Evaluation of Electrophoretic Protein Extraction and Database-Driven Protein Identification from Marine Sediments

Intact proteins comprise a major component of organic carbon and nitrogen produced globally and are likely an important fraction of organic matter in sediments and soils. Extracting the protein component from sediments and soils for mass spectral characterization and identification represents a substantial challenge given the range of products and functionalities present in the complex matrix. Multiple forms of gel electrophoresis were evaluated as a means of enhancing recovery of sedimentary protein before proteomic characterization and compared with a direct enzymatic digestion of proteins in sediments. Resulting tryptic peptides were analyzed using shotgun proteomics and tandem mass spectra were evaluated with SEQUEST. Multiple databases were then evaluated to examine the ability to confidently identify proteins from environmental samples. Following evaluation of electrophoretic extraction of proteins from sediments, the recovery of an experimentally added standard protein (BSA) from older (>1 ky) sediments was optimized. Protein extraction from sediments via direct electrophoresis of a slurry mixture and the specified extraction buffer resulted in the greatest number of confident protein identifications and highest sequence coverage of the BSA standard. Searching tandem mass spectral data against larger databases with a higher diversity of proteomes did not yield a greater number of, or more confidence in, protein identifications. Regardless of the protein database used, identified peptides correlated to proteins with the same function across taxa. This suggests that while determining taxonomic-level information remains a challenge in samples with unknown mixed species, it is possible to confidently assign the function of the identified protein. *Corresponding author: E-mail: [email protected] †These authors contributed to the work equally. Acknowledgments This work was supported through a collaborative research award by the Chemical Oceanography program of NSF to HRH (OCE-1128776), BLN and DRG (OCE-0825790). MOGEL contribution No. 11-004 of the Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University. We thank Sonia Ying Ting and Shannon Yihsuan Tsai for bioinformatic support. DOI 10.4319/lom.2012.10.353 Limnol. Oceanogr.: Methods 10, 2012, 353–366 © 2012, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and OCEANOGRAPHY: METHODS sedimentary minerals appears to be surface adsorption (Mayer 1994; Mayer et al. 2002). Various mechanisms have been proposed to regulate adsorption of organic matter to mineral surfaces including van der Waals interactions (Rashid et al. 1972), ligand exchange (Davis 1982), cation bridges (Greenland 1971), cation (Wang and Lee 1993) and anion exchange (Greenland 1971), and hydrophobic effects (Nguyen and Harvey 2001). These mechanisms of interaction between protein, sedimentary minerals, and organics often include some form of charge interaction. This electrokinetic phenomenon was first observed by Reuss (1809) when the application of a constant electric field caused migration of aqueous clay particles in water. Fractionation of mineral species by electrophoresis was later demonstrated by Dunning et al. (1982). This principle of mobilization by an electric field was incorporated in the study design to assist the liberation of proteins from sediments. Gel electrophoresis has been widely used for decades as a protein separation and visualization technique. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and related approaches separate proteins based primarily on their molecular weights (Laemmli 1970). The wide application of SDS-PAGE and its ability to solubilize and immobilize proteins have made it a standard analytical technique for protein separation and isolation across the fields of biochemistry, cell biology, and medical sciences (e.g., Reisfeld et al. 1962; Laver 1964; Shapiro et al. 1967; Fairbanks et al. 1971; Maizel 2000; Pederson 2008). This includes electrophoretic separation to purify proteins from cell cultures before analysis with mass spectrometry (Tran and Doucette 2009; Botelho et al. 2010). The focus here was to develop and validate a modified electrophoretic approach as an extraction and preparative technique for complex environmental samples before high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, allowing us to accomplish the goal of identifying proteins or peptides in marine sediments. Gel electrophoresis as a preparatory method is founded on the visualization of protein bands by staining, followed by the excision and enzymatic digestion of the proteins bound within the bands. This method is a standard tool for protein identification using HPLC-MS/MS (Hirano et al. 1992; Shevchenko et al. 1996; Kuster, et al. 1998). Rather than using gel electrophoresis as a means for visualizing the isolated proteins, however, we employed the SDS-PAGE technique to 1) enhance protein solubilization (i.e., see Botelho et al. 2010) from the sediment matrix, 2) isolate sediment particles from soluble components, 3) stabilize and retain proteins while rinsing away unwanted contaminants, and 4) suspend denatured proteins (Fairbanks et al. 1965) for enzymatic digestion. Although the application of sediments to SDS-PAGE is unorthodox, the method provided multiple benefits in addition to being a standard technique that is frequently employed for the digestion of proteins for tandem mass spectrometry analysis. Advancements in proteomic use of HPLC-MS/MS have increased sensitivity and detection limits, providing the user with an increased ability to identify peptides from complex mixtures (Schulze et al. 2005; Morris et al. 2010; Dong et al. 2010). The extraction, isolation, and analysis of proteins and peptides using MS/MS are only the first steps, however, that provide the researcher with spectral data. To interpret peptide mass spectral data, it must be correlated with peptides and proteins from a user-provided proteomic database. Many of the current databases are not yet mature, and bioinformatic challenges for identifying proteins retained in soils and sediments include the high diversity of unknown taxonomic contributors, the incomplete availability of protein databases, and the diverse mixture of proteins, which yield limiting concentrations of specific sequences for detection and identifications (Graves and Haystead 2002; Quince et al. 2008; Bastida et al. 2009). The goals of this work were two-fold. The first goal was to optimize the extraction of proteins from marine sediments with a broadly applicable methodology. The second and equally important goal was to assess the effectiveness of proteomic database complexity on environmental samples. For the first goal, we evaluated two methods that employed a SDSPAGE clean-up step: 1) a more traditional method where the buffer-solubilized material is separated from particles and loaded directly onto gels, and 2) a novel slurry extraction method where the buffer-solubilized material remains with the sediment particles and is loaded as a composite material together. Two electrophoresis gels were compared, including preparatory tube gels and standard one-dimensional flat gels with multiple combinations of extraction buffers for each. To address the second goal, mass spectra were searched against five databases of varying size to evaluate database-driven protein identifications from multiple species using probabilistic scoring. As model sediments, continental shelf surface and deeper core sediments from the Bering Sea were used as the test matrix since this area is one of the world’s most productive ecosystems (Sambrotto et al. 1986; McRoy 1987; Walsh et al. 1989), and is known to be diatom-dominated during spring blooms. A high carbon export flux (Chen et al. 2003) in the spring, coupled with rapid transit times, elevates the amounts of diatom derived organic material reaching sediments. These factors make the Bering Sea a realistic system to explore sedimentary protein extraction and evaluate information from multiple database searches. Materials and procedures Protein extraction Bering Sea surface sediments were extracted using a buffer followed by SDS-PAGE. The buffer consisted of 7 M urea, 2 M thiourea, 0.01 M Tris-HCl, 1 mM EDTA, 10% v/v glycerol, 2% CHAPS, 0.2% w/v ampholytes (Fluka BioChemika, high resolution pH 3-10, 40% in water), 2 mM Tributyl-phosphine (Kan et al. 2005). The mixture includes chaotropic agents, detergents, denaturants, and salts, thus proteins are solubilized and Moore et al. Protein identification in sediments