by Mike May  The ultraflex III MALDI TOF/TOF with smartbeam laser provides top-down sequencing. (Source: Bruker Daltonics) |
With so many proteins to be characterized, proteomic scientists really feel the need for speed. That's just what matrix-assisted laser desorption ionization mass spectrometry, or MALDI MS, provides.
"Sample preparation is straightforward and once this is done, you can acquire data in literally seconds," says Richard Caprioli, PhD, Stanley Cohen Professor of Biochemistry and director of the Mass Spectrometry Research Center at Vanderbilt University School of Medicine in Nashville, Tenn. But that's just the beginning of why so many scientists study proteomics with MALDI.
In fact, MALDI unveils a range of protein features. According to Scott Kuzdzal, PhD, life sciences business leader at Shimadzu Scientific Instruments in Columbia, Md., "MALDI MS has a very wide range of proteomics applications, including peptide and protein intact-mass analyses, quality control of a variety of sample types, peptide-mass fingerprinting for protein identification, analysis of post-translational modifications and—more recently—tissue imaging and biomarker discovery."
Getting the full benefits of MALDI, though, takes adding some twists to this technology. Moreover, MALDI MS often works best when combined with other approaches.
Sensitivity and sequencing
 At the Vanderbilt University School of Medicine MALDI MS gets used for a variety of purposes, such as tracking protein patterns in diseases, including cancer. (Source: Richard Caprioli) |
For most any biological technology these days—or even these decades—scientists keep wanting more sensitivity, and MALDI MS provides that. For example, Mari Prieto Conaway, PhD, product marketing specialist at Thermo Fisher Scientific in San Jose, Calif., says, "Our MALDI LTQ XL is a MALDI ionization source on our most sensitive linear ion trap." In addition, she adds that Thermo Scientific's MALDI Orbitrap XL and MALDI LTQ Orbitrap Discovery provide mass accuracy to one to three parts per million.
Thermo Scientific's MALDI products also work with multiple MS, or MS
n. As Conaway says, "MS
n means that our instruments don't stop at MS/MS. MS
3 is crucial for detecting post-translational modifications in proteins, and it also makes all the difference for elucidation of isobaric species upon precursor isolation."
MALDI also provides other benefits. For example, says Conaway, "The analysis of membrane proteins or protein-associated compounds that are hard to analyze by traditional LC/MS approaches—such as proteins found in plants or lower organisms—is another application for which MALDI is ideally suited."
Beyond getting more sensitive, MALDI MS can also be used for sequencing. In Bremen, Germany, Bruker Daltonics makes its MALDI-TDS, which stands for top-down sequencing, available on its ultraflex III MALDI TOF/TOF with smartbeam laser. In short, a top-down approach pulls out specific protein sequences without any proteolytic digestion, whereas a bottom-up technique digests all of the proteins in a sample and then uses databases to put together the pieces. "The MALDI-TDS sequences undigested proteins in the mass range of 5 to 100 kilodaltons," says Detlev Suckau, PhD, head of MALDI applications development and proteomics at Bruker Daltonics. Moreover, he notes that this instrument provides direct access to information about N- and C-termini, protein isoforms, post-translational modifications, and proteolytic
in vivo events. He adds, "Only top-down analysis provides access to such events, which are important in various diseases, such as cancer." This is also the reason that tissue imaging using MALDI is highly relevant for biomarker discovery: The top-down approach monitors proteolytic events not accessible by shotgun proteomics. Here the large mass range of axial MALDI-TOF instruments provides for imaging capabilities up to 20 kilodaltons and higher.
Although MALDI MS provides a fast view of proteins, it can also follow other compounds. For example, Kuzdzal says, "The Shimadzu AXIMA-QIT can be used for phosphoproteomics and glycomics." Moreover, Shimadzu makes MALDI MS instruments that provide collision-induced dissociation, which Kuzdzal says, "gives highly informative MS/MS spectra for a variety of compounds, including peptides, sugars, lipids, small molecules, etc."
Advances in imaging
 The AXIMA Perfortmance is a MALDI TOF/TOF mass spectrometer that can explore proteomics and other areas of biology, including glycomics. (Source: Shimadzu) |
Beyond just identifying proteins, MALDI MS can also locate them. Instead of grinding up an entire sample before MS, MALDI imaging looks for proteins in specific parts of the tissue. In some cases, the resolution can be as high as five micrometers, which is smaller than most mammalian cells. "It's not Buck Rogers science," says Caprioli, "but you have to control experimental parameters to get highly reproducible results."
