The chief purpose of this experiment is to analyze the assorted methods used for protein and peptide word picture which includes Matrix Assisted Laser Desorption/Ionization – Time of Flight – Mass Spectrometry ( MALDI-TOF-MS ) and Liquid Chromatography – Mass Spectrometry/Mass Spectrometry ( LC-MS/MS ) . Peptide synthesis to qualify proteins shall be analysed in item and the differences in qualifying and synthesising peptides compared to oligonucleotides would so be discussed.
3 high bunchs were obtained from the mass spectra of MALDI-TOF-MS as shown in B2.1 ( JX u will attach vitamin D graph subsequently? ) The molecular weight ( MW ) of the protein sample can be calculated utilizing the expression M/Z. Since the ions in the MALDI-TOF-MS has a charge of +1 ( i.e Z=1 ) , the MW of the sample is given by Hence, the MW of protein sample is calculated to be The difference of the highest extremum in the 1st bunch and that of the n-th bunch gives the ion nowadays in the protein sample. The ions identified are
The shorter extremums found shacking beside the highest extremum can be explained by the being of the isotopes of the elements found in the protein sample.
2. Analysis of MS spectra from LC-MS/MS
The mass spectra from LC-MS/MS provided us with 4 chief bunch groups, located at 677.8221, 688.8134, 696.7956 and 699.8036. ( B2.2 ) It is observed from the spectra that peptides of the highest extremum have a charge of + 2 ( i.e Z=2 ) . Using the same expression, , we calculated the MW of protein to be
Can you advert the peptide name given to us over here? I lost that sheet of paper!
Proteomicss, the survey of proteins, involves finding of construction and maps of the protein and its interactions within and outside its construction. The consequence obtained from the mass spectrometer is a mass spectrum, which shows the sum of M/Z at a peculiar case. After obtaining the mass spectrum, an analysis grapevine with a sequence of new algorithms is cardinal to a successful designation of protein.
PSD ( Post beginning Decay ) is one of the techniques which uses MALDI TOF MS to place the protein construction. In this method, the protein is foremost fragmented into smaller peptides utilizing a optical maser. The peptides are observed from the minute they enter the TOF part ( or the minute when they leave the ion beginning ) till the clip they enter the ion mirror. Since the proteins are fragmented, the kinetic energy of the disconnected ions ( mass m ) differs mostly from the kinetic energy of the original ions ( mass M ) . To integrate the big fluctuation in kinetic energies, the ions are accelerated to energies much higher than the initial energy of the ion. The mass declaration for PSD can be improved by utilizing gridless curving field mirror. This procedure is repeated several clip and the complete PSD spectrum can be obtained by sewing all the obtained spectra together. Mass spectra analyses the multitudes of the different fragments present in the protein sequence. Since most aminic acids have different multitudes, the corresponding amino acid can be found out from the mass recorded. In this manner, the individuality and sequence of aminic acids can be obtained, and therefore the protein construction can be determined.
PMF ( Peptide Mass Fingerprinting ) is a 2nd sort of technique used for placing the protein. This technique involves spliting the unknown protein into smaller peptides. MALDI TOF mass spectrometer is used to analyse the multitudes of these peptides.
The procedure so starts with the natural informations coming from the mass spectrometer. Initially, this information is deionized to ease its subsequent stairss of the analysis. Following this, the baseline is detected and removed. Then the spectrum is normalized to obtain a common degree, so it can be compared to other spectra. Now the existent sensing is performed, which identifies the extremums in the spectrum. To accomplish this, possible start and terminal points are sort. In most state of affairss, their curves overlap, therefore the curves need to be recovered. Once found, each individual extremum is analyzed and its belongingss, such as place tallness and country are stored. In the following measure, the consequences obtained therefore far are overlapped or compared to the immense protein databases like Swissprot or genbank which contain protein sequence information. A comparing is made between the peak list of the measured peptides and all the multitudes from the deliberate peptides. The consequences obtained are analyzed and the possible lucifers are tabulated. When ciphering the molecular weight, there will be some mistake in measuring. The grade of mistake depends on the efficiency of the experimenter and the mass spectrometer.
