MALDI TOF TOF – A Speedy Protein Inventory

by Mary Martialay on January 10, 2018

How is it that a shark can sense electric fields generated by its prey? To find out, glycoproteins expert Robert Linhardt turned to an instrument called a MALDI TOF TOF mass spectrometer in the Rensselaer Center for Biotechnology and Interdisciplinary Studies (CBIS), and in this post we’re going to talk about MALDI TOF TOF (we’ll get to the name), how it works, and how that makes it a valuable instrument in translational medicine.

But first, back to sharks. Sharks use an organelle – called the ampullae of Lorenzini – lining their heads to sense electrical fields generated by fish and other animals around them in the water. To understand how the ampullae of Lorenzini translates electrical fields in the water to electrical signals in their brain, Linhardt first needs a full accounting of the proteins in the organelle.

One way to compile that list would be to look at the shark’s genome, in which are encoded directions for all the proteins the shark can produce. But in this case, the shark’s genome hasn’t been sequenced. So the next best thing is to sample and identify each protein present in the organelle. The MALDI provides a rapid, semi-quantitative structure and sequence of each protein, allowing researchers to compare what they’ve found to well-documented proteins in a related, better known, organisms, and compile their list. Here’s how Linhardt puts it:

When you have a biological organ and you want to understand what proteins are present, you do MALDI TOF TOF. Without a lot of set-up, MALDI gives you a really quick interpretation of what proteins are present. If you have a lot of samples, you put them on the target and you get a lot of results. It’s not ultra-sensitive, but it gives enough information to explain the structure of the proteins that are there and some of their interesting properties.

And how does it do that? Like other forms of mass spectrometry, MALDI TOF TOF takes advantage of the simple truth that atoms of each element, as well as ions and isotopes of those elements, have a unique mass. Therefore molecules – made up of atoms, ions, and isotopes – also have a unique mass.

Mass spectrometers in general determine that mass by propelling a molecule through a vacuum using an electric or magnetic field, and obtaining data that can be translated into the identity of the chemical species. But to enter the vacuum the molecule must be evaporated into the gas state and ionized (possess a positive or negative charge). A variety of instruments have been developed that best analyze particular classes of chemicals by using different techniques to vaporize, ionize, and measure the mass of the molecule.

And so while MALDI TOF TOF is a mass spectrometer, it’s full name — Matrix Assisted Laser Desorption/Ionization Time-of-Flight Time-of-Flight — indicates the ionization and vaporization as well as how masses of ions are measured.

Dmitri Zagorevski, director of the Proteomics Core, where the MALDI lives, explained that in MALDI, samples to be tested are mixed in a large quantity of matrix, a liquid typically consisting of crystallized molecules, such as sinapinic acid. A droplet of the sample in matrix is dropped onto a metal plate and loaded into the instrument. When a laser is pulsed at the droplet, most of its energy is absorbed by the matrix, which vaporizes together with the target sample. Molecules in the plume of resulting gas are ionized and then accelerated into the vacuum. The instrument measures the time of flight across a fixed distance in two directions, hence Time-of-Flight Time-of-Flight.

As a vaporization and ionization technique, MALDI is well suited for analysis of fragile organic molecules – particularly non-volatile biological molecules like proteins, peptides, and oligosaccharides – that would disintegrate under conventional ionization methods.

The MALDI is one of four mass spectrometers in the Proteomics Core, which is itself one of ten research cores – each housing specialized equipment and facilities — within CBIS. Each of the mass spectrometers is suited to a specific role and each supports research that is elucidating the complexities of biological processes, like our circadian rhythms, tracking down the causes of diseases like Alzheimer’s, and producing innovations in areas such as regenerative medicine.

For some researchers, like Wilfredo Colón, professor and head of the Department of Chemistry and Chemical Biology, MALDI is indispensable. Colón uses the MALDI to identify hyperstable proteins in various organisms and biological systems. Hyperstable (i.e., difficult to degrade) proteins play important biological and pathological roles, and the Colón lab has developed a unique technique that isolates these proteins, allowing them to be identified via MALDI. Without the MALDI, his work wouldn’t be possible. Here’s how he put it:

As useful as our method is for separating hyperstable proteins from most of the proteins in a sample, without MALDI we would not be able to quickly identify the proteins, and this is where the exciting discoveries lie. Our research without MALDI is like getting a Christmas gift and not knowing what’s inside – the latter is nice, but unwrapping the gift is the exciting part. MALDI is already yielding exciting “knowledge gifts” in our research and more are on the way.

A MALDI instrument was part of the original suite of equipment installed in CBIS when it opened in 2004. It’s the only one of its kind at Rensselaer, and one of just a few in the Capital Region. Recently, the original MALDI was replaced with a newer, faster, and better version. The CBIS website lists the other instruments within the core as: a Thermo LTQ-Orbitrap mass spectrometer and a Thermo TSQ Quadrupole mass spectrometer, both coupled with micro-flow Agilent HPLC systems, and a Shimadzu gas chromatograph mass spectrometer.

In addition to the Proteomics Core Facility, CBIS has additional research equipment and facilities in analytical biochemistry and nanobiotechnology, bioimaging, bioresearch, cell and molecular biology, flow cytometry, microbiology and fermentation, microscopy, nuclear magnetic resonance, and stem cell research. The cores are available to Rensselaer faculty, staff and students, and also to external academic and industrial collaborators and researchers. As one of the most advanced research facilities in the nation, CBIS promotes innovation and discovery at the interface of the life sciences, physical sciences, and engineering.