JoVE Guest Blog Post – Visualising Scientific Experiments

JoVE October blog

This is the first in a series of guest posts from JoVE, the Journal of Visualized Experiments. Each month we’ll be featuring a different peer-reviewed video article with insights from their team. Like Mendeley, JoVE is using technology to make science more open, and we were really happy to have their Director of Marketing Rachel Greene join us at Social Media Week London for the Mendeley Masterclass on How Technology is Changing Research. You can watch the video of her presentation, talking about how visualising experiments enhance reproducibility on the Mendeley YouTube channel, and as usual let us know what you think in the comments below!

MALDI-TOF MS, an Adaptable Method for Protein Characterization, Visualized in a JoVE-Chemistry Video Article

(J. Vis. Exp. (79) e50635, doi:10.3791/50635 (2013))

By Val Buntrock, Ph.D.

Journal Development Editor, JoVE

A recent video-article published in JoVE, the Journal of Visualized Experiments, by a research group at the Centre National de la Racherche Scientifique (CNRS) captures the process of analyzing intact proteins using mass spectrometry (MS). In their video article, Signor et al. demonstrate how to accurately measure large proteins using matrix assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS. Often, when describing MALDI-TOF MS procedures in text, essential information is omitted, leading to poor reproducibility. Part of a new trend in publishing, this video demonstration records the subtleties and nuances of this complex technique. Employing proper technique and variable consideration, this research group identifies an intact, large protein (> 100 kDa) with high sensitivity using a small amount (0.5 pmoles) of protein sample. Using these video instructions, research groups around the globe can modify this flexible technique to identify an infinite number of large proteins.

Phosphoregulation

Characterizing proteins is important for determining the current state of the protein, which has severe implications to several biological processes. The significance of proteins switching between active and inactive forms via protein kinase phosphorylation events has been recognized and applied to cellular and molecular research for several decades. Researchers have gone on to show that protein folding, as determined by phosphorylation, either exposes or protects structural motifs that serve as binding sites for effector molecules. Further, the binding between protein and effector molecule controls protein function. Therefore, the initial protein phosphorylation event regulates the activity level of the protein.

Protein activity or function plays a role in switching on or off a large number of cellular processes, such as cell communication and cell replication. As structural biologists identify a growing number of disease states related to malfunctioning protein modifications and subsequent de-regulation, understanding and identifying the differences between the two states of the protein (active or inactive) has become a priority.

Protein Characterization

A simple, fast, and common method of determining the presence or absence of phosphorylation in proteins is by determining their mass using mass spectrometry (MS). MS ionizes the molecule of interest, generating a charged species, and measures its mass-to-charge or m/z ratio.  The m/z ratio is determined by the isotopic distribution of each element present, meaning that each molecule or protein has its own unique isotopic pattern or fingerprint.

Two MS techniques are typically used to ionize heavy and labile biomolecules, such as proteins: electrospray ionization (ESI MS) and matrix assisted laser-desorption ionization time-of-flight (MALDI-TOF MS). ESI MS analysis requires dissolution of the sample in a pure solvent for direct ionization from the solution mixture. MALDI-TOF MS utilizes a co-crystallization method wherein the protein is crystallized with an ultraviolet (UV) absorbing organic species. This organic molecule is referred to as the matrix molecule or substance.

While ESI MS is more sensitive and accurate, the instrument compatible solvents or buffers typically contain significant amounts of substances, such detergents and salts, which interfere with the desired protein pattern. Additionally, ESI MS data is more difficult to interpret given that ESI MS spectra are riddled with multiple overlapping charge states from a single protein. A more gentle technique, MALDI-TOF MS produces fewer multiply-charged species, leading to a much cleaner spectra that is easier to analyze. This is especially true for larger biomolecules, such as proteins, which can fragment into numerous multiply charged species using ESI MS. For these reasons, MALDI-TOF MS is the preferred technique for protein analysis.

Optimizing MALDI-TOF MS Technique

Matrix

Purity at every stage of MALDI-TOF MS analysis is crucial to obtaining the highest quality MS spectra or protein fingerprint. For this reason, Signor et al. provide detailed instructions for how to effectively 1) clean the MALDI plate that holds the matrix and protein of interest and 2) purify the matrix substances using standard recrystallization techniques. Further, they employ two different matrix systems to compare which one yields the best results for the protein of interest. A single matrix and a mixed matrix system are used, demonstrating the influence of the matrix on the resulting spectra. In their work, Signor et al. found that the mixed matrix system yielded a higher signal to noise and a greater sensitivity than the single matrix system.