A proteome is the aggregation of modified translated proteins in the cell. Proteomics involves qualitative and/or quantitative comparing of proteomes under different conditions to unknot its construction and maps. Mass spectroscopy plays a really of import function in proteomics. MALDI TOF MS is widely used for mass spectroscopy. It involves ciphering the charge to mass ratio of an ion based on the clip it takes to make a peculiar mark. The basic MALDI TOF construction involves a sample home base with a combination of a matrix and the analyte ( sample molecules ) molecules. A optical maser is bladed across the sample. The analyte and the matrix molecules together are known as the co-crystalline. Laser exposure to the co-crystalline molecules consequences in the vibrational excitement of the matrix molecules. This is followed by a vibrational relaxation, ensuing in the release of energy, which so excites and ionizes the sample molecules. The terminal consequence is both charged matrix and analyte molecules.
The conventional diagram of a simple, additive TOF mass spectrometer is as shown below: ( can Maldi tof images be here itself? Or do you desire to set it in the appendix? )
The sample investigation is bladed with a optical maser which excites and ionizes the matrix and sample molecules, which, driven by a electromotive force possible start speed uping towards the impetus part. In this impetus part, the ions separate or move with different velocities depending on their charge to mass ratio. The sensor nowadays at the terminal of the impetus part is a clip of flight sensor which so detects the clip taken by the molecule to float across the part. The optical maser does non break up the molecules. This allows for analysis of big biomolecules such as proteins, peptides, oligonucleotides, etc.
While the additive TOF spectrometer is used for proteins with high molecular weight, when ions with really close charge to mass to bear down ratio must be separated, reflecting TOF Mass Spectrometer is used. The conventional diagram of a reflecting TOF is as shown below:
By utilizing these individual or double phase reflectron, mass declaration and preciseness of the multitudes obtained is improved. With this TOF reckoner, it is made certain that all the atoms are aimed at the sensor. It provides the best declaration as it compensates for the difference in flight times for the same m/z ions ( caused by the broad scope of kinetic energies exhibited by these ions at the exit terminal ) . However, signal loss is much lesser in a additive TOF. It is cheaper and easy to manage. Hence, based on the protein, and the experimental conditions, linear or reflecting MAALDI-TOF-MS is used.
As observed, the MWs obtained from MALDI TOF and LC-MS/MS are different. However, UPLC or any other chromatographic technique is considered more accurate as compared to MALDI TOF.
Molecular weights will change if there is even a little sum of dross nowadays in the sample. The drosss are ionized along with the analyte molecules, and therefore will impact the observations.
Mistake may happen if matrix and analyte are non assorted decently, or in other words, are non in a homogeneous solid solution.
If the frequence of optical maser shootings is excessively small, it will impact the declaration of the extremums. Excessive optical maser shootings will take to decomposition of the peptides. This will increase the figure of extremums, but will cut down its strength, therefore cut downing the truth of the obtained consequences.
Sing all the above mentioned mistakes, the most accurate manner of obtaining the MW of peptide is to happen out the arithmetic mean MW obtained from the 3 graphs.
PROTEIN CHARACTERISATION ( Heading ) Briefly describe the assorted methods to qualify proteins and compare their advantages and disadvantages.
Proteins can be isolated based on differences in their physical and chemical belongingss, e.g size, mass, charge & A ; adhering affinity. Methods adopted to qualify proteins include the Matrix Assisted Laser Desorption/Ionization – Time of Flight – Mass Spectrometry ( MALDI-TOF-MS ) , Liquid Chromatography – Tandem Mass Spectrometry ( LC-MS/MS ) & A ; Nuclear Magnetic Resonance ( NMR ) . The elaborate operation procedure of MALDI-TOF-MS was elucidated above.
LC-MS/MS is an analytical technique using the usage of Ultra Performance Liquid Chromatography ( UPLC ) & A ; Tandem Mass Spectrometry ( MS/MS ) . An enzyme is foremost used to digest the protein sample into peptides. UPLC is so employed to divide the mixture of peptides harmonizing to size, charge and hydrophobicity of the peptides. The footing of separation is the different surface assimilation coefficients of the analyte and the solid polymers that line the column. The detached elements so emerged consecutive. An of import advantage of UPLC is its ability to deprive the sample of drosss, leting a more accurate analysis of the sample. The pure sample is so vaporized and ionized utilizing the Electrospray Ionization ( ESI ) . After which, girl ions of a specific mass/charge ( M/Z ) ratio ( which is pre-set at the MS1 ) is allowed entry into the hit cell where Collision-Induced Dissociation ( CID ) , atomization of gaseous ions, occurs. Last, the disconnected girl ions are transported to MS2 where mass spectroscopy takes topographic point, bring forthing a mass spectrum. This spectrum is so compared with literature values to place the protein molecule. A conventional diagram is shown in B2.3
Nuclear Magnetic Resonance ( NMR )
Nuclear Magnetic Resonance is another normally used technique for the word picture of proteins. The immense advantage lies in the fact that it is a non-destructive method as opposed to MALDI-TOF-MS which involves break uping the protein sample. As such, NMR is a good pick for analyzing unsafe samples. The footing of NMR is the magnetic belongings of the atom ‘s karyon. The spinning of a nucleus generates a magnetic minute which will take on either alpha or beta spin province when an external magnetic field is applied. There is a difference in energy degree between the alpha and beta spin, which is relative to the applied external magnetic field. The karyon is so subjected to a pulsation of Electromagnetic Radiation which transfers energy to the karyon. The alpha spin province will absorb the energy from the electromagnetic radiation and theodolite to the beta spin province, obtaining resonance as shown in B2.4. A resonance spectrum for a molecule is obtained by maintaining the magnetic field invariable and changing the frequence of the electromagnetic radiation. An illustration of a resonance spectrum is shown in B2.5.
COMPARISON OF METHODS ( Pros and Cons ) :
Geting informations and database hunt take a few proceedingss merely.
Used for singly charged ions, hence analysis is straightfoward
Capable of analyzing species of big molecular weight of up to 500 kDa.
Ease of operation
Limited to pure protein samples merely
Destructive as samples are ionized.
Unable to supply information on secondary and third proteins.
Unable to observe proteins in really low copiousness due to ion suppression effects.
Able to place and qualify in complex analyte.
Able to bring forth information on peptide primary construction ( sequence ) , therefore increasing protein designation.
Protein sample can incorporate drosss since it will be gotten rid during the procedure
Higher sensitiveness. Able to observe proteins in lower copiousness due to UPLC.
Destructive as samples are ionized.
Unable to supply information on secondary and third proteins.
Data acquisition, informations hunt and consequence analysis take a great trade of clip since it involves ions of assorted charges.
Nuclear magnetic resonance
Capable of distinguishing between structural isomers and supply information on secondary and third construction of protein.
Non-destructive. Protein sample remains non-fragmented
Protein sample can non incorporate drosss
Data readings are more complex and clip consuming.
Requires one to work in a high-magnetic field environment.
Maximum molecular size of NMR is 30kDa. And therefore unable to analyze complex protein sample
PROTEIN AND OLIGONUCLEOTIDE CHARACTERISATION
Proteins are additive polymers built of monomer units called amino acids, which are linked terminal to stop. Amino acids are the edifice blocks of proteins. The sequence of linked amino acids is calledA the primary construction. Structures are determined by the sequence of aminoA acids in the protein polymer. It is this sequence that has to be determined while characterizing the protein. When people characterize protein, people chiefly search for the amino acids. The mechanism of protein synthesis involves finding the codon nowadays in the protein, which can so be identified as a specific amino acid. is a processA called interlingual rendition because the four-letter alphabet of nucleic acids isA translated into the wholly different twenty-letter alphabet of proteins.
Deoxyribonucleic acid has the signifier of dual spiral. A dual spiral separated into two singleA strands can be replicated. Each strand serves as a templet on whichA its complementary strand can be assembled.
Differences in word picture
Peptides ( Proteins )
Oligonucleotides ( DNA )
Aim of word picture
finding of the amino acid sequence
Determination of the deoxynucleoside triphosphates sequence on the DNA strand.
20 different amino acids, peptide word picture can be seen to be a more complicated procedure.
Merely 4 different sorts of nitrogen-bearing bases present in DNA, viz. , Adenine ( A ) , Cytosine ( C ) , Guanine ( G ) , Thymine ( T )
3D conformations of peptides with bonding and intermolecular forces the construction of proteins varies as the amino acid sequence
3D dual spiral construction
Word picture Techniques
Peptide Mass Fingerprinting ( PMF ) , MALDI-TOF-MS, LC-MS/MS, Nuclear Magnetic Resonance ( NMR ) .
MALDI-TOF-MS, capillary gel cataphoresis, Electrospray Ionization-Fourier Transform Ion Cycylotron Resonance mass spectroscopy. Southern Blot.
When utilizing the MALDI-TOF-MS technique
Desalinating non required No necessitate to be desalted
Have to be desalted ( i.e. Metal ions nowadays in the compounds have to be replaced with ammonium or trialkyklammonium ions )
PROTEIN AND OLIGONUCLEOTIDE
Deoxyribonucleic acid synthesis ( Oligonucleotide synthesis )
Oligonucleotides are short nucleotide sequences with fewer than 20 bases. Chemical DNA synthesis is done in 3 ‘ to 5 ‘ way, while enzyme synthesis of DNA is done in the opposite way. Deoxyribonucleic acid has the signifier of dual spiral. A dual spiral separated into two singleA strands can be replicated because each strand serves as a templet on whichA its complementary strand can be assembled. To accomplish faithful reproduction, each strand withinA the parent dual spiral Acts of the Apostless as a templet for the synthesis of a new DNAA strand with a complementary sequence. The edifice blocks for the synthesis of the new strands are deoxyribonucleoside triphosphates. They areA added, one at a clip, to the 3 ‘ terminal of an bing strand of DNA. The two strands replicate to acquire another two strands of DNA, with one stands continuously replicate. The other strand, called the lagging strand, is replicated as Okazaki fragments, which are subsequently joined together utilizing the enzyme ligase. ( Fig B2.6 )
( Why does it look like its transcript pasted from a web site? )
TheA polymerase concatenation reactionA ( PCR ) is a biochemical engineering inA molecular biologyA toA amplifyA a individual or a few transcripts of a piece of DNAA across several orders of magnitude, bring forthing 1000s to 1000000s of transcripts of a particularA DNA sequence.
1 Initialization measure: Heat the reaction to temperature 94-96a„?
2 Denaturation measure: This measure cause DNA runing.high temperature disrupt the H bond and acquire individual strand.
3 Annealing measure The reaction temperature is lowered to 50-65A A°C for 20-40 seconds leting tempering of the primers to the single-stranded DNA templet.
4 Extension/elongation measure: At this measure the DNA polymerase synthesizes a new DNA strand complementary to the DNA templet strand by adding dNTPs that are complementary to the templet in 5 ‘ to 3 ‘ way, distilling the 5′-phosphate groupA of the dNTPs with the 3’-hydroxyl groupA at the terminal of the nascent ( widening ) Deoxyribonucleic acid strand.A
The above procedure is repeated several times to obtain the Deoxyribonucleic acid concatenation. The procedure has been described utilizing the figure B2.7course circulate to acquire DNA quickly
Peptides are additive polymers formed by peptide bond which links the I±-carboxyl group of one amino acid to the I±-amino group of another amino acid. Chemical peptide synthesis starts from the carboxyl end point to the amino end point, while protein biogenesis is in the opposite way.
Chemical peptide synthesis
Liquid stage peptide synthesis: Liquid-phase peptide synthesis is a classical attack to peptide synthesis. It has been replaced in most labs by solid-phase synthesis ( Fig B2.8 ) . However, it retains its usefulness in large-scale production of peptides for industrial intents.
Solid stage peptide synthesis is a procedure in which man-made reactions are carried out on a solid support. Solid stage synthesis can be used for many intents. Synthesis of peptides and proteins is one such illustration. in many ways for illustration to make saccharides, peptides and oligonucleotides. And is most normally used technique for synthesising peptides in the lab. The procedure involves two basic stairss.
1. Peptide concatenation assembly with protected amino acid derived functions on a polymeric support.
2. The cleavage of the peptide from the rosin support with the coincident cleavage of all side concatenation protecting groups to give the petroleum free peptide.
Once the amino acid derivative and the solid support are selected, the activation and barricading procedure is begun. A protecting group is attached to the amino terminal which prevents it from responding with itself. The carboxyl group is automatically activated one time aminic group is blocked. Fmoc is the widely used reagent for this intent. The following measure, the yoke procedure, involves attaching an amino acid residue to the bing concatenation. Once the amino acid is added, deprotection ( to take the protecting reagent ) is done. This procedure is repeated several times to obtain the peptide sequence.
1 choice amino acids derivative and solid support
2 activation and blocking
Either the amino or carboxyl group is reacted with a reagent to forestall the amino acid from responding with itself. Fmoc is the protect group we use widely
The protected amino acid is reacted with the amino acid attached to the polymer.
4 repetition stairss for each amino acids attached
The above stairss repetition to increase the concatenation
5 concluding block
6final measure is to acquire free peptides
The obtained merchandise is non pure. Hence isolation of peptide utilizing cleavage solution followed by sublimation and purification leads to the needed protein. To acquire the merchandise, many stairss are needed, such as isolation of peptide from cleavage solution, solubilisation in dissolvers, and purification of the merchandise
Differences in synthesis
Peptides ( Proteins )
Oligonucleotides ( DNA )
Nucleotides ( a sugar, a phosphate and one of the 4 bases )
Chemical bonds formed during synthesis
Amide bonds are formed between the amino end point and the carbonyl end point of next amino acids
Phosphodiester bonds are formed between the 5 ‘ and the 3 ‘ terminals of next bases
Protection during synthesis
Not necessary for protein synthesis.
Note: Deprotection merely occurs one time, at the start of peptide synthesis
The oligonucleotide polymer that is being formed has to be capped for protection to forestall it from being deleted during the rinsing procedure.
Similarly, deprotection occurs merely one time at the start of synthesis.
PCR as an alternate method for synthesis
PCR can non be used for protein synthesis.
Oligonucleotides can be instead synthesized via the PCR method, which is faster, less clip devouring and less boring.
In this experiment, we learnt the working of MALDI-TOF, LC-MS/MS and how they can be used in proteomics to find the protein construction. The complexness of mass spectroscopy and chromatographic techniques were illustrated. The graphs or spectra obtained from the spectrometer were used to find the mass of the protein. Importance of systems such as FinMod was illustrated by happening the individuality of the protein.
Berg, J.M. , Tymoczko, J.L. & A ; Stryer. L. Biochemistry, 7th edition, W. H. Freeman and Company, USA ( 2012 )
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MALDI matrix assisted laser desorption/ionization
MS mass spectroscopy
PCR Polymerase Chain Reaction
LC-MS/MS Liquid Chromatography – Mass Spectrometry/Mass Spectrometry
PSD Post beginning Decay
PMF Peptide Mass Fingerprinting
UPLC Ultra Performance Liquid Chromatography
NMR Nuclear Magnetic Resonance