Proteomics was introduced by Marc Wilkins and his colleagues. This novel branch of Biology involves the study of proteome and genomic protein complements. It provides information about identification and quantification of proteins and also helps in determining their function, localisation and interaction etc . Analysis of protein combines separation science which separates the peptides or proteins, analytical science which helps in identification and quantification of peptides and finally the bioinformatics tool for data analyses . Because of the high complexity of proteomes, easier and practically feasible approaches are needed to deal with it. Proteins are involved in almost all biological activities so their analysis demands a lot of advanced and highly sensitive techniques.
Mass spectrometry is an in indispensible tool for identification and characterisation of novel proteins. It involves the conversion of peptides to ions and their analyses according to the mass/charge (m/z) ratio. Mass spectrometer consists of three parts: Source obtains ions from sample and mass analyser sorts or separates ions on the basis of their mass/charge ratio and detector detects separated ions .The sensitivity is very high as the ions having a very small difference in mass/charge ratio even up to .001amu can be easily be distinguished by this instrument.
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Figure 1: parts of mass spectrometer .
Antimicrobial peptides are an important class of peptides and play an important role in the immune system of animals. Dermaseptin( a biologically active, 28-34 amino acid long peptide present in amphibian skin secretions) is an important antimicrobial peptide. These peptides are generally released during stress conditions or in response to infections . Various protein analysis techniques are available like Tandem Mass Spectrometry (MS/MS), Quadrupole-Time Of Flight (QToF), Matrix Assisted Laser Desorption/Ionization- Time Of Flight (MALDI-TOF), ), High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS), Edman Degradation Protein Sequencing and FMOC peptide synthesis to deal with such peptides. These techniques help in proper isolation, effective identification and structural analysis of such proteins, despite the presence of other compound in the mixture .
Steps involved in isolation and identification of peptides:
ï¿½ Crude sample is taken
ï¿½ High Performance Liquid Chromatography performed
ï¿½ Biological assay of peptide fraction
ï¿½ Selected fraction subjected to MALDI-TOF
ï¿½ MS-QTOF or EDMAN degradation is performed
ï¿½ BLAST Search confirms the peptide.
HPLC is the best and a reliable method for peptide separation which offers a high resolution because of varieties in stationary phase used for analysis. Analytic proteomics involve different combinations of HPLC separation modes. The serial combination of these modes is called tandom HPLC. Reverse phase High pressure liquid chromatography is an advanced form of HPLC used for purification of biologically active compounds (peptides). Chromatographic column C-18 is a commonly used column. Depending on the solvent mixture, HPLC is monitored at different wavelengths, 214nm or 280 nm.
Peptides below 10000Da can be purified efficiently by HPLC . Some organisms secrete Cytolytic peptides for innate immunity . Skin secretions of frogs and other amphibians attract researchers as the secretions involves proteins like phylloseptins, dermaseptins, dermatoxins, phylloxins and Gly-Leu rich peptides  showing antimicrobial  anticancer and antibiotic properties. The main organisms are leaf frog Hylomantis leimur  Rana nigrovittata , and Hylarana guentherior .
According to colon et al 2007, complex skin peptides can rapidly be analysed by MALDI. Fractions within a sample are separated by HPLC, and then fed into MALDI where molecular weight of peptides is determined. Amino acid sequences are identified by subjecting fragments to EDMAN degradation. Databases search or BLAST further verifies the sequences. The results from these methods are then correlated and structure of peptides can be determined. If sample contains phylloseptin or dermaseptin, data obtained will be correlated with the database of peptide to confirm the peptide.
Purification of proteins by HPLC-MS
Crude sample is subjected to HPLC-MS for separation of fractions. Miscible combinations of methanol and H2O in a fixed ratio are used for loading the sample onto column. High pressure is set up which helps in fast resolution. UV absorbance monitors the eluents of the column and after regular intervals fractions are collected .This combination of HPLC-MS generates the fragmentation patterns which on comparing with database identify the peptides. Database GENEBANK, SWISSPROT are commonly used for this purpose. Composition of mobile phase can be varied, we can use 5% methanol as starting concentration.
