Protein And Peptide Characterization Biology Essay

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

The main aim of this experiment is to analyse the various methods used for protein and peptide characterization 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 characterize proteins shall be analysed in detail and the differences in characterizing and synthesizing peptides compared to oligonucleotides would then be discussed.

3 high clusters were obtained from the mass spectra of MALDI-TOF-MS as shown in B2.1 (JX u will attach d graph later?) The molecular weight (MW) of the protein sample can be calculated using the formula 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 peak in the 1st cluster and that of the nth cluster gives the ion present in the protein sample. The ions identified are

The shorter peaks found residing beside the highest peak can be explained by the existence 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 main cluster 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 peak have a charge of + 2 (i.e Z=2). Using the same formula, , we calculated the MW of protein to be

Can you mention the peptide name given to us over here? I lost that sheet of paper!


Proteomics, the study of proteins, involves determination of structure and functions of the protein and its interactions within and outside its structure. The result obtained from the mass spectrometer is a mass spectrum, which shows the amount of M/Z at a particular instance. After obtaining the mass spectrum, an analysis pipeline with a sequence of new algorithms is key to a successful identification of protein.

PSD (Post source Decay) is one of the techniques which uses MALDI TOF MS to identify the protein structure. In this method, the protein is first fragmented into smaller peptides using a laser. The peptides are observed from the moment they enter the TOF region (or the moment when they leave the ion source) till the time they enter the ion mirror. Since the proteins are fragmented, the kinetic energy of the fragmented ions (mass m) differs largely from the kinetic energy of the original ions (mass M). To incorporate the large variation in kinetic energies, the ions are accelerated to energies much higher than the initial energy of the ion. The mass resolution for PSD can be improved by using gridless curved field mirror. This process is repeated several time and the complete PSD spectrum can be obtained by stitching all the obtained spectra together. Mass spectra analyses the masses of the different fragments present in the protein sequence. Since most amino acids have different masses, the corresponding amino acid can be found out from the mass recorded. In this way, the identity and sequence of amino acids can be obtained, and hence the protein structure can be determined.

PMF (Peptide Mass Fingerprinting) is a second kind of technique used for identifying the protein. This technique involves cleaving the unknown protein into smaller peptides. MALDI TOF mass spectrometer is used to analyze the masses of these peptides.

The process then starts with the raw data coming from the mass spectrometer. Initially, this data is deionized to facilitate its subsequent steps of the analysis. Following this, the baseline is detected and removed. Then the spectrum is normalized to obtain a common level, so it can be compared to other spectra. Now the actual detection is performed, which identifies the peaks in the spectrum. To achieve this, potential start and end points are sort. In most situations, their curves overlap, hence the curves need to be recovered. Once found, each single peak is analyzed and its properties, such as position height and area are stored. In the next step, the results obtained thus far are overlapped or compared to the huge protein databases like Swissprot or genbank which contain protein sequence information. A comparison is made between the peak list of the measured peptides and all the masses from the calculated peptides. The results obtained are analyzed and the possible matches are tabulated. When calculating the molecular weight, there will be some error in measurement. The degree of error depends on the efficiency of the experimenter and the mass spectrometer.


A proteome is the collection of modified translated proteins in the cell. Proteomics involves qualitative and/or quantitative comparison of proteomes under different conditions to unravel its structure and functions. Mass spectrometry plays a very important role in proteomics. MALDI TOF MS is widely used for mass spectrometry. It involves calculating the charge to mass ratio of an ion based on the time it takes to reach a particular target. The basic MALDI TOF structure involves a sample plate with a combination of a matrix and the analyte (sample molecules) molecules. A laser 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 results in the vibrational excitation of the matrix molecules. This is followed by a vibrational relaxation, resulting in the release of energy, which then excites and ionizes the sample molecules. The end result is both charged matrix and analyte molecules.

The schematic diagram of a simple, linear TOF mass spectrometer is as shown below: (can Maldi tof pictures be here itself? Or do you want to put it in the appendix?)

The sample probe is bladed with a laser which excites and ionizes the matrix and sample molecules, which, driven by a voltage potential start accelerating towards the drift region. In this drift region, the ions separate or move with different speeds depending on their charge to mass ratio. The detector present at the end of the drift region is a time of flight detector which then detects the time taken by the molecule to drift across the region. The laser does not fragment the molecules. This allows for analysis of large biomolecules such as proteins, peptides, oligonucleotides, etc.

