Mass Spectrometry & Proteomics Core Facility

Ms. Spec's Corner

 

Good way to start Interactome analysis?

 



Dr. Dragana Noe sharing some useful knowledge and tips you should consider before planning a new interactome analysis project. Check on the new video and her YouTube channel Ms. Spec!
You can follow Ms. Spec as she will be posting informative videos and step-by-step tutorials of different mass spectrometry protocols, data analysis, tips and more. 


 

 

Researchers In The MSPCF Spotlight
RESEARCH LEADERS
Early Career Leaders

Meet Dr. Gwenn Skar

About me: I am an Assistant Professor in the Department of Pediatrics at UNMC that specializes in infectious diseases. I did my medical school, pediatric residency and pediatric infectious disease fellowship at UNMC and was fortunate to join the faculty in 2017.

Our research: My laboratory is focused on improving the clinical care of cerebrospinal fluid shunt infections. Around 1 million people in the United States have hydrocephalus, a buildup of the cerebrospinal fluid (CSF) within the brain. This is most commonly treated by placing a CSF shunt which drains the fluid to another body site where it can be absorbed. Unfortunately, these devices can be complicated by infection which can lead to seizures and other neurologic consequences. The goal of our lab is to improve the care of individuals with shunt infection and we have two main avenues of investigation: improving diagnosis and understanding the underlying mechanisms for the neurologic consequences that are associated with shunt infections. Currently the gold standard of diagnosis for shunt infections is microbiologic culture, however, these can be falsely negative and take many days to obtain results. Our goals is to discover host biomarkers that can be used for diagnosis.

How the Mass Spec Core Helped Our Research: We have collaborated with the Mass Spec Core on a number of projects to characterize the CSF proteome during in a rodent model of cerebrospinal fluid shunt infections. Our first study demonstrated that animals with sterile and Staphylococcus epidermidis infected catheters had similar protein profiles in the CSF at day 1 post-infection, however, at day 5 post-infection there was an increase in the number of differentially expressed proteins . Of particular interest there were a number of complement proteins elevated at day 5 post-infection, indicating a potential role in the neurologic consequences seen in these infections. Additionally, we have examined the proteome in Cutibacterium acnes shunt infection and found that there are long term changes in the proteome of animals with infected shunts as far as 1 month out from infection.


Meet Dr. Premila Leiphrakpam

About me: I am a Postdoctoral researcher in the Department of Surgery-Acute Care Surgery, College of Medicine, University of Nebraska Medical Center (UNMC). I received my Ph.D. degree from UNMC in 2017. During my doctoral program my research was focused on the identification of potential signaling molecules and pathways involved in cell survival and apoptosis related to human diseases, and I was awarded program of excellence fellowship for my thesis project. In 2019, I was recruited by Dr. Keely Buesing for my expertise in basic and translational research.

My Research: Our lab has been focused on understanding the pathophysiology and molecular mechanism of wood smoke exposure-related acute lung injury, and the effect of oxygen microbubble (OMB) treatment. Since 2014, our lab has been collaborating with external researchers to investigate the therapeutic potential of OMB therapy in small and large animal models of smoke inhalation and intra-tracheal lipopolysaccharide (LPS) induced acute lung injury. My main responsibility is to establish in-vitro research projects in the lab, and my research background in both basic and translational sciences provided me the necessary expertise. We are employing proteomic and genetic approaches to identify potential molecules differentially expressed in lung tissues samples after smoke inhalation and with OMB treatment. With proteomic analysis of lung tissue samples, we have seen promising preliminary results of partial reversal of wood smoke-mediated differential global protein expression with OMB treatment.

How the Mass Spec Core Helped My research: We utilized resources available in the Mass Spectrometry & Proteomic Core Facility Core for our analyses to determine differential global protein expression in wood smoke inhalation induced lung injury and the potential of reversing these protein expressions with OMB treatment. TMT method was utilized to label our pig lung tissue samples to directly compare protein abundance between control animals and smoke exposed animals, with and without OMB treatment. These labeled samples are then analyzed by the core using their Orbitrap Fusion Lumos mass spec. Our collaboration with MSPCF have resulted in the submission of an NIH R01 grant and a research manuscript draft is under preparation for publication in a peer reviewed journal. We greatly appreciate MSPCF Core for their fast turnaround time and for providing technical expertise in troubleshooting our experiments and data analysis. We look forward to working with them in our upcoming projects.

How much protein is needed for the mass spec analysis?

 
Our mass specs need nanograms to a couple of micrograms of the peptides for those fancy looking peaks.
However, while our specs don't like too much of the material,
WE DO!
Sample preparation in our core is not a one-day process. There are many steps involved, while we are turning those proteins into peptides, and we like to start with a rather generous amount of proteins.
That being said, bring around 100 micrograms of proteins per sample, especially if you are doing TMT!


For more questions:
             mspcf@unmc.edu
Six conditions with three biological replicates, and TMT quantitation? 

                           
Now possible! 
Introducing TMT18plex
 
With 2 additional TMT channels, TMTpro-134C and TMTpro-135N, Li and co-authors showed that TMT18plex is as efficient as TMTpro16 for sample multiplexing. That means that we can quantify a set of 18 TMT-tagged samples in one run!

Read more
 

Tip Of The Month

The most POSITIVE thing to see in your mass spec project would be that clean polished NEGATIVE control

University of Nebraska Medical Center