The purpose of this experiment is to utilize MALDI-TOF and bioinformatics to qualify the fragments produced after the trypsin digestion of Carbonic Anhydrase II. Carbonic Anhydrase II is an enzyme belonging to a household of carbonaceous anhydrases and are classified as metalloenzymes as they contain a Zn ion in the active site. Their primary map is to keep the pH of the blood and tissues by catalysing the transition of C 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 non all tissue. [ 1 ]
Proteases are an indispensable constituent for the digestion of proteins, where the peptidases act to breakdown proteins down to their monomeric units. The ability to interrupt down proteins is indispensable for their consumption into the being. The enzyme being employed for digestion of carbonaceous anhydrase II is trypsin. Trypsin is a serine peptidase which specifically cleaves peptide bonds on the carboxyl side of Arginine and Lysine, except when bonded to proline ( likely due to the conformation of proline as a consequence of its ring construction ) . [ 2 ]
MALDI-TOF is an highly valuable mass spectroscopy instrument capable of finding the molecular weight of compounds. The acronym MALDI-TOF bases for matrix assisted optical maser desorption/ionization – clip of flight spectroscopy. This instrument basically works by using a optical maser to ionise a sample which is placed on a chromium steel steel home base. This ionisation takes topographic point in a chamber under vacuity which is attached to a flight tubing. Once the sample is ionized by the optical maser, 20,000 Vs are applied through an electric field in order to speed up the sample down the flight tubing. The sum of clip ( clip of flight ) the sample requires to go the distance of the flight tubing is relative to its mass. Typically 50-100 optical maser shootings are done to accomplish the mean ion multitudes. The mass to bear down ratio is plotted against the figure of entire ions of the prevailing species. Other ions present are represented as a per centum relation to the prevailing coinage.
Unlike other techniques ( such as electrospray ionisation ) , MALDI-TOF utilizations soft ionisation and relies on a matrix to absorb the ionisation energy of the optical maser ( typically 337 nanometer ) and reassign it the sample ( which normally have hapless optical density at 337nm ) . The benefit of the soft ionisation is that it prevents atomization of the sample. The general standard for a good matrix is: its ability to transform non-absorbing molecules into absorbing molecules ; solubility in H2O ; non excessively volatile ; and non chemically aggressive. The matrix besides aides in the crystallisation of the sample on the sample home base. The usage of matrix was foremost found to be utile when aromatic compounds aided in ionisation of biomolecules when added to the sample of involvement. The first most successful matrix was nicotinic acid, which is still used today for larger compounds ( & gt ; 100kDa ) . Other normally used matricies include? -cyano-4-hydroxycinnamic acid, sinnapinic acid, dihydroxybenzoic acid. The penchant of one matrix to the other is normally due to the molecular weight scope of the sample of involvement. Without matrix, MALDI is limited to compounds of 1000 Da or less. The usage of matrix has been become standard pattern, nevertheless, the existent grounds as to why the usage matrix works is unknown, despite the legion theories sing its mechanism. [ 3 ]
Bioinformatics is the application of computing machine scientific disciplines to work out jobs of molecular biological science. Of the many possibilities for bioinformatics, databases of DNA, RNA, and Proteins, are the most outstanding. The information stored in these databases comes from assorted research groups, and informations is continually added and modified. Proteomics is the big graduated table survey of proteins. In this peculiar experiment, the database of involvement is in respects to proteins. These protein databases include many maps, but once more, in respects to this experiment, the information of involvement concerns fragments generated from trypsin digestions. By set uping a database of proteins, surveies of future unknown proteins can profit by correlating known sequences to construction and map. A large ground for the survey of proteomics is to find little drug molecules which can inactive proteins. The benefit of proteomics comes from the ability to make “ practical ligand showing ” , where a big set of possible inhibitors can be screened utilizing three-dimensional computing machine mold of the enzyme and inhibitor. This would evidently cut down the clip and cost involved in the showing of big libraries of inhibitor campaigners. [ 4 ]
The carbonaceous anhydrase II will be digested utilizing trypsin in a 20:1 ratio, severally. The carbonaceous anhydrase II solution will dwell of 1 mg/mL in 500? L of 100mM ammonium hydrogen carbonate ( 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 phial, 5? L of each solution will be added, and vortexed. The solution will so be incubated for 2 hours at 37 & A ; deg ; C.
The readying of the matrix will dwell of 1 milligram of? -cyano-4-hydroxycinnamic acid in 100? L of acetonitrile: H2O: trifluoroacetic acid ( 50:50:0.1 ) . In a separate eppendorf phial, 1? L of matrix solution and 1? Lof protein digest solution are added. From this mixture, 1? L is placed on the MALDI-TOF home base and allowed to dry at room temperature. The home base is so inserted into the MALDI-TOF instrument, sample figure is selected, and spectra is acquired.