While the linear TOF spectrometer is used for proteins with high molecular weight, when ions with very close charge to mass to charge ratio must be separated, reflecting TOF Mass Spectrometer is used. The schematic diagram of a reflecting TOF is as shown below:

By using these single or dual stage reflectron, mass resolution and precision of the masses obtained is improved. With this TOF calculator, it is made sure that all the particles are aimed at the detector. It provides the best resolution as it compensates for the difference in flight times for the same m/z ions (caused by the wide range of kinetic energies exhibited by these ions at the exit end). However, signal loss is much lesser in a linear TOF. It is cheaper and easy to handle. 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 alter if there is even a small amount of impurity present in the sample. The impurities are ionized along with the analyte molecules, and hence will affect the observations.

Error may occur if matrix and analyte are not mixed properly, or in other words, are not in a homogenous solid solution.

If the frequency of laser shots is too little, it will affect the resolution of the peaks. Excessive laser shots will lead to disintegration of the peptides. This will increase the number of peaks, but will reduce its intensity, hence reducing the accuracy of the obtained results.

Considering all the above mentioned errors, the most accurate way of obtaining the MW of peptide is to find out the arithmetic mean MW obtained from the 3 graphs.

PROTEIN CHARACTERISATION (Heading) Briefly describe the various methods to characterize proteins and compare their advantages and disadvantages.

Proteins can be isolated based on differences in their physical and chemical properties, e.g size, mass, charge & binding affinity. Methods adopted to characterize proteins include the Matrix Assisted Laser Desorption/Ionization - Time of Flight - Mass Spectrometry (MALDI-TOF-MS), Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) & Nuclear Magnetic Resonance (NMR). The detailed operation process of MALDI-TOF-MS was elucidated above.


LC-MS/MS is an analytical technique employing the use of Ultra Performance Liquid Chromatography (UPLC) & Tandem Mass Spectrometry (MS/MS). An enzyme is first used to digest the protein sample into peptides. UPLC is then employed to separate the mixture of peptides according to size, charge and hydrophobicity of the peptides. The basis of separation is the different adsorption coefficients of the analyte and the solid polymers that line the column. The separated elements then emerged sequentially. An important advantage of UPLC is its ability to strip the sample of impurities, allowing a more accurate analysis of the sample. The pure sample is then vaporized and ionized using the Electrospray Ionization (ESI). After which, daughter ions of a specific mass/charge (M/Z) ratio (which is pre-set at the MS1) is allowed entry into the collision cell where Collision-Induced Dissociation (CID) , fragmentation of gaseous ions, occurs. Lastly, the fragmented daughter ions are transported to MS2 where mass spectrometry takes place, producing a mass spectrum. This spectrum is then compared with literature values to identify the protein molecule. A schematic diagram is shown in B2.3

Nuclear Magnetic Resonance (NMR)

Nuclear Magnetic Resonance is another commonly used technique for the characterization of proteins. The huge advantage lies in the fact that it is a non-destructive method as opposed to MALDI-TOF-MS which involves fragmenting the protein sample. As such, NMR is a good choice for analysing dangerous samples. The basis of NMR is the magnetic property of the atom's nucleus. The spinning of a nucleus generates a magnetic moment which will take on either alpha or beta spin state when an external magnetic field is applied. There is a difference in energy level between the alpha and beta spin, which is proportional to the applied external magnetic field. The nucleus is then subjected to a pulse of Electromagnetic Radiation which transfers energy to the nucleus. The alpha spin state will absorb the energy from the electromagnetic radiation and transit to the beta spin state, obtaining resonance as shown in B2.4. A resonance spectrum for a molecule is obtained by keeping the magnetic field constant and varying the frequency of the electromagnetic radiation. An example of a resonance spectrum is shown in B2.5.






Acquiring data and database search take a few minutes only.

Used for singly charged ions, hence analysis is straightfoward

Capable of analysing species of large molecular weight of up to 500 kDa.

Ease of operation

Limited to pure protein samples only

Destructive as samples are ionized.

Unable to provide information on secondary and tertiary proteins.

Unable to detect proteins in very low abundance due to ion suppression effects.


