Application of MALDI-TOF
The aim of this experiment is to use MALDI-TOF and bioinformatics to characterize the fragments produced after the trypsin digestion of Carbonic Anhydrase II. Carbonic Anhydrase II is an enzyme belonging to a family of carbonic anhydrases and are classified as metalloenzymes as they contain a zinc ion in the active site. Their primary function is to maintain the pH of the blood and tissues by catalyzing the conversion of carbon dioxide to bicarbonate. These enzymes are among the fastest of all enzymes, and are typically limited by the diffusion rate of their substrates. The molecular weight is 29kDa and is found in the cytosol of most if not all tissue. 
Proteases are an essential component for the digestion of proteins, where the proteases act to breakdown proteins down to their monomeric units. The ability to break down proteins is essential for their uptake into the organism. The enzyme being employed for digestion of carbonic anhydrase II is trypsin. Trypsin is a serine protease which specifically cleaves peptide bonds on the carboxyl side of Arginine and Lysine, except when bonded to proline (probably due to the conformation of proline as a result of its ring structure). 
MALDI-TOF is an extremely valuable mass spectrometry instrument capable of determining the molecular weight of compounds. The acronym MALDI-TOF stands for matrix assisted laser desorption/ionization – time of flight spectrometry. This instrument essentially works by utilizing a laser to ionize a sample which is placed on a stainless steel plate. This ionization takes place in a chamber under vacuum which is attached to a flight tube. Once the sample is ionized by the laser, 20,000 volts are applied through an electric field in order to accelerate the sample down the flight tube. The amount of time (time of flight) the sample requires to travel the distance of the flight tube is proportional to its mass. Typically 50-100 laser shots are done to achieve the average ion masses. The mass to charge ratio is plotted against the number of total ions of the predominant species. Other ions present are represented as a percentage relative to the predominant specie.
Unlike other techniques (such as electrospray ionization), MALDI-TOF uses soft ionization and relies on a matrix to absorb the ionization energy of the laser (typically 337 nm) and transfer it the sample (which usually have poor absorbance at 337nm). The benefit of the soft ionization is that it prevents fragmentation of the sample. The general criteria for a good matrix is: its ability to transform non-absorbing molecules into absorbing molecules; solubility in water; not too volatile; and not chemically aggressive. The matrix also aides in the crystallization of the sample on the sample plate. The use of matrix was first found to be useful when aromatic compounds aided in ionization of biomolecules when added to the sample of interest. The first most successful matrix was nicotinic acid, which is still used today for larger compounds (> 100kDa). Other commonly used matricies include ?-cyano-4-hydroxycinnamic acid, sinnapinic acid, dihydroxybenzoic acid. The preference of one matrix to the other is usually due to the molecular weight range of the sample of interest. Without matrix, MALDI is limited to compounds of 1000 Da or less. The use of matrix has been become standard practice, however, the actual reasons as to why the use matrix works is unknown, despite the numerous theories regarding its mechanism. 
Bioinformatics is the application of computer sciences to solve problems of molecular biology. Of the many possibilities for bioinformatics, databases of DNA, RNA, and Proteins, are the most prominent. The data stored in these databases comes from various research groups, and data is continually added and modified. Proteomics is the large scale study of proteins. In this particular experiment, the database of interest is in regards to proteins. These protein databases include many functions, but again, in regards to this experiment, the data of interest concerns fragments generated from trypsin digestions. By establishing a database of proteins, studies of future unknown proteins can benefit by correlating known sequences to structure and function. A big reason for the study of proteomics is to determine small drug molecules which can inactive proteins. The benefit of proteomics comes from the ability to do "virtual ligand screening", where a large set of possible inhibitors can be screened using 3-dimensional computer modeling of the enzyme and inhibitor. This would obviously reduce the time and cost involved in the screening of large libraries of inhibitor candidates. 
The carbonic anhydrase II will be digested using trypsin in a 20:1 ratio, respectively. The carbonic anhydrase II solution will consist of 1 mg/mL in 500?L of 100mM ammonium bicarbonate (pH 9.5). The 20?L of trypsin solution will be prepared in 1mM HCl with a trypsin concentration of 1mg/mL. In an eppendorf vial, 5?L of each solution will be added, and vortexed. The solution will then be incubated for 2 hours at 37°C.
The preparation of the matrix will consist of 1 mg of ?-cyano-4-hydroxycinnamic acid in 100?L of acetonitrile:water:trifluoroacetic acid (50:50:0.1). In a separate eppendorf vial, 1?L of matrix solution and 1?Lof protein digest solution are added. From this mixture, 1 ?L is placed on the MALDI-TOF plate and allowed to dry at room temperature. The plate is then inserted into the MALDI-TOF instrument, sample number is selected, and spectra is acquired.
