The Microbial Communities

METAPROTEOMICS

Microorganisms are the predominant form of life which first came into existence. Microorganisms are present almost in all types of environment. They play a major role in various biogeochemical cycles. Various enzymes are involved in the process of the above said cycles. Enzymes in turn are proteins. Microorganisms are present in the nature in the form of natural communities called as Microbial Communities. Proteomics is the term defined to the study of different protein samples in a given community whereas Metaproteomics is the analysis of various protein samples from mixed microbial communities.

Proteomics gives us a much more detail understanding of the organism as it identifies the protein structure. Proteomics also gives us evidence of gene expression under a given condition. Proteomics is widely used for the better understanding of the microbial function of genes of a microbial community. Proteomics is also divided into two different areas. One being functional proteomics whereas the other being Comparative proteomics. In functional proteomics the attention being given on studying the structures of protein & its complexes by the use of variety of techniques. Comparative proteomics on the other hand aims on identification of proteins from various complexes and then comparing most of the protein samples by the methods such as two dimensional (2D) gel electrophoresis for identification of proteins followed by Mass spectrometry in which the proteins identified are further quantified.

Most of the microbial samples are studied as pure cultures in the laboratory. But unfortunately these does not give us detail of how a particular gene is expressed in the mixed microbial community.Metaproteomic study applies the 2D-PAGE method of proteomic analysis in this case to study the functional dimensions of the community. The process of Metaproteomics is as follows. Firstly it collects various mixed microbial samples from the environment the next step being separation of proteins by the process of 2D gel electrophoresis followed by Mass Spectrometry method for identification & quantification of protein. Because of Metaproteomics a lot of information is obtained to the microbiologists regards of the different microbial communities .Also after the application of Metaproteomics microbiologists are having a keen interest in knowing more about the gene expression which can be studied in a microbial community of having minimum amount of the microbial diversity. In short Metaproteomics have proved its potential of providing insight structure of the microbial population within a microbial community[1].

In this essay I m going to talk further regards of the various techniques of Metaproteomics, the difference between Metagenomics & Metaproteomics how both the approaches are different from one another, the case studies done in the field of metaproteomics, some of the challenges faced in the field of metaproteomics & the future prospects of the field.

Differences between Metagenomics & Metaproteomics:-

Metagenomics is the study of the culture independent genomic analysis of various microbial communities. Because of the use of Metagenomics we now have a huge data of metagenomic sequences. The study of Metagenomics gives access to the microbiologists to study million of microbes which are not studied before. In the process of Metagenomics DNA is cloned into a host & further genomic analysis is carried out. Metagenomics is also referred to as Community genomics. A lot more is known about the genetic diversity present within the sample because of metagenomics.

Metaproteomics is the process in which protein samples from mixed microbial communities are analysed to identify the protein structure & to study the various functional genes present in the sample.Metaproteomics is also referred to as Community proteomics or Environmental proteomics. We can come to know about the complete protein profile of a

Sample by the process of Metaproteomics.

Techniques / Methods of Metaproteomics:-

The various techniques used for protein identification from microbial communities are as follows

Two Dimensional (2D) gel electrophoresis:-

Two Dimensional gel electrophoresis is the widely used high resolution method for identification of individual proteins from complex protein mixtures. This method is also used to know about the amount of proteins present in the sample of mixed microbial community & to detect changes in gene activity by various quantitative methods.

This method separates protein from the mixture by the process of Isoelectric focusing (IEF) & by use of Sodium Dodecyl Sulphate (SDS) gel electrophoresis. It uses IEF in the first dimension whereas SDS in the second dimension. The proteins are separated in the first dimension on the basis of their isoelectric point (pI) & in the second dimension on the basis of their molecular weights. Usually Two dimensional gel electrophoresis is coupled with Mass spectrometry for identification of proteins. Also image analysis of proteins can be done by using various softwares available.To name a few are ProXPRESS, PHORETIX, MELANIE etc.

