Bovine albumin (10mg/ml) in H2O solution. Unknown protein sample. Recombinant Porcine Trypsin. Stock solution 25mg/ml in 0.9% NaCl. 20mM potassium phosphate solution, pH9. 200mM potassium phosphate, pH9. Thermal Block. Bruker Autoflex III MALDI mass spectrometer. Matrix solution (a-Cyano-4-Hydroxycinnamic Acid, 5mg/ml solution in methanol supplied by agilent. Acetone and 1% Acetic Acid). Microcentrifuge tube. The website protein prospectus and mascot was used to identify unknown protein sample.
Trypsin digestion of proteins which were analysed by the MALDI-TOF MS provided the mass to charge ratios of the fragmented peptides. Proteins and peptides have the general amino acid formula of H2NCHRCOOH, but differing in their R side chains . The peptide bonds in comparison to the other types of bonding in amino acids have weaker bond energy which makes them easier to fragment at that specific point. The fragmentation of peptides gives the unique peak pattern on mass spectrum. The MALDI-TOF MS was arranged to only measure positive ions, this means that the sample could have adopted a H+ indicated by (M+H+)+ or Na+ which was present in the stock solution.
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Peptide mass fingerprinting was achieved using protein prospector . The masses of the fragments where submitted into protein prospector and compared to the database. The database used was Swiss-prot because it showed high accuracy in matching peptide mass/charge ratio to correct protein.
Table 3 was produced using spot E13 (on MALDI plate) which was the positive control for the experiment and once it was submitted to protein prospector, the protein identified was Bovine serum albumin, which was used in the experiment, the tolerance was set at 10 and the minimum number of peptides was set to 5. The data field ââ‚¬" missed cleavages ââ‚¬" was set to one, because as the number of miss cleavage increases so does the possibilities of different combinations of peptides resulting in an inaccurate protein analyses. Moreover, the field ââ‚¬" tolerance ââ‚¬" corresponds to the accuracy of the MALDI-TOF MS used, increasing the tolerance widens the range and so allows for more proteins to be matched, however, widening the range also increases the number of incorrect matches of proteins. Furthermore, the minimum number of peptides matched (mpm) varied between different samples, the higher the mpm the more confidence we were of the correct matched protein.
This technique of using peptide mass fingerprinting has a major limitation. Protein prospector or Mascot can only match proteins that have had their genomic genes sequenced and stored on the database . However, if the proteins that is being identified is not stored on the database, the possibility of matching the correct protein become impractical.
The analysis of unknown protein that was supplied was determined to be fibrinogen alpha chain (table.4). Using MALDI-TOF MS spot E14 (on MALDI plate) to determine the unknown protein. This was the closest spot next to the calibrated spot (E13) and this was used to increase the accuracy of the matched protein. It was observed that the further away the spot was analysed relative to e13 the more inaccurate the results became. As the distance increases away from e13 there is the possibility that the surface can become elevated or the depth of samples becomes thicker or thinner, which negatively contribute to the accuracy of the masses produced. The spot E1 was analysed which was supposed to contain albumin, but on analysis the mass spectra was shifted to the left slightly in comparison to the spectra of E13, giving different fragment masses. Several attempts using protein prospectus had to be made to determine this protein, with each attempt having slightly different tolerance and minimum peptide matches figures. The tolerance that this protein was observed at was 40 and the minimum peptide match was 8.
The unknown masses of protein that was supplied; unique to each individual was determined to be Human actin-binding protein anillin (table 5). Tolerance range and minimum peptide match where set at 50 and 6, respectively.
The protein samples that were analysed using MALDI-TOF MS showed a pattern which is similar to spectra 1. The peak patterns are due to samples containing isotopes. The first peak illustrates the monoisotopic mass which is the mass of the most abundant isotope in the element. This highest peak suggests that there is only C12 atom present in the fragment.
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The second highest peak suggests that there is one C13 and the rest C12. Similarly, the third highest peak suggests that there is two C13 and the rest C12 because the peaks differ in mass by 1.
MALDI-TOF MS produces masses of peptide fragments which can then be identified using peptide mass fingerprinting. Nevertheless, it does not identify the specific amino acids which constitute the N or C terminus. However, peptide sequencing by Edman degradation reacts a Phenyl isothiocyanate with the N-terminus (that acts as a nucleophile) of the peptide producing phenylthiohydantoin derivative which includes the N terminus amino acid subunit Fig.1. The derivative can be identified using electrophoresis or chromatography. 
Similarly, carboxypeptidases can be applied to the peptide fragment. This enzyme removes a amino acid from the C-terminus one at a time. By measuring the relative rate at which the amount of free amino acids released against time, the C-terminus amino acid can be deduced. The combination of this method and the method above for sequencing N and C terminus with MALDI-TOF can increase sensitive and allows for the sequencing of polypeptides to be more efficient.
During the experiment, some deviations from the protocol occurred which could have negatively affected our results. The incubation time for Trypsin digestion was only limited to 1 hour as opposed to 10 hour. This limited the time for Trypsin to fully digest proteins due to the peptides being enclosed in protein structure may cause an inaccurate reading on mass spectrometer. The lack of negative control for the positive control was a limitation in the method that was adopted. To generate a negative control, randomly generated masses could be produced and analysed using protein prospector or mascot and the mowse score for the matched protein can be determined.
Systematic errors could have arisen due to the incorrect calibration of the apparatus. Because the digestion time was limited to 1 hour, the sample which was used to calibrate the MALDI-TOF MS (Bovine albumin) could have contained undigested peptides. This systematic error will have negatively skewed the results of the entire experiment.
To further this experiment, it can be repeated using chromatography and MS/MS. To ensure that the protein sample is pure and free of contaminants, chromatography be used to purify proteins and ensure the sample is pure . Moreover, this method can be combined with tandem MS which breaks down peptide fragments into smaller fragments allowing more peptide mass fingerprinting to locate more matches of proteins.
The use of protein mass fingerprinting in conjunction with MALDI-TOF MS gives a powerful combination which can be used to identify proteins. The ability of this method to perform this became evident during the experiment as the unknown sample was identified to be fibrinogen alpha chain. The ability to identify proteins and their structures can be extremely useful in evolutionary studies of proteins.