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Resolution of peaks can be refined by progressively increasing the concentration. The progressive increase in concentration refines the resolution of peaks- by gradient elution.
Fig 2: Schematic representation of HPLC-MS.
Fig 3 :This is image of Chromatogram obtained by HPLC for complex mixture .
High resolution, improved reproducibility, rapid analysis, stationary phase (column) reuse and automated instrumentation are the important features of this technique, which make it an efficient analytic tool in proteomics.
Biological assay: Biological activities of eluted fractions is determined by performing a number of assays such as antimicrobial assay vasolidation assay, insulin secretion assay etc. We are only concerned with dermaseptin an AMP, so antimicrobial assay using zone inhibition analysis is best suited for it. Culture clearance in the plate shows the potential antimicrobial activity of the chemical .
The fraction exhibiting antimicrobial activity is selected for MALDI-TOF.
Determination of molecular mass: (MALDI-TOF analysis).
Figure:4 Schematic representation of MALDI-TOF MS.
Fraction is then subjected to MALDI-TOF, which helps in obtaining homogeneity and purity of fractions. MALDI-TOF is an ionisation technique for analysis of bio molecule (proteins), where ionization reaction is initiated by a beam of nitrogen laser striking the matrix. On absorbing the laser energy the matrix get ionised. Matrix then transfers some part of its charge to the bio molecule and thus protects it from the denaturing effects of the laser beam. Commonly used matrix is a-cyano-4-hydroxycinnamic acid. Refined spectra are obtained by using variable laser intensities which target the wells . Ion mirrors are present in MALDI-TOF, where ions are deflected by an electric field causing the flight path to increase thereby enhancing the resolution. The ions in the flight tube are detected at the top of the flight tube.
Fig 5. m/z spectrum of a peptide mixture using alpha-cyano-4-hydroxycinnamic acid as matrix in MALDI (Thompson et al., 2007).
The results obtained by MALDI-TOF guides us towards the original sample. It provides a single neutral molecule M with a protonated ion.
QTOF-Tandem Mass Spectrometry for sequence analysis
For determining the sequences of peptides, fractions are subjected to de novo sequencing using QTOF MS/MS technique . QTOF is described as triple quadrupole instrument, where the last section is replaced by a TOF analyser. Q0, Q1, Q2 are the quadrupoles in the instrument followed by the mass analyser. `Product ion Scan` is the simplest operational mode in MS/MS, where all fragment ions which belongs to a specific precursor ion are detected to provide structural information of the sample  .QTOF-MS is a popular proteomic tool because of high sensitivity and high mass resolution.This method is used for peptide sequencing and BLAST confirms the results.
B). To isolate and characterise the novel peptides, firstly database search is performed to determine the respective peaks in MALDI TOF and the peptides with unidentified masses are searched for the match. Then the class of protein is identified in which the novel peptide falls, which further helps in determining the biological assay for the peptide. The primary structure of peptides is identified by a technique known as Edman degradation.
In this method the amino acids are cleaved one by one from the amino terminal. Phenyl thiocynate is commonly used for the process which on reaction with amino acid forms Phenythiohydantoin (PTH) - amino acid. In mild acidic conditions PTHï¿½one terminal residue is released and HPLC identifies the compound. This is an efficient technique to sequence the proteins without any breakage of bonds between residues. The method is unsuitable for larger peptides because of longer degradation time.
Figure: 5 Edman degradation
This method provides resolutions even for isoform such as leucine and isoleucine, which is not possible by mass spectrometry analysis. This is quite an expensive method but provides refined resolutions. A software PDB Deep View Programme is used for predicting 3 D structures of peptide .