Process of Two Dimensional (2D) gel electrophoresis:-

Firstly the sample is loaded on an IEF gel having a pH gradient or on an Immobilized pH gradient (IPG) strips. An electric current is applied across the gel because of which the proteins start moving in a continuous pH gradient and are separated on the basis of their isoelectric point. Isoelectric focusing is a major step in the process of two dimensional (2D) gel electrophoresis.IPG strips are also used for this step.IPG strips of narrow pH ranges are preferred.The next step in 2D gel electrophoresis is SDS PAGE in which the IPG strips are placed on the top of the SDS gels. The electric current is applied to the gel which results in the separation of proteins on the basis of their molecular weight. The proteins having low molecular weight settles at the bottom of the gel whereas the proteins with high molecular weight stays at the top at the gel. The proteins separated are detected as spots & hence to visualize the protein spots present on the gel various dyes are used. Out of which the Coomassie blue and Silver stain methods are often used [2].

Drawbacks of 2D gel electrophoresis:-

Although the method is widely used for the identification of proteins still it possess some limitations which are as follows:-

  • Some proteins cannot be detected by this method.
  • The very high & low molecular mass proteins are poorly being resolved.
  • It is a time consuming process
  • 2D cannot be used in analyzing the membrane bound proteins

Mass Spectrometry:-

Mass spectrometry is the method used for measuring the molecular mass of the sample. Mass spectrometry is also used in determination of mass to charge ratios of the molecules. This method uses mass spectrometers for determining the mass. In the method of Mass spectrometry the proteins are digested into small peptide molecules. In Tandem Mass spectrometry the smaller peptide molecules are recorded for getting information on partial or complete sequence information of the proteins. The common methods used in mass spectrometry are Matrix Assisted Laser Desorption Ionization (MALDI) & Electrospray Ionization (ESI).

Process of Mass spectrometry:-

Ion source, Mass analyzer & an Ion detector are the main components of a Mass spectrometer. The mass analyzers commonly used are: Time of Flight (TOF), Ion trap, Triple Quadrupole etc. In the process of mass spectrometry the sample solution is introduced in the Ion source in which it is released into a gas phase. Inside the Ion source the samples are ionised as Ions as they are easy to handle. Immediately after the process of Ionisation Ions reaches to Mass analyzer where they are separated according to their mass-to-charge (m/z) ratios. The separated ions is then directed towards the detector where the signal is send to a data system for storage of m/z ratios [3].

MALDI Spectrometry:-

MALDI process is a soft ionization technique. In the process of MALDI solid mixtures having our interest which are to be analysed are pushed onto a target plate by use of pulsed laser beam. Because of the use of pulsed laser beam the molecules present in the mixture are being heated & as a result of this ion are being desorbed into gas phase. MALDI is often coupled with the mass analyzer Time-of-Flight (TOF) commonly called as MALDI-TOF. Short focuses of the pulsed laser beam are applied on the mixture so as to desorb the proteins of interest into gas phase. The proteins separated are then passed to a mass analyzer for determination of mass spectrum or as to trap the ions for further mass analysis.TOF are the mass analyzers having large m/z ranges.

Electrospray Ionization (ESI):-

Electrospray Ionization is also a soft ionization technique in which a no of multiple charged ions are produced from the mixture of biological samples. In the process of ESI, our sample of interest which is present in the solution is passed through a Electrospray capillary & a high voltage is applied across the capillary. Because of this voltage ions are generated in the capillary. The ions generated may be positive or negative charged. The ions are then being guided to the mass analyzer region. ESI method is interfaced with various separation methods like Liquid Chromatography. Also ESI is coupled with Quadrupole mass spectrometer[3].

3. Protein Microarray:-

Protein Microarrays are viewed as an upcoming method for the study of protein samples from a complex protein mixture. Protein microarrays are the arrays which consist of various capture reagents like Antibodies, Recombinant proteins, Peptides etc. Capture reagents are the ones which are being printed on to the microarray on which the protein samples of our interest are loaded. They are usually labelled by various fluorescent tags. Hybridization of proteins takes place on the capture reagents which are present on the array. In this process many proteins bind to the capture reagents (e.g. Antibodies) while the ones remaining having fluorescent tags are being washed off. The next step is of scanning in which the hybridized proteins are scanned on to a Microarray scanner & different protein levels are being checked.

