Bio Patrika interviews Dr. Sethi on her thoughts about “Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis”

Dr. Manveen K Sethi’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. Sethi serves as Instructor of Biochemistry at the Boston University School of Medicine, USA. She did her Ph.D. at Macquarie University (MQ), Australia, under the primary supervision of Dr. Morten Thaysen-Andersen, an internationally recognized researcher in Glycobiology. During her Ph.D., she forayed into colorectal cancer (CRC) research. After her Ph.D., she was offered a postdoctoral research fellowship at Boston University School of Medicine (BUSM), USA, under Prof. Joseph Zaia, an internationally-acclaimed leader in mass spectrometry (MS) and Glycoscience, where she is currently employed in a research faculty-track position of Instructor. Her research work involves identifying and characterizing biomolecules such as proteins and glycans using mass spectrometry techniques and utilizing this information to understand biomolecular deregulation in human diseases, such as cancer and Alzheimer’s disease. Here, Manveen talks about her work titled “Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis” published as a first author in Molecular Omics (2020).

How would you explain your paper’s key results to the non-scientific community?

This article utilized sophisticated and holistic mass spectrometry glycomics and proteomics profiling together to determine the structural changes in key brain extracellular matrix (ECM) biomolecules such as glycosaminoglycans (GAGs), including hyaluronan (HA), chondroitin sulfate (CS), heparan sulfate (HS), proteoglycans (PGs), glycoproteins, and proteins, known to play a pivotal role in in the neuropathophysiology. This project’s critical and novel aspect is that it integrates glycomics and proteomics to analyze multiple classes of biomolecules, including the overlooked GAGs and PGs that are analytically challenging to study due to their high sulfation and multi anionic nature. The In solution digestion protocol presented here is an analytical workflow that follows a filter-aided samples preparation (FASP) type sequential in-solution exhaustive digestion of tissue samples using multiple enzymes, releasing different biomolecules at each step that could be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as shown in the figure below. In the current paper, we used mouse brain lysate and various standard PGs and proteins to target different biomolecules. Each biomolecule required overnight incubation and thus, took one day of sample processing time. We identified HA, CS, and HS unsaturated and saturated disaccharides from the standard proteoglycans and complex samples. In addition, proteoglycans including syndecan and neurocan were identified with good coverage, and mouse brain tissue lysate identified about 1500 and 800 proteins, with 1 and 2 unique peptides, respectively.

“The protocol could be used for simultaneous processing of 24 samples at a given time despite a five day processing time that yields four different biomolecules.”

What are the possible consequences of these findings for your research area?

The protocol is an efficient, high-throughput, economical, and robust way of processing brain tissues. The protocol could be used for simultaneous processing of 24 samples at a given time despite a five day processing time that yields four different biomolecules. The overarching aim here is to establish a streamlined scaffold for the identifying, and characterizing important ECM-related biomolecules in different neuropsychiatric disorders to understand their underlying molecular functions in neuropathology.

What was the exciting moment (eureka moment) during your research?

Performing LC-MS/MS analysis could be challenging, and highly sulfated and anionic molecules like GAGs make it a much daunting task. The most exciting moment for us was to capture, identify, and characterize each biomolecule at each step of the protocol and then reproduce the process with a different batch one week apart.

What do you hope to do next?

I plan to utilize this protocol to explore of the structure and biology of PGs and GAGs in neurodegenerative disorders, such as Alzheimer’s disease (AD). The end goal is to improve our understanding of neuropathogenesis and provide markers and therapeutics drug targets for brain disorders.

Where do you seek scientific inspiration?

I have been fortunate to be trained and mentored by renowned experts and leaders in the field of Mass Spectrometry, proteomics, and Glycoscience, including Dr. Morten Thaysen-Andersen, Dr. Susan Fanayan, Prof. Nicolle H. Packer, Prof. Mark Baker, Prof. William S. Hancock, Prof. Joseph Zaia, and Prof. Catherine Costello. You get inspired every day when surrounded by such super achievers, curious minds, and humble and modest scientists. Notably, the women leaders like Prof. Nicolle H. Packer, and Prof. Catherine Costello, who have laid the foundation for young women scientists like me, and have taught me that a balance in scientific and personal life is essential to embrace life both as a scientist and a woman with dedication and diligence.

How do you intend to help Indian science improve?

A basic essence of science and research is to collaborate. Indian science and scientists are incredible, with limited financial and resources compared to the West, they have shown phenomenal growth. I wish to collaborate with peers from the Indian science community, and I hope that by this little contribution by collaboration, I will assist in improving Indian science.

Reference

Sethi MK, Downs M, Zaia J. Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis. Mol Omics. 2020 Aug 1;16(4):364-376. doi: 10.1039/d0mo00019a. Epub 2020 Apr 20. PMID: 32309832; PMCID: PMC7423595.

Email: msethi@bu.edu

Website: https://www.bumc.bu.edu/biochemistry/profiles/manveen-sethi/

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s

%d bloggers like this: