Evaluation of electrophoretic protein extraction and database‐driven protein identification from marine sediments

Abstract

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.

Electrophoretic Extraction and Proteomic Characterization of Proteins Buried in Marine Sediments

“…The identification of algal peptides and proteins in the deep marine sediment core section demonstrates that gel electrophoresis can be an effective method for isolating proteins present in complex matrices, but further improvements can be made in the total recovery of material. The results obtained by loading sediment buffer mixture directly to the gel interface supports previous findings by Moore et al [16] that an electric field applied directly to sediment particles enhances protein extraction. The number of proteins identified was increased by running the gel for a longer period of time, allowing the ion front to move further down the gel (7 cm vs. 2 cm) and the electric field to mobilize proteins out of the sediment.…”

“…These identified proteins represent material that may have been preserved after burial [12,[54][55][56][57] or mixed from the surface into deeper sediments by bioturbators [58][59][60]. Thus, it is expected that fewer proteins, with potentially lower sequence coverage, would be identified in the Bering Sea 8-to 10-cm core section than in surface sediments observed by Moore et al [16,29]. Age estimation of these sediments can be uncertain, because the surface sedimentary mixed layer can extend from 0 to 16.5 cm on the outer Bering Sea shelf [61].…”

“…Identified sedimentary peptides possessed amino acid sequences that matched peptide sequences within the proteomes of the above mentioned species. This database was chosen after extensive comparison revealed that larger databases, including the NCBI non-redundant database containing over 11.9 million protein sequences and the Global Ocean Survey Combined Assembly Protein database [44,45], did not enhance the number of proteins identified, added limited species diversity to identified proteins and had 95% functional agreement with proteins identified from Bering Sea sediment with the smaller database [16].…”

“…Identifying intact proteins and peptides buried in marine sediments would give valuable information towards understanding marine biogeochemical cycles and reconstructing algal/microbial populations [9,10]. The complex matrix effects of sediments [11][12][13], however, have made extracting buried proteins for further analysis a challenge [14][15][16]. New bioseparation methods are needed to successfully extract proteins from deep sediments for subsequent analyses.…”

“…Modified SDS-PAGE methods have been used to purify proteins from cell cultures before mass spectrometry analysis [25,26] and to separate proteins from soil trichloroacetic acid (TCA) extracts [27,28]. Recent studies by Moore et al [16,29] found that intact algal proteins and peptides could be tracked and identified from the marine water column and extracted from surface sediments in the Bering Sea using modified SDS-PAGE tube gels where sediment-buffer mixtures were loaded directly to the gels for separation. In-gel protein extractions were then performed, and peptides were identified using proteomic mass spectrometry and database searching.…”