Opening the Black Box of MALDI-TOF MS

July 16, 2018

MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) is an increasingly popular method for identifying bacteria and yeasts in the clinical microbiology laboratory. Spectra that can be interpreted to identify a pathogen can be generated very quickly without any a priori knowledge about the organism (no need for a Gram stain). 

This results in quicker turnaround times compared to traditional identification techniques, especially for less commonly encountered pathogens, which otherwise may require setup of additional biochemical tests if they cannot be definitively identified by the lab’s identification system. Reduced turnaround time may also result in faster time to initiation of appropriate antimicrobial therapy and to discontinuation of antibiotics for blood culture contaminants. In addition to these advantages, MALDI-TOF MS may also result in significant cost savings by reducing supply costs for conventional identification.

Despite its popularity, those of us trained in biological sciences may have relatively little exposure to the underlying technology, as it has roots in physics and chemistry. In this post, we will discuss what’s “under the hood” of a typical MALDI-TOF instrument.

Getting Molecules from Cells in One Piece 

In the simplest terms, MALDI-TOF allows us to detect molecules (typically proteins) present in cells. These results are then compared to a database of known organisms to predict the identity of our unknown organism. So, how do we obtain the molecules (the analytes of MALDI) that we need for this analysis?

The first step in the MALDI-TOF procedure is to place a spot of the sample (usually whole bacteria or yeast, but sometimes cell extracts) onto the surface of a metal plate (Figure 1-1). During analysis, the spot is removed from the surface in a process called desorption (the opposite of absorption). Being scientists, we like to use lasers whenever we can, so we use a “laser desorption” technique (Figure 1-3). However, blasting our sample with a laser directly would reduce our analyte to pieces so tiny they would be of no use. 

To avoid this, we shield our sample from the laser by mixing it with small organic molecules (exact composition varies), which are dried to form a matrix (Figure 1-2). The matrix is analogous to the shell of a piñata, which protects the candy inside from the stick while still allowing it to fly out after being struck. During the desorption process, the matrix also donates protons to the analyte, imparting a positive charge (ionization). Therefore, the matrix assists in the laser desorption and ionization (MALDI) of our analyte. 

Weighing Molecules

Now that we have our analytes, we need to measure their mass. MALDI-TOF measures the mass of molecules by determining how long they take to fly a specific distance (time of flight). Since this isn’t the most intuitive concept, let’s start with an analogy using a pitching machine

Figure 1. Sample preparation and laser desorption. Culture and matrix (1 and 2) are dried on the surface of a plate, then removed from the surface (in a process called desorption) using a laser (3).

The molecules’ time of flight (TOF) is recorded and (using some math) their mass can be calculated. Finally, a spectrum is created (Figure 2, right side) that shows the relative abundance of molecules at the indicated molecular weight. In practice, results are reported as the mass to charge ratio (m/z) but since the charge on the majority of molecules is +1, m/z is typically assumed to equal the molecular weight.These machines are very good at applying consistent force to propel a ball towards a batter. If a baseball (about 145 grams) reaches the batter in 1 second, a softball (about 180 grams) will have less acceleration and take slightly longer to arrive. Similarly, a tennis ball (about 58 grams) will have a greater acceleration and arrive more quickly.

The same observation that heavier objects accelerate more slowly also holds true at the molecular level. In fact, the earliest precursor to the modern MALDI-TOF mass spectrometer was called an “ion velocitron,” alluding to the differential acceleration of molecules. Of course, moving individual molecules  is more difficult than moving a baseball. To do this, we take advantage of the molecules’ charge to direct and accelerate them using an electric field. After acceleration, they are allowed to fly on their own (with no electric field adding force) until they reach the detector, which registers an electric charge when the molecule hits (Figure 2, left side). 
Figure 2. Detection and output. Desorbed molecules are accelerated into a “time of flight” tube guided by an electric field (left side). Based on their time of flight, relative mass can be calculated and represented as a spectrum (right side).

Analyzing Spectra

Prominent peaks in the spectrum are identified (such as in this spectrum from Klebsiella pneumoniae) and compared to a database of peaks from known organisms. This database is a critical component of the identification process - without it, the spectrum is impossible to interpret. We are interested in the subset of those peaks that are unique to a specific organism. Luckily, manual inspection of spectra is not necessary as comparison is performed automatically by software provided with MALDI-TOF instruments that are used in the clinical microbiology lab . What users see is simply an identification with an associated confidence level—no mass spectrometry knowledge required.

MALDI-TOF is rapidly becoming a standard technology in the clinical microbiology laboratory. Although we will likely never physically “open the hood” on our MALDI-TOF instruments ourselves, understanding how they work may help us better interpret the data they provide. For example, knowing the basics of how spectra are produced and interpreted makes it clear why we can only identify organisms in our reference database or why E. coli and Shigella (organisms with very similar protein compositions and therefore nearly identical spectra) are indistinguishable to the instrument. 

What other technologies in the clinical laboratory seem to be “black boxes?” Send us a message and we’ll be happy to address them in future posts!

The above represent the views of the author and does not necessarily reflect the opinion of the American Society for Microbiology.

Author: Kenneth (K.P.) Smith

Kenneth (K.P.) Smith
Dr. Kenneth (K.P.) Smith is a postdoctoral fellow at Beth Israel Deaconess Medical Center. His research interest is in development of drugs and diagnostics for multidrug-resistant gram-negative bacteria.