Deposition Method

The deposition method is another technique variable that impacts the quality of MALDI-TOF MS results. The two most commonly employed deposition methods are the droplet and thin layer method. Using a droplet technique, a mixture of the protein analyte and matrix solution is “dropped” onto the target substrate, and the solvent is evaporated, yielding a crystalline mixture of the matrix and protein. Slightly more controlled, the thin layer technique is composed of layers of matrix sandwiching the protein analyte layer. While the droplet method suffers from poor resolution and an inability to observe larger proteins (> 100 kDa), the thin layer deposition yields a protein fingerprint for large proteins, sharper peaks, and a higher signal to noise.

As protein analysis becomes a more vital component of studying protein modifications, mastering protein characterization techniques is increasingly important. In this video-article, Signor et al. provide a detailed overview of the steps involved in utilizing MALDI-TOF MS to analyze proteins. They also provide essential considerations and modifications to guide beginners and experts alike through tailoring this powerful technique to study different target proteins. Shown in video format, the necessary level of details, such as how to properly perform multiple deposition methods, is captured and relayed to the viewer for increased transparency.

 

 

 

 

How is Technology Changing Research? Let’s Find Out!

SMW London badge v2

Social Media Week London will soon be upon us and Mendeley is really excited to be hosting a Masterclass in the heart of Covent Garden. This year’s SMW theme is “Open and Connected” and  that’s literally what Mendeley is all about. So we thought we’d bring together a few like-minded people to talk about how social media and technology are changing the way we research and what this means not only for the academic community, but for everybody.

Research is not about a “walled garden” any more. It’s global, open and collaborative (the fact that Mendeley just reached 2.5 million users proves that) If you’re a student, have a professional interest in science or academia, are one of the millions of people who have contributed in some way to a citizen science project or thought about supporting research through crowdfunding, then you’re part of this story. And if you aren’t yet, then chances are that you soon will be. People want to participate in science, creating communities around interesting projects and helping to advance the pace of discovery in the process, and now they can even do this by playing Facebook games.

The dynamic format of the event will have 9 high-profile international speakers presenting their ideas in 5-minute PechaKucha-style presentations followed by a 45-minute open discussion between the panellists and the audience (both in the venue and online) mediated by Nicolai Humphreys, Web Editor of the Lancet. We have a very impressive cross-section line-up, and they’ll explore, from a variety of angles, how technology and social media are enabling scientific research and making science more open for everyone. We will look at how games can leverage the brainpower of millions of citizen scientists, how crowdfunding initiatives have helped raise funds for finding new cures for cancer, and how global collaborative platforms are helping to bring together researchers to help solve some of the biggest problems facing us today.

The space in the venue is limited to only few guests and VIP pass holders, but we’ll be streaming everything live, so wherever you are in the world you can watch the sessions and send in your comments.

So if you have a question (whether it’s for on of the panelists or a general issue about technology and research you’d like to explore) get in touch via Twitter using the hashtag #smwSciTech or send an email to alice.bonasio@mendeley.com and we’ll try our best to include it!

See you there!

Event Details

Social Science Masterclass: How Technology is Changing Research

Thursday 26th September 2013

11:00am to 1:00pm

Covent Garden, London

Speakers:

Jan Reichelt, President and Co-Founder at Mendeley, dubbed the “Facebook for Scientists” – a collaboration platform with 2.5 million users worldwide that also offers researchers workflow and content management tools to accelerate the pace of scientific discovery. In April 2013 Mendeley was acquired by Elsevier, one of the world’s largest scientific publishers

Nicolai Humphreys Web Editor, The Lancet, one the world’s best known, oldest and most respected medical journals, founded in 1823 and published by Elsevier since 1991

Rachel Greene – Director of Marketing at JoVE (Journal of Visualized Experiments) the first peer-reviewed and internationally recognised Scientific Journal that uses video content

IJsbrand Jan Aalbersberg Senior Vice President Journal and Content Technology at Elsevier . He heads the Article of the Future project, which is an initiative that looks at how the traditional paper/pdf based article will evolve to accommodate the wealth of multimedia content that researchers will increasingly rely upon to build scholarly conversation

Margaret Gold Director and Co-Founder of the Mobile Collective,which includes projects such as Citizen Cyberlab, looking to build tools and platforms for citizen science as well as collaborative environments and software tools that stimulate creative learning.

Emma Cooper, Business Development Director at Team Cooper, the developers of Facebook game Fraxinus which harnesses the power of citizen science to find a cure for ash dieback disease

Liz Wald, Head of International at Indiegogo, the largest crowdfunding platform on the web which has successfully enabled scientific research via projects such as uBiome and iCancer which raised funds as well as awareness through their highly successful campaigns.

Rayna Stamboliyska, Board Member of the Open Knowledge Foundation France, working at the Centre for Research and Interdisciplinarity in Paris which focuses on open digital tools for science and has prototyped a platform for citizen science projects in synthetic biology.

Robert Simpson, Researcher and Developer at Zooniverse  (a platform that connects citizen scientists with projects they can contribute to)