58
proteins can be isolated by normal or two-dimensional (2-D) gel electrophoresis.
2-D gel electrophoresis offers better segregation of proteins, and these proteins can
be further analysed by a mass spectrometric analysis-based database search; however, the approach has its own limitations such as the infeasibility of consistently
monitoring low abundance (Gygi et al. 2000), and extremely hydrophobic, extremely
acidic, or extremely basic proteins (Lee 2001). There is considerable development
of numerous technologies for proteomics studies, such as chromatography or/capillary electrophoresis separation (Lee 2001; Yates 2004) and protein microarray
developments (Ramachandran et al. 2004). Protein microarray allows robust studies
of protein mixtures and promotes its application for analysis of samples from
diverse environments. After identification of the proteome from particular environments, they are linked to their genes through reverse genetics, which is a key objective of metaproteomics. However, the technological advances are still inadequate
and need optimization (Maron et al. 2007; Schulze et al. 2004). Many physicochemical methods have been reported for protein extraction, depending on proper
cell lysis of the buffer application using detergents (SDS, CHAPS, Triton X-100)
(Wilmes and Bond 2004; Chourey et al. 2010), chaotropic agents (urea, guanidine
hydrochloride) (Wilmes and Bond 2004; Verberkmoes et al. 2009), reducing agents
[dithiothreitol (DTT), tributylphosphine] (Kan et al. 2005), and other compounds
(phenol, NaOH) (Keiblinger et al. 2012; Leary et al. 2012; Benndorf et al. 2007),
along with heat treatment (Singleton et al. 2003; Ogunseitan 1997; Schneider et al.
2012), mechanical disruption (Wilmes and Bond 2004; Keiblinger et al. 2012;
Kolmeder et al. 2012), and sonication (Tang et al. 2014). Once the protein is isolated, compounds that may hinder the digestion, chromatographic separation, or
mass spectrometric studies require urgent removal. Generally, this is accomplished
by precipitating the protein by addition of acetone, trichloroacetic acid, and other
compounds to the cellular extract (Chourey et al. 2010; Benndorf et al. 2007; Leary
et al. 2012), and the pelletized protein is further resuspended in buffer (Fic et al.
2010; Jiang et al. 2004). An additional efficient approach is to carry out onedimensional electrophoresis separation of proteins and their subsequent digestion,
which helps in capturing the hindrance causing chemicals in the gel and provides
protein in sliced gels (Kolmeder et al. 2012; Ferrer et al. 2013). However, despite its
efficiency, the approach is a time- and labour intensive process with lower reproducibility, thus less preferred (Choksawangkarn et al. 2012). One of the latest alternatives is the filter-aided sample preparation (FASP), which offers better cleanup and
enzymatic cleavage, done in a ultracentrifugal filter (Wiśniewski et al. 2009). It was
successfully applied in microbe samples and reported to be better than other
approaches, accounting for less protein content in the samples (Tang et al. 2014).
Moreover, sample complexity should be reduced for better insight through MS
studies. In earlier metaproteomics studies, it was achieved by fractionation of proteins (Kolmeder et al. 2012; Perez-Cobas et al. 2013) or peptides (Verberkmoes
et al. 2009; Schneider et al. 2012; Ram et al. 2005). However, additional fractionation requires more sample concentration, increased MS time length, with tougher
reproducibility; for example, the preferred approach, that is, 2D-LC-MS, is technically challenging and demands longer time duration for analysis (Köcher et al.
2012; Verberkmoes et al. 2009).
5 Metatranscriptomics and Metaproteomics for Microbial Communities Profiling
proteins can be isolated by normal or two-dimensional (2-D) gel electrophoresis.
2-D gel electrophoresis offers better segregation of proteins, and these proteins can
be further analysed by a mass spectrometric analysis-based database search; however, the approach has its own limitations such as the infeasibility of consistently
monitoring low abundance (Gygi et al. 2000), and extremely hydrophobic, extremely
acidic, or extremely basic proteins (Lee 2001). There is considerable development
of numerous technologies for proteomics studies, such as chromatography or/capillary electrophoresis separation (Lee 2001; Yates 2004) and protein microarray
developments (Ramachandran et al. 2004). Protein microarray allows robust studies
of protein mixtures and promotes its application for analysis of samples from
diverse environments. After identification of the proteome from particular environments, they are linked to their genes through reverse genetics, which is a key objective of metaproteomics. However, the technological advances are still inadequate
and need optimization (Maron et al. 2007; Schulze et al. 2004). Many physicochemical methods have been reported for protein extraction, depending on proper
cell lysis of the buffer application using detergents (SDS, CHAPS, Triton X-100)
(Wilmes and Bond 2004; Chourey et al. 2010), chaotropic agents (urea, guanidine
hydrochloride) (Wilmes and Bond 2004; Verberkmoes et al. 2009), reducing agents
[dithiothreitol (DTT), tributylphosphine] (Kan et al. 2005), and other compounds
(phenol, NaOH) (Keiblinger et al. 2012; Leary et al. 2012; Benndorf et al. 2007),
along with heat treatment (Singleton et al. 2003; Ogunseitan 1997; Schneider et al.
2012), mechanical disruption (Wilmes and Bond 2004; Keiblinger et al. 2012;
Kolmeder et al. 2012), and sonication (Tang et al. 2014). Once the protein is isolated, compounds that may hinder the digestion, chromatographic separation, or
mass spectrometric studies require urgent removal. Generally, this is accomplished
by precipitating the protein by addition of acetone, trichloroacetic acid, and other
compounds to the cellular extract (Chourey et al. 2010; Benndorf et al. 2007; Leary
et al. 2012), and the pelletized protein is further resuspended in buffer (Fic et al.
2010; Jiang et al. 2004). An additional efficient approach is to carry out onedimensional electrophoresis separation of proteins and their subsequent digestion,
which helps in capturing the hindrance causing chemicals in the gel and provides
protein in sliced gels (Kolmeder et al. 2012; Ferrer et al. 2013). However, despite its
efficiency, the approach is a time- and labour intensive process with lower reproducibility, thus less preferred (Choksawangkarn et al. 2012). One of the latest alternatives is the filter-aided sample preparation (FASP), which offers better cleanup and
enzymatic cleavage, done in a ultracentrifugal filter (Wiśniewski et al. 2009). It was
successfully applied in microbe samples and reported to be better than other
approaches, accounting for less protein content in the samples (Tang et al. 2014).
Moreover, sample complexity should be reduced for better insight through MS
studies. In earlier metaproteomics studies, it was achieved by fractionation of proteins (Kolmeder et al. 2012; Perez-Cobas et al. 2013) or peptides (Verberkmoes
et al. 2009; Schneider et al. 2012; Ram et al. 2005). However, additional fractionation requires more sample concentration, increased MS time length, with tougher
reproducibility; for example, the preferred approach, that is, 2D-LC-MS, is technically challenging and demands longer time duration for analysis (Köcher et al.
2012; Verberkmoes et al. 2009).
5 Metatranscriptomics and Metaproteomics for Microbial Communities Profiling