A Challenge For Making Hard-to-fly Stuffs Fly: An Introduction Of Simple And Effective Methods For Protein MALDI-TOF MS
Mass spectrometry (MS) has been a powerful tool for analyzing the molecular weights of the biological components comprising our body. Biochemists routinely utilize the information of the analyzed molecular weights for detecting pathological changes (or lesions) of patients. In order to detect in MS, an analyte must be excised for ionization with the application of excessive energy, which, ironically, means that components with higher molecular weights, such as proteins, are exposed to harsh degrading environments, eventually disrupting the MS detection completely.
Because such biological components could not be detected through the classical MS method, in the early era of the development for biomedical use, alternative methods fine-tuned for the biological macromolecules were extensively sought. Dr. Koichi Tanaka (Nobel Laureate in Chemistry in 2002) found an elegant method for detecting the protein mass, now called soft laser desorption. In this method, analyte protein can be ionized without losing its structure with the aid of additives, comprising cobalt and glycerol pre-mixed with the analyte. He successfully “flew” a biomolecule with the molecular weight of over 10,000.
Then, groups led by Drs Franz Hillenkamp and Michael Karas individually developed a more versatile and excellent MS method for biological macromolecules, called MALDI (Matrix-Assisted Laser Desorption Ionization), in which “cinnamic acid” derivatives are used to relay the pulsed electron to the analyte macromolecules in a “bland” manner. This MALDI technique for ionizing biological specimen has been broadly utilized until now.
Today, MALDI combined with TOF MS (Time Of Flight Mass Spectroscopy) have been daily and routinely employed for biomedical use. In TOF MS, the time(s) of ionized analyte required for reaching a certain distance is measured wherein the time is proportional to the molecular weight of the analyte, thus providing the information of the precise molecular weight of the analyte, further giving essential scientific insights into fine molecular structures.
Interestingly, this MALDI-TOF MS technology has been now combined to microscopes (imaging mass microscope), enabling to analyze the molecular mass(es) of a specific portion of the microscopic image. Application of this brand-new technology is envisaged for providing, for example, pharmacokinetic data, and paving the way for biomarker research characteristic to each disease, all of which are hoped to promise future medical diagnostics, especially early diagnosis of certain illness including neurodegenerative disorders and cancers etc.
It seems to be commonly acknowledged by researchers that MALDI-TOF MS is an “established” method (or “almighty” method) for analyzing biomolecules, capable of flying all of the macromolecules. However, it depends on the molecule species of analyte whether the molecule can correctly “fly” or not. In our case, we encountered by chance a molecular species (protein) that “never flies” in the course of our biomedical study. We, of course, tried common troubleshooting solutions, such as concentrating the analyte or purifying the analyte rigorously, lowering the salt concentration of the sample, unfortunately resulting in no detection after all.
We realised that our protein solution turned whitish upon adding the matrix solution. We then carefully collected this white pellet by centrifugation, directly deposited onto the MS sample plate, and tried the MS detection, surprisingly leading to successful peak detection. It is technically acknowledged that the good formation of “mixed crystals” between analyte and matrix is prerequisite for the ionisation of analyte in MS detection. Based on this principle in MALDI, we suspect that this white precipitant should contain (some sort of) mixed crystal or seeds of mixed crystals, which could confer the successful MS detections. We are now planning to pursue the molecular mechanism of this phenomenon, which can be hopefully applied to future advances in biomedical research.
We here introduced very simple and effective method for MALDI−TOF MS detection called “direct deposition method” (loading the precipitant directly onto the sample plate which feasibly allows the stuffs that does never fly to “fly”), leading to fine detections in the MS.
These findings are described in the article entitled A simple and effective method for detecting precipitated proteins in MALDI−TOF MS, recently published in the journal Analytical Biochemistry. This work was conducted by Hiroyuki Oshikane and Toshio Nakaki from the Teikyo University School of Medicine and Masahiko Watabe from Teikyo University. We would like to thank Dr. Kazue Kikuchi-Utsumi from Teikyo University School of Medicine for helpful comments in drafting the manuscript.