MALDI-TOF spectra were obtained for both the intact and fragmented (after trypsin digestion) carbonic anhydrase II protein. Sixteen major peaks (those with relative intensities of >~20% ) were identified as displayed in figures 3, 4, & 5. The protein sequence of bovine carbonic anhydrase II was found from the UniProtKB/Swiss-Prot database under accession number P00921 (CAH2_BOVIN). Of the 16 peaks, 9 (peaks 1, 2, 4, 5, 6, 9, 10, 13, & 14) were within 5 daltons of the fragment masses obtained from the MS-Digest ProteinProspector 5.3.2 web server at the University of California at San Francisco. These differences between the experimental data obtained and the theoretical data found on the server are most likely caused by instrument calibration. Peaks 3, 7, 11, & 12 had masses corresponding to the fragment masses found on MS-digest server with the addition of sodium, with peak 15 containing a potassium atom instead of sodium. Peak 8 had a mass corresponding to a fragment mass found on the MS-digest server plus a water molecule. Peak 16, that of the intact carbonic anhydrase II, had an experimental mass of 31135.55 and a theoretical mass of 30351.6187, with a difference of 801.9313.
This experiment relied on trypsin for the digestion of carbonic anhydrase II to produce fragments which were cleaved at the carboxyl side of lysine and arginine residues. Once digested, a sample of the products was prepared for MALDI-TOF using ?-cyano-4-hydroxycinnamic acid as the matrix. Several unsuccessful attempts were made to obtain MALDI-TOF spectra of both the intact carbonic anhydrase II protein as well as its fragments.
A few explanations come to mind when trying to determine the cause of the problem. The first is in regards to the concentration of the carbonic anhydrase II solution. Perhaps the concentration of protein was too small (unlikely as MALDI-TOF is sensitive to the picomolar range), or the source of carbonic anhydrase II was contaminated or mislabeled (also unlikely). Perhaps solutions of various concentrations could be prepared for analysis. The next possibility is that the carbonic anhydrase II solution contained too much salt and hindered the ionization process. In order to de-salt the solution, the use of C-18 zip tip micropipette tips could be used. Alternatively, dialysis could be used, however, this would be much more time consuming. These reasons explain some possibilities as to why the intact protein did not ionize and fly.
Since determining the mass of the intact protein was a control (to establish that the protein solution did in fact contain carbonic anhydrase II), and was undetectable, this could explain why the trypsin digestion did not work. On the other hand, if carbonic anhydrase II was in fact present in the solution, and given that trypsin was active, digestion fragments should have been observed in the MALDI-TOF spectra, which was not in fact the case. A related scenario exists where there is insufficient trypsin for digestion, and perhaps increasing the amount of trypsin would result in useful spectra. Perhaps the amount of ammonium bicarbonate in the carbonic anhydrase II solution or the HCl concentration in the trypsin solution were insufficient for the proper activity of trypsin (as recommended by a professor). Another possibility exists that the MALDI-TOF instrument was not operating properly, however, other researchers had successfully obtained spectra of their compounds.
Since useful spectra were not obtained, spectra from previous digestions were used (figures 1, 2, 3, &4). The major peaks (those with relative intensities of >~20% with masses greater than about 1000Da) were correlated with the possible fragments found on the proteomics database. Even though most of the peaks were found (including those with some modification including the addition of sodium, potassium, and water) there were some peaks which had no matching fragments from the database. These peaks include those with mass-charge ratios of 8305.00 and 8464.25. There were some peaks with masses in the 8-9kDa range, and it is indeed possible these peaks correspond to the experimental values obtained (given that they had multiple modifications), however, these results would be inconclusive. Lastly, as previously mentioned, Peak 16 (the intact carbonic anhydrase II), had an experimental mass of 31135.55 and a theoretical mass of 30351.6187, with a difference of 801.9313. This difference of close to 1000 Da is concerning since it has several implications. It could be that the protein was just modified by several atoms or molecules of sodium, potassium, or water, and it is indeed the same protein. The other possibilities include that the protein used for the experiment was not bovine carbonic anhydrase II, or if it was, perhaps it was the zymogen form which is possible since the difference in mass could equate to about 6 amino acids.
The purpose of this experiment was to determine the masses of the fragments created from the trypsin digestion of carbonic anhydrase II. Carbonic anhydrase II is an ubiquitous enzyme which catalyzes the reaction of carbon dioxide to bicarbonate in order to maintain the pH of blood and tissues. Trypsin is a protease which specifically cleaves peptide bonds at the carboxyl end of lysine and arginine.
This experiment highlighted the extreme usefulness of MALDI-TOF as a spectrometric technique. This technique is extremely easy, accurate, sensitive, and quick in obtaining the mass of a compound, especially when compared to gel filtration. It was a disappointment that we could not get any of the samples to fly in the MALDI-TOF. Future repetitions of this experiment should be performed with several different changes in variables such as enzyme, substrate, and buffer concentrations. Furthermore, the samples should be desalted prior to the loading of the sample onto the MALDI-TOF plate. Even though MALDI-TOF is a relatively new technique (created in the late 1980's) it has many useful applications especially in the realm of biochemistry, and more so when coupled to bioinformatics.
The techniques learned in this experiment could be useful in further experiments where the MALDI-TOF and bioinformatics could be coupled to HPLC fractionation of the fragments to obtain specific amino acid sequences from a larger protein. This would serve as an alternative to solid-phase peptide synthesis of the peptide fragment desired. Overall, the experiment illustrated some of the key points in protein digestion and their subsequent characterization.
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