The unknown peptide can be confirmed by synthesizing the same peptide by solid phase peptide synthesis. For this the sequence information is taken from Edman degradation sequencing method .The same biological assay is used for this artificially synthesize peptide. The same biological activity confirms the sequence of the peptide and biological properties. In 2006, Zhou et al. identified a novel AMP named AMP1-5, which was present in skin secretions of Hylarana guentheri. Edman degradation and Q-TOF MS / MALDI-TOF MS were used to determine the primary structure of these peptides. Inhibition zone assay and minimum inhibitory concentration confirmed the antimicrobial activity of this peptide .
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According to Chang et al., 2001, some limitations such as reading N-terminal derivatised proteins and posttranslational modifications are associated with Edman degradation. At present, ESI-Q-TOF is helping researchers in determination of primary structure of peptides.
Chang et al in year 2001, faced some problems in identifying the novel proteins from Bacillus pumilus using Edman degradation method. They used de novo MS/MS using Q-TOF for identification of novel protein .
C.) Identification of large sized proteins needs deviation from general MS technology protein characterisation protocol. Let us take an example of Kininogen. The protein is firstly digested by the enzyme trypsin which cleaves at arginine and lysine residues resulting in a mixture of peptides. This fragmentation of large proteins prior to HPLC increases the chance for efficient identification of peptides . After HPLC the fraction is subjected to MALDI-TOF which is able to analyse peptides or proteins up to 100kDa. Alternatively the peptide fraction is subjected to MS-QTOF for analysis.
Using Peptide mass fingerprinting:
PMF involves enzymatic cleavage, giving a set of peptides which serve as a fingerprint. The MALDI-TOF determine m/z value of peptides and proteins are identified by matching their resulting mass with theoretical peptide mass on database e.g. SWISS PROT and NCBI. For sequencing, the enzymatically cleaved peptides are subjected to MS/MS and finally the peptide masses are matched using a relevant database. PMF offers advantages over MS/MS, as post translational modifications does not affect its scoring and matching as they do in MS/MS analysis. PMF uses multiple peptides for identification of proteins thus results in positive identification .
Using FT-ICR MS (Fourier transform ion cyclotron resonance mass spectrometry):
As we know the measurement of accurate mass is an advantage for identification and characterization of protein using PMF. In terms of high accurate mass measurement under specific conditions, FT-ICR is found to be an efficient tool. Dynamic range and high mass accuracy of this instrument make it an effective tool for analysis .
Figure 6: Automated PMF by using FT-ICRMS .
Multidimensional protein identification technology (Mud PIT): .
This technique used for analysing the complex sample and the large scale proteome. It combines 2D-LC with tandom MS. In this technique protein mixtures are reduced, alkylated and finally digested to obtain peptide mixture. Pressure loading is done to load peptide on 100 ï¿½m i.d. fused silica column which is packed with 10cm of resin C18 followed by resin SCX (3 cm). Sample containing high salt concentration is desalted and loaded on biphasic column or sample directly loaded on to triphasic column and then it is mounted to platform connected to mass spectrometer. Quaternary HPLC pump supplying 4 different buffers is interfaced with the column.3 buffers which are mainly used for separation are buffer A, B , C.
Buffer A: 5% Acetonitrile ( ACN), .11 % Formic acid
Buffer B: 80% ACN, .1% Formic acid.
Buffer C: 50 mM ammonium acetate.5% ACN, .1% Formic acid.
Firstly buffer A is used to wash the column, then buffer C is applied in small quantity followed by buffer B treatment. In each step the concentration of buffer C is increased because of its application the peptides from SCX resin move to C18 resin. Reverse phase gradients used for separation of peptides on C18 resin .
Peptides having same iso-electric points, which are transferred from SCX to C18 resin are separated according to their size and hydrophobic nature. After separation and removal of peptides from micro-capillary column, they enter MS for ionization and on the basis of m/z ratio they are isolated. Once after generation of TM spectra, they are compared with protein database by using SEQUEST and thus determining the closely related peptide mathematically .
Submitted by: Avinash Thakur
University of Ulster