With that control, though, Caprioli keeps turning up surprises. In looking at the margins of tumors, for example, Caprioli found patterns in the localization of proteins. "We can measure hundreds of proteins," he says, "and we find that some proteins involved in invasive mechanisms of cancer can penetrate into normal tissues further than we expected." This molecular information could be used to identify a cancer that is likely to recur after surgery versus one that is not.
Caprioli has also used MALDI imaging to track a drug and its metabolites in whole mice. He says, "We can look at where the drug ends up and how it metabolizes." He can also examine changes in surrounding proteins at the drug's destination.
Although Caprioli initially developed his own imaging instrument, some companies offer products for MALDI imaging. The MALDI Molecular Imager from Bruker Daltonics, for example, provides a resolution of 30 to 50 micrometers, according to Suckau. He adds that "correlation with multivariate statistics provides a powerful biomarker detection platform to identify those marker candidates and to use the data in pathology and clinical research and development."
In addition, Thermo Fisher's MALDI tools work in imaging studies. "You can see if a drug that is being developed for cancer is going to the specific tumor of interest," says Amy Zumwalt, proteomics marketing programs manager at Thermo Fisher Scientific. MALDI imaging can also be applied to other areas of pharmaceutical research. In searching for drugs to treat neurodegenerative diseases, for example, "you can take a brain slice and see the entire subsection of brain and where the drug has gone," says Zumwalt.
Covering all angles
 MALDI MS can localize proteins in samples. Here a human brain-tumor was grafted into a mouse, and MALDI MS imaging shows a protein from the tumor (red) and one from normal tissue (green). (Source: Richard Caprioli) |
Despite the benefits of using MALDI ionization with MS, it does not find every protein in every sample. "To identify as many proteins as possible, scientists might want to consider MALDI together with electrospray ionization," says Ronan O'Malley, MALDI Synapt product manager at Waters in Milford, Mass.
"Some instruments don't do both," says O'Malley. With MALDI Synapt, though, researchers can apply both forms of ionization, which means users can access the full range of electrospray-based proteomics solutions, effectively getting two research platforms in one.
The MALDI Synapt High Definition MS also provides other benefits. For one thing, it uses orthogonal geometry so measurements are determined by the time-of-flight of ions from pusher to detector. In other words, you don't have to take into account time of flight from the source, so any energy spread at the source has no detrimental effect on resolution and, therefore, mass measurement. As a result, orthogonal systems provide constant resolution over the entire mass range, better mass accuracy (two parts per million), and better calibration stability over time than do axial systems. "The use of a quadrupole for ion selection makes it more likely to select the ion you want to do MS/MS on," says O'Malley. "That provides a higher degree of confidence that the fragments that you are searching are coming from the precursors that you selected." In addition, this instrument includes multi-stage fragmentation and ion-mobility separation, which is integrated in the body of the MS. "For the first time with a MALDI instrument," says O'Malley, "you can separate by size, shape, and charge, as well as mass." He adds, "With ion mobility a scientist can separate isobaric ions that were previously unresolvable. So you can identify more peptides in the sample, and therefore more proteins."
Moreover, scientists know that ion-mobility separation will add some new capabilities. "For MALDI imaging," says O'Malley, "ion mobility is particularly important here for separating the classes of components from interfering ions, as well as separating individual molecular species." He adds, "That extra dimension of separation might transfer tissue imaging to something where you get genuinely biologically-relevant and significant answers."
Integrating options
Although MALDI MS provides many benefits and researchers and companies continue to add new ones, it does not provide answers to every question in proteomics. "If anyone tells you that any technology is without limitations," says Caprioli, "don't believe them." When it comes to proteomics, though, he does find lots of uses for MALDI MS, but he adds that "LC/MS/MS has some great advantages, too." In fact, different techniques tend to grab different parts of the overall protein population in a sample.
In reality, the diversity of techniques adds strength to our overall understanding of the proteome. One technique might show something that another misses. Likewise, one technique can confirm the findings of another. As Caprioli concludes, "We are all looking at little pieces of the proteome pie and trying to understand it." He adds, "MALDI shows us a picture of one piece of that pie." To understand the complete proteome pie, though, researchers must combine a variety of techniques.