One more approach in the protein microarrays is that two protein samples (out of which one referred to as Standard & the other having our protein interest referred to as experimental sample) are labelled & are analyzed in parallel microarrays. This approach is referred to as the comparative analysis of both the samples in which both are labelled by various fluorescent tags as they do in the normal process of Microarrays. These two labelled samples are then hybridized together on the same microarray, then washed & scanned so as to detect the protein expression of the samples. The detection for this approach is done by normalization in which the different signals are calculated by the normalization ratio. Normalization ratio is the ratio in which both the ratios (1 & 2) are calculated. In this ratio one is of the Standard sample whereas ratio two is of the experimental sample used [4].

Advantages of Protein Microarray:-

  • Because of the wide range of capture reagents available today we can produce protein microarrays of different content i.e. Protein microarrays are flexible.
  • When large no of protein samples are to be detected we can use the approach of using samples in parallel microarrays which help in cost reduction.

Stable Isotope Labelling:-

Stable Isotope labelling is the method in which proteins are labelled with stable isotope & then are being analyzed by using Mass spectrometry. The stable isotopes used are 15N, 18O,13C etc.

Isotope labelling can be either done by in vitro labelling or by in situ labelling.

In vitro labelling methods includes Isotope coded affinity tags (ICAT),18O labelling method etc. Whereas In situ labelling includes 15N incorporation from Ammonium salts & Incorporation of Isotope labelled amino acids.

In the method of Isotope coded affinity tags, the proteins to be analyzed are isotope labelled at specific sites by various isotopic reagents. Most of the proteins gets denatured because of the reagents. The proteins which are denatured are then combined & washed in case if any excess reagent is remaining. After washing the proteins are digested by the use of trypsin. The peptides which are having the tags are further analyzed by Mass spectrometry. The most commonly used labelling method used for the isotope labelling is Post isolation chemical isotope tagging of the proteins in which we can do the selective isolation and analysis of the protein.[5]

Case studies in the field of Metaproteomics:-

Various case studies have been studied to know more about the mixed microbial communities & various bacterial communities as well.

Metaproteomic approach to study the functionality of Microbiota in the Human Infant gastrointestinal tract:-

This study was done by Klassens, Willem M.de Vos & Elaine E. Vaughan.

They took the uncultured infant fecal microbiata for their study as the infants fecal is said to be conquered by bifidobacteria. This Bifidobacteria was then studied by various protein separation procedures i.e two dimensional (2D) gel electrophoresis & MALDI-TOF mass spectrometry. As fecal samples of the infants were required in the study the permission from their infants respective parents were obtained at the first. Then in their study they collected the infants fecal samples having high content of bifidobacteria by a specific fluorescent in situ hybridization. The fecal samples were collected of infants at two different stages one infant being 8 days old whereas the other of 117 days old. A huge content of bifidobacteria were present in both the stages. Then Denaturing gradient gel electrophoresis was carried out in order to know about the presence of 16s rRNA gene in the bifidobacteria. Also it was confirmed that huge amount of bifidobacteria present in the infants fecal samples was because of the presence of 16s rRNA gene in the bifidobacteria.

Now in their further study they tooked the fecal samples from two infants at different stages & referred the infants as A & B. The fecal samples from infant A were being collected on the days 8, 24 & 41 whereas that from infant B was collected on the days 103,117 & 144.

