Lab on a Chip Blog

Researchers at York University in Toronto have developed an integrated microfluidic device to facilitate analysis of modified protein structures after biochemical reactions.  

The platform guides a protein right through from a controlled reaction, functional labeling, and fragmentation zones to a coupled mass spectrometer for determination of the protein’s secondary structure. Using this device, the group conducts the first dynamic structural analysis of the β-lactamase enzyme TEM-1 to identify key regions of the enzyme that affect catalytic activity.  TEM-1 is the enzyme that provides bacterial resistance to β-lactam antibiotics.

Protein binding events crucial to protein function can change protein shape directly at the binding site or through another site (an allosteric mechanism). One example of allostery is when the binding of oxygen to one heme site in hemoglobin increases oxygen affinity at other heme sites due to structural changes. For hemoglobin, functional labeling during binding events and mass spectrometry (MS) protein structure analysis was used to find the 3 sets of residues responsible for the allosteric mechanism at each heme site.¹ The integrated microfluidic platform described here by Derek J. Wilson’s group enables similar experiments with faster sample transfer to electrospray ionization for MS analysis.

This integrated microfluidic platform first incubates the protein of interest with a reactant for a controlled time. Then, the amide protons of the backbone are functionally labeled with deuterium and the labeled protein is digested in a pepsin-functionalized region. Finally, the fragments are ionized and transferred into a time-of-flight mass spectrometer (Q-TOF MS). Labeled peptides are quickly delivered to the spectrometer resulting in high retention of the proton label.

Evaluating TEM-1 after incubation with its inhibitor clavulanate, Wilson’s group notes that regions of the protein which undergo slow changes in deuterium uptake correspond to allosteric transitions and occur on the protein periphery. Rapid changes occur to reposition specific residues, “loosening” some structures and “locking up” others near the active site.

These measurements are the first to analyze TEM-1 inhibition dynamics and demonstrate the great utility of this microfluidic platform to facilitate exploration of allosteric mechanisms and other dynamic structural changes affecting protein function.

1. I. A. Kaltashov and S. J. Eyles, in Mass Spectrometry on the Frontiers of Molecular Biophysics and Structural Biology: Perspectives and Challenges, Wiley Online Library, 2012, pp.186-208.

An electrospray MS-coupled microfluidic device for sub-second hydrogen/deuterium exchange pulse-labelling reveals allosteric effects in enzyme inhibition
Tamanna Rob, Preet Kamal Gill, Dasantila Golemi-Kotra, and Derek J. Wilson
DOI: 10.1039/C3LC00007A

Sasha is a PhD student at Stanford University working with Professor Beth Pruitt’s Microsystems Lab. Her research interests focus on designing microscale devices for studying cell mechanobiology and the biophysical underpinnings of cell-cell and cell-substrate interactions.

Researchers at the University of Illinois led by Brian Cunningham use an iPhone camera as a spectrometer to detect biomolecules.

A custom-designed cradle holds the smartphone in place so that all of the optical components are perfectly aligned for detection in the path of a photonic crystal biosensor made on a plastic substrate.

The smartphone’s computational capabilities and simple user interface enable it to guide a user through the steps of the assay using touchscreen commands via an iPhone App. The phone processes the image converting a sequence of photos into a spectrum and then converts this spectrum into a value for the resonant wavelength of the photonic crystal.

The team are now working to expand the range of assays possible. They envision that this device could be made even more practical by incorporating microfluidic channels for wet samples.

Label-free biodetection using a smartphone
Dustin Gallegos, Kennet D. Long, Hojeong Yu, Peter P. Clark, Yixiao Lin, Sherine George, Pabitra Natha and Brian T. Cunningham
DOI: 10.1039/C3LC40991K

Do you know how chemical scientists can tackle global challenges in Human Health? If so, the RSC is running a one minute video competition this summer for young researchers such as PhD and Post-doc students; get involved and innovate the way scientists share their research. Your video should communicate your own personal research or an area of research that interests you, highlighting its significance and impact to Human Health.

Five videos will be shortlisted by our judging panel and the winner will be selected during the ‘How does chemistry keep us healthy?’ themed National Chemistry Week taking place 16-23 November. 

A £500 prize and a fantastic opportunity to shadow the award winning video Journalist, Brady Harran, is up for grabs for the winner.

The judging panel will include the makers of The Periodic Tale of Videos, Martyn Poliakoff and Brady Harran, and RSC Division representatives.

Check out our webpage for further details of the competition and an example video. 

The competition will open 02 April 2013 and the closing date for entries is 01 July 2013. Please submit your entries to rsc.li/take-1-video-competition.