MALDI-TOF spectra were obtained for both the integral and fragmented ( after trypsin digestion ) carbonic anhydrase II protein. Sixteen major extremums ( those with comparative strengths of & gt ; ~20 % ) were identified as displayed in figures 3, 4, & A ; 5. The protein sequence of bovine carbonaceous anhydrase II was found from the UniProtKB/Swiss-Prot database under accession figure P00921 ( CAH2_BOVIN ) . Of the 16 extremums, 9 ( extremums 1, 2, 4, 5, 6, 9, 10, 13, & A ; 14 ) were within 5 Daltons of the fragment multitudes obtained from the MS-Digest ProteinProspector 5.3.2 web waiter at the University of California at San Francisco. These differences between the experimental informations obtained and the theoretical information found on the waiter are most likely caused by instrument standardization. Peaks 3, 7, 11, & A ; 12 had multitudes matching to the fragment masses found on MS-digest waiter with the add-on of Na, with peak 15 incorporating a K atom alternatively of Na. Top out 8 had a mass matching to a fragment mass found on the MS-digest waiter plus a H2O molecule. Peak 16, that of the integral carbonaceous 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 carbonaceous anhydrase II to bring forth fragments which were cleaved at the carboxyl side of lysine and arginine residues. Once digested, a sample of the merchandises was prepared for MALDI-TOF utilizing? -cyano-4-hydroxycinnamic acid as the matrix. Several unsuccessful efforts were made to obtain MALDI-TOF spectra of both the integral carbonaceous anhydrase II protein every bit good as its fragments.
A few accounts come to mind when seeking to find the cause of the job. The first is in respects to the concentration of the carbonaceous anhydrase II solution. Possibly the concentration of protein was excessively little ( improbable as MALDI-TOF is sensitive to the picomolar scope ) , or the beginning of carbonaceous anhydrase II was contaminated or mislabeled ( besides unlikely ) . Possibly solutions of assorted concentrations could be prepared for analysis. The following possibility is that the carbonaceous anhydrase II solution contained excessively much salt and hindered the ionisation procedure. In order to de-salt the solution, the usage of C-18 zip tip micropipette tips could be used. Alternatively, dialysis could be used, nevertheless, this would be much more clip devouring. These grounds explain some possibilities as to why the integral protein did non ionise and wing.
Since finding the mass of the integral protein was a control ( to set up that the protein solution did in fact contain carbonaceous anhydrase II ) , and was undetectable, this could explicate why the trypsin digestion did non work. On the other manus, if carbonaceous anhydrase II was in fact nowadays in the solution, and given that trypsin was active, digestion fragments should hold been observed in the MALDI-TOF spectra, which was non in fact the instance. A related scenario exists where there is deficient trypsin for digestion, and possibly increasing the sum of trypsin would ensue in utile spectra. Possibly the sum of ammonium hydrogen carbonate in the carbonaceous anhydrase II solution or the HCl concentration in the trypsin solution were deficient for the proper activity of trypsin ( as recommended by a professor ) . Another possibility exists that the MALDI-TOF instrument was non runing decently, nevertheless, other research workers had successfully obtained spectra of their compounds.
Since utile spectra were non obtained, spectra from old digestions were used ( figures 1, 2, 3, & A ; 4 ) . The major extremums ( those with comparative strengths of & gt ; ~20 % with multitudes greater than approximately 1000Da ) were correlated with the possible fragments found on the proteomics database. Even though most of the extremums were found ( including those with some alteration including the add-on of Na, K, and H2O ) there were some extremums which had no fiting fragments from the database. These extremums include those with mass-charge ratios of 8305.00 and 8464.25. There were some extremums with multitudes in the 8-9kDa scope, and it is so possible these extremums correspond to the experimental values obtained ( given that they had multiple alterations ) , nevertheless, these consequences would be inconclusive. Last, as antecedently mentioned, Peak 16 ( the intact carbonaceous 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 stopping point to 1000 Da is refering since it has several deductions. It could be that the protein was merely modified by several atoms or molecules of Na, K, or H2O, and it is so the same protein. The other possibilities include that the protein used for the experiment was non bovid carbonaceous anhydrase II, or if it was, possibly it was the proenzyme signifier which is possible since the difference in mass could compare to about 6 aminic acids.
The intent of this experiment was to find the multitudes of the fragments created from the trypsin digestion of carbonaceous anhydrase II. Carbonic anhydrase II is an omnipresent enzyme which catalyzes the reaction of C dioxide to bicarbonate in order to keep the pH of blood and tissues. Trypsin is a peptidase which specifically cleaves peptide bonds at the carboxyl terminal of lysine and arginine.
This experiment highlighted the utmost utility of MALDI-TOF as a spectrometric technique. This technique is highly easy, accurate, sensitive, and promptly in obtaining the mass of a compound, particularly when compared to gel filtration. It was a letdown that we could non acquire any of the samples to wing in the MALDI-TOF. Future repeats of this experiment should be performed with several different alterations in variables such as enzyme, substrate, and buffer concentrations. Furthermore, the samples should be desalted prior to the burden of the sample onto the MALDI-TOF home base. Even though MALDI-TOF is a comparatively new technique ( created in the late 1980 ‘s ) it has many utile applications particularly in the kingdom of biochemistry, and more so when coupled to bioinformatics.
The techniques learned in this experiment could be utile in farther experiments where the MALDI-TOF and bioinformatics could be coupled to HPLC fractional process of the fragments to obtain specific amino acerb sequences from a larger protein. This would function as an alternate to solid-phase peptide synthesis of the peptide fragment desired. Overall, the experiment illustrated some of the cardinal points in protein digestion and their subsequent word picture.
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