59
Recently, introduction of another simple process based on NanoLC-ESI-MS/
MS allows detection of more than thousands of proteins in a single reaction
(Köcher et al. 2012; Yu et al. 2014; Tanca et al. 2014). It is imperative to have the
complete genome sequence of the sample under consideration for proteogenomic
comparison of obtained protein/peptide data for identification and characterization (Hettich et al. 2013). Proteogenomics act as a connecting link between
genomics and proteomics (Beverley et al. 2002; Hettich et al. 2013). The technical necessities for proteomic studies are robust processing, efficient protein/peptides detection, wide range, capacity of complex sample analysis, and structural
determination of the peptide sequences that are largely offered by mass spectrometry (MS) (Hettich et al. 2013). Initial studies in proteomics were conducted
with 2-D gel electrophoresis (Klose 1975; O’Farrell 1975), frequently followed
by MS detection, which is supported by use of multiple dyes in gel electrophoresis (Unlu et al. 1997). The capacity to interface multidimensional liquid chromatographic separation with MS allowed better insight in intricate samples
(Delahunty and Yates 2007; Peng et al. 2003). The introduction of robust MS
methods, such as the quadrupole time-of- flight mass spectrometry (Q-TOF-MS),
linear trapping quadrupole-Fourier transform ion cyclotron resonance-MS (LTQFTICR-MS), and linear-trapping-quadrupole (LTQ)-Orbitrap-MS led to
enhanced ability for quick scanning and better resolution of a wide range of
protein/peptide mass, allowing high-throughput proteomic analysis (Hettich
et al. 2013). Computational analysis, optimized sample processing accompanied
by MS identification, seeks special attention in proteomics studies (Tanca et al.
2014). The efficacy of the method appropriate for the shotgun proteomic study of
intricate samples depends basically on these processes:
• Protein isolation: an all-inclusive protein content of the complete set of microbes
from the sample is required
• Cleanup: detergents should be used for removing hindrance-causing compounds
before digestion of proteins
• Pre-fractionation: effective separation of peptides/proteins before MS identification to reduce sample complexity and enhance analysis depth (Tanca et al. 2014).
5.2.3 Metaproteomics and Soil Microbial Communities
Soil is highly heterogeneous in nature and harbours a highly diverse vast microbial
biomass, more so than its other counterparts in the environment (Mocali and
Benedetti 2010; Keiblinger et al. 2012).
• Metaproteomics study of the soil environment is an audacious work as it provides a wealth of information about soil proteins. Sample processing is the critical step in the proteome analysis for better resolution (Wang et al. 2006).
However, there are many technical challenges in soil metaproteomics studies
because the samples are intricate (Bastida et al. 2009; Nannipieri and Smalla
5.2 Metaproteomics
Recently, introduction of another simple process based on NanoLC-ESI-MS/
MS allows detection of more than thousands of proteins in a single reaction
(Köcher et al. 2012; Yu et al. 2014; Tanca et al. 2014). It is imperative to have the
complete genome sequence of the sample under consideration for proteogenomic
comparison of obtained protein/peptide data for identification and characterization (Hettich et al. 2013). Proteogenomics act as a connecting link between
genomics and proteomics (Beverley et al. 2002; Hettich et al. 2013). The technical necessities for proteomic studies are robust processing, efficient protein/peptides detection, wide range, capacity of complex sample analysis, and structural
determination of the peptide sequences that are largely offered by mass spectrometry (MS) (Hettich et al. 2013). Initial studies in proteomics were conducted
with 2-D gel electrophoresis (Klose 1975; O’Farrell 1975), frequently followed
by MS detection, which is supported by use of multiple dyes in gel electrophoresis (Unlu et al. 1997). The capacity to interface multidimensional liquid chromatographic separation with MS allowed better insight in intricate samples
(Delahunty and Yates 2007; Peng et al. 2003). The introduction of robust MS
methods, such as the quadrupole time-of- flight mass spectrometry (Q-TOF-MS),
linear trapping quadrupole-Fourier transform ion cyclotron resonance-MS (LTQFTICR-MS), and linear-trapping-quadrupole (LTQ)-Orbitrap-MS led to
enhanced ability for quick scanning and better resolution of a wide range of
protein/peptide mass, allowing high-throughput proteomic analysis (Hettich
et al. 2013). Computational analysis, optimized sample processing accompanied
by MS identification, seeks special attention in proteomics studies (Tanca et al.
2014). The efficacy of the method appropriate for the shotgun proteomic study of
intricate samples depends basically on these processes:
• Protein isolation: an all-inclusive protein content of the complete set of microbes
from the sample is required
• Cleanup: detergents should be used for removing hindrance-causing compounds
before digestion of proteins
• Pre-fractionation: effective separation of peptides/proteins before MS identification to reduce sample complexity and enhance analysis depth (Tanca et al. 2014).
5.2.3 Metaproteomics and Soil Microbial Communities
Soil is highly heterogeneous in nature and harbours a highly diverse vast microbial
biomass, more so than its other counterparts in the environment (Mocali and
Benedetti 2010; Keiblinger et al. 2012).
• Metaproteomics study of the soil environment is an audacious work as it provides a wealth of information about soil proteins. Sample processing is the critical step in the proteome analysis for better resolution (Wang et al. 2006).
However, there are many technical challenges in soil metaproteomics studies
because the samples are intricate (Bastida et al. 2009; Nannipieri and Smalla
5.2 Metaproteomics