large data is equal to finding a needle in a haystack.
To make this technology very amenable for analyzing large-scale protein interactions, two areas of research are very important:
1) Design of the effective chemical cross-linker with innovative features, which will help reduce the complexity of mass-spectrometry data of large-scale protein interactions.
2) Develop easy to analyze software tools.

[Source: Anal Chem. 2009 Jul 1;81(13):5524-32. doi: 10.1021/ac900853k. ]

Lipid posttranslational modifications

Proteolytic cleavage

[Source:Anal Chem. 2015 Feb 17;87(4):2178-86. doi: 10.1021/ac503794s. Epub 2015 Feb 5.
Rapid Commun Mass Spectrom. 2014 Mar 30;28(6):635-44. doi: 10.1002/rcm.6820.]

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