Able to identify and characterize in complex analyte.

Able to generate information on peptide primary structure (sequence), thus increasing protein identification.

Protein sample can contain impurities since it will be gotten rid during the process

Higher sensitivity. Able to detect proteins in lower abundance due to UPLC.

Destructive as samples are ionized.

Unable to provide information on secondary and tertiary proteins.

Data acquisition, data search and result analysis take a great deal of time since it involves ions of various charges.


Capable of differentiating between structural isomers and provide information on secondary and tertiary structure of protein.

Non-destructive. Protein sample remains non-fragmented

Protein sample cannot contain impurities

Data interpretations are more complex and time consuming.

Requires one to work in a high-magnetic field environment.

Maximum molecular size of NMR is 30kDa. And thus unable to analyse complex protein sample



Proteins are linear polymers built of monomer units called amino acids, which are linked end to end. Amino acids are the building blocks of proteins. The sequence of linked amino acids is called the primary structure. Structures are determined by the sequence of amino acids in the protein polymer. It is this sequence that has to be determined while characterising the protein. When people characterize protein, people mainly search for the amino acids. The mechanism of protein synthesis involves determining the codon present in the protein, which can then be identified as a specific amino acid. is a process called translation because the four-letter alphabet of nucleic acids is translated into the entirely different twenty-letter alphabet of proteins.


DNA has the form of double helix. A double helix separated into two single strands can be replicated. Each strand serves as a template on which its complementary strand can be assembled.

Differences in characterization

Peptides (Proteins)

Oligonucleotides (DNA)

Aim of characterization

determination of the amino acid sequence

Determination of the deoxynucleoside triphosphates sequence on the DNA strand.

Building blocks

20 different amino acids, peptide characterization can be seen to be a more complicated process.

Only 4 different kinds of nitrogenous bases present in DNA, namely, Adenine (A), Cytosine (C), Guanine (G), Thymine (T)


3D conformations of peptides with bonding and intermolecular forces the structure of proteins varies as the amino acid sequence

3D double helix structure

Enzymes required


Restriction enzymes

Characterization Techniques

Peptide Mass Fingerprinting (PMF), MALDI-TOF-MS, LC-MS/MS, Nuclear Magnetic Resonance (NMR).

MALDI-TOF-MS, capillary gel electrophoresis,Electrospray Ionization-Fourier Transform Ion Cycylotron Resonance mass spectrometry. Southern Blot.

When using the MALDI-TOF-MS technique

Desalting not required No need to be desalted

Have to be desalted (i.e. Metal ions present in the compounds have to be replaced with ammonium or trialkyklammonium ions)


DNA synthesis (Oligonucleotide synthesis)

Oligonucleotides are short nucleotide sequences with fewer than twenty bases. Chemical DNA synthesis is done in 3' to 5' direction, while enzyme synthesis of DNA is done in the opposite direction. DNA has the form of double helix. A double helix separated into two single strands can be replicated because each strand serves as a template on which its complementary strand can be assembled. To achieve faithful replication, each strand within the parent double helix acts as a template for the synthesis of a new DNA strand with a complementary sequence. The building blocks for the synthesis of the new strands are deoxyribonucleoside triphosphates. They are added, one at a time, to the 3' end of an existing strand of DNA. The two strands replicate to get another two strands of DNA, with one stands continuously replicate. The other strand, called the lagging strand, is replicated as Okazaki fragments, which are later joined together using the enzyme ligase. (Fig B2.6)

(Why does it look like its copy pasted from a website?)

The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

1 Initialization step: Heat the reaction to temperature 94-96℃

2 Denaturation step: This step cause DNA melting .high temperature disrupt the hydrogen bond and get single strand.

3 Annealing step The reaction temperature is lowered to 50-65 °C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template.

4 Extension/elongation step: At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. 

The above process is repeated several times to obtain the DNA chain. The process has been described using the figure B2.7course circulate to get DNA rapidly

Peptide synthesis

Peptides are linear polymers formed by peptide bond which links the α-carboxyl group of one amino acid to the α-amino group of another amino acid. Chemical peptide synthesis starts from the carboxyl terminus to the amino terminus, while protein biosynthesis is in the opposite direction.

Chemical peptide synthesis

Liquid phase peptide synthesis: Liquid-phase peptide synthesis is a classical approach 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 purposes.

Solid phase peptide synthesis is a process in which synthetic reactions are carried out on a solid support. Solid phase synthesis can be used for many purposes. Synthesis of peptides and proteins is one such example. in many ways for example to create carbohydrates, peptides and oligonucleotides. And is most commonly used technique for synthesizing peptides in the lab. The process involves two basic steps.

1. Peptide chain assembly with protected amino acid derivatives on a polymeric support.

2. The cleavage of the peptide from the resin support with the concurrent cleavage of all side chain protecting groups to give the crude free peptide.

Once the amino acid derivative and the solid support are selected, the activation and blocking process is begun. A protecting group is attached to the amino end which prevents it from reacting with itself. The carboxyl group is automatically activated once amino group is blocked. Fmoc is the widely used reagent for this purpose. The next step, the coupling process, involves attaching an amino acid residue to the existing chain. Once the amino acid is added, deprotection (to remove the protecting reagent) is done. This process is repeated several times to obtain the peptide sequence.

1 select amino acids derivative and solid support

2 activation and blocking

Either the amino or carboxyl group is reacted with a reagent to prevent the amino acid from reacting 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 repeat steps for each amino acids attached

The above steps repeat to increase the chain

5 final block

6final step is to get free peptides

The obtained product is not pure. Hence isolation of peptide using cleavage solution followed by sublimation and purification leads to the required protein. To get the product , many steps are needed , such as isolation of peptide from cleavage solution, solubilisation in solvents, and purification of the product

Differences in synthesis

Peptides (Proteins)

Oligonucleotides (DNA)

Building block

Amino Acids

Nucleotides(a sugar, a phosphate and one of the 4 bases)

Bonds formed during synthesis

Amide bonds are formed between the amino terminus and the carbonyl terminus of adjacent amino acids

Phosphodiester bonds are formed between the 5' and the 3' ends of adjacent nucleotides

Protection during synthesis

Not necessary for protein synthesis.

Note: Deprotection only occurs once, at the start of peptide synthesis

The oligonucleotide polymer that is being formed has to be capped for protection to prevent it from being deleted during the washing process.

Similarly, deprotection occurs only once at the start of synthesis.

PCR as an alternative method for synthesis

PCR cannot be used for protein synthesis.

Oligonucleotides can be alternatively synthesized via the PCR method, which is faster, less time consuming and less tedious.


In this experiment, we learnt the working of MALDI-TOF, LC-MS/MS and how they can be used in proteomics to determine the protein structure. The complexity of mass spectrometry and chromatographic techniques were illustrated. The graphs or spectra obtained from the spectrometer were used to determine the mass of the protein. Importance of systems such as FinMod was illustrated by finding the identity of the protein.


Berg, J.M., Tymoczko, J.L. & Stryer. L. Biochemistry, 7th edition, W. H. Freeman and Company, USA (2012)

Cohen. A, MALDI LC/MS Interface for continuous sample deposition from reverse phase HPLC (n.d.). Retrieved on January 27, 2013 from:

Effasy (2007), Solid phase synthesis (SPPS) Retrieved on January 30, 2013 from

High Power liquid chromatography- HPLC (n.d.) Retrieved on January 25, 2013, from

Nuclear Magnetic Resonance (n.d.). Retrieved January 26, 2013, from Wikipedia:

Proteomics (n.d.), Retrieved on January 27, 2013 from:

Peptide Mass Fingerprinting (PMF) (n.d.); Retreived on 29th January 2013, from:

Post-source decay (PSD) (n.d.); Retrieved on 29th January 2013, from

Solid Phase Synthesis, Retrieved on January 30, 2013 from

Trauger. A, Webb. W, Siuzdak. G. (2002). Peptide and Protein analysis with mass spectrometry, Retrieved on January 25, 2013, from


MALDI matrix assisted laser desorption/ionization

TOF time-of-flight

MS mass spectrometry

PCR Polymerase Chain Reaction

LC-MS/MS Liquid Chromatography - Mass Spectrometry/Mass Spectrometry

PSD Post source Decay

PMF Peptide Mass Fingerprinting

UPLC Ultra Performance Liquid Chromatography

NMR Nuclear Magnetic Resonance