The fecal samples were then washed by discharging the microbial cells from it. Further for the extraction of protein from the infants fecal sample the bead beating method was chosen. Then a standard protocol of bead beating method was followed in order to get a soluble protein. For each stage/day a separate 2D gel electrophoresis was carried out. Further two dimensional gel electrophoresis was carried out. In which first the protein samples were loaded on an IPG strip. Then isoelectric focussing was carried out by applying an electric current across the strip. The IPG strip was then placed on the top of the SDS gel. Electrophoresis was carried out resulting in a no. of protein spots. These spots were then determined by various staining methods. The three resulting 2D gels were then gathered so as to compare the results by the use of PDQuest software. Also the mean coefficient of variation (CV) was checked for each gel which showed that there was a change in the no. of protein spots as well as in their intensities for the period of days but they resembled a similar pattern throughout. An enormous number of protein spots were seen on a gel. Out of which few were excised & digested by trypsin in order to be further analyzed by MALDI-TOF mass spectrometry. The final results showed that there was a high similarity between a protein spot which contained Bifidobacterium Infantis with the Bifidobacterium longum transaldolase gene. Thus it was confirmed that Bifidobacterial transaldolase protein is present in the faeces of newborn infants in dominance. Finally they concluded that in the future metaproteomics approach would be helpful in order to study more about the functionality of microbiota in human faeces [6].

More case studies have been done in the field of metaproteomics on Microbial biofilm.The other case study is in which they have done Metaproteomic analysis of Chesapeake Bay Microbial community.

Challenges involved in the field of Metaproteomics:-

The challenges in Metaproteomics are:-

  • Protein extraction from a variety of complex microbial communities such as soil, seawater etc is difficult because of the reason that proteins consist of 20 amino acids & it exists in different structures & has different physical as well as biological confirmations. Also the main reason in having difficulty in extracting the proteins from the above said complex microbial communities is that there is no fixed protocol for the extraction of proteins.
  • The current metaproteomic methods are not sufficient to identify all the proteins present in all the microbial communities. All the proteins from the communities having vast range of diversities cannot be identified & extracted by the methods viz.Two dimensional (2D) gel electrophoresis & Mass spectrometry. For instance the microbial ecologists having deep interest in studying the microbial communities of Soil or Sea water. They can only detect a few no. of proteins using the current metaproteomic protein extraction methods.
  • By the use of Metaproteomics, one cannot explain the functions of all proteins which are present in a microbial community. [1]

Conclusion:-

Inspite of the challenges currently faced during investigations of the protein expression in complex microbial communities such as Soil & Seawater still it is clear in the near future Metaproteomics has an immense potential in itself. In order to study the microbial diversity of the microbial populations in situ the microbial ecologists have found some methods like FISH-MAR, Functional microarrays, Stable isotope probing etc. In the near future if all these methods can be combined together then we will be able to get a more detailed knowledge of the microbial functions expressed within the microbial communities. Today we can get a large amount of metagenomic sequences because of the study of metagenomics. These metagenomic sequences contain various genes of unknown function. Metaproteomics in this case in the future can be an important tool in investigating the unknown function of genes. Even if all the functional proteins present within the microbial community are not cleared Metaproteomics in the near future can be focused only on the studies of the proteins expressed within the community because of which can lead to the development of the field.

References:-

  1. Paul Wilmes & Philip L.Bond (2006); Metaproteomics: studying functional gene expression in microbial ecosystems, Trends in Microbiology.
  2. Paul Gromov, Irina Gromova & Julio E.Celis(2006);Proteomic analysis by Two-Dimensional Polyacrylamide Gel electrophoresis,Proteomics for Biological Discovery.
  3. Timothy D. Veenstra(2006); Mass Spectrometry: The foundation of proteomics,Proteomics for Biological Discovery.
  4. Cassio Da Silva Baptista & David J.Munroe(2006); Protein Microarrays, Proteomics for Biological Discovery.
  5. Kristy J.Brown & Catherine Fenselau(2006);Isotope labelling in quantitative proteomics, Proteomics for Biological Discovery.
  6. Eline S.Klassens,Willem M.de Vos,Elaine E.Vaughan(2006);Metaproteomic approach to study the functionality of the Microbiota in the Human infant gastrointestinal tract, Applied & environmental microbiology.

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