Comparison of Sample Preparation Methods for Shotgun Proteomic Studies in Aquaculture Species

Abstract

Proteomics has been recently introduced in aquaculture research, and more methodological studies are needed to improve the quality of proteomics studies. Therefore, this work aims to compare three sample preparation methods for shotgun LC–MS/MS proteomics using tissues of two aquaculture species: liver of turbot Scophthalmus maximus and hepatopancreas of Mediterranean mussel Mytilus galloprovincialis. We compared the three most common sample preparation workflows for shotgun analysis: filter-aided sample preparation (FASP), suspension-trapping (S-Trap), and solid-phase-enhanced sample preparations (SP3). FASP showed the highest number of protein identifications for turbot samples, and S-Trap outperformed other methods for mussel samples. Subsequent functional analysis revealed a large number of Gene Ontology (GO) terms in turbot liver proteins (nearly 300 GO terms), while fewer GOs were found in mussel proteins (nearly 150 GO terms for FASP and S-Trap and 107 for SP3). This result may reflect the poor annotation of the genomic information in this specific group of animals. FASP was confirmed as the most consistent method for shotgun proteomic studies; however, the use of the other two methods might be important in specific experimental conditions (e.g., when samples have a very low amount of protein).

In Vitro Toxicity Evaluation of Cyanotoxins Cylindrospermopsin and Microcystin-LR on Human Kidney HEK293 Cells

“…The assessment of proteins, genes and other biomolecules through the use of OMICS techniques, such as proteomics, allows the study to the molecular pathways and metabolism of organisms in a more comprehensive manner [ 61 ]. In environmental sciences, proteomics enables to investigate and disclose the toxicity of many xenobiotics or pollutants in the biota [ 62 ].…”

“…In environmental sciences, proteomics enables to investigate and disclose the toxicity of many xenobiotics or pollutants in the biota [ 62 ]. In addition, the existence of new methods of sample preparation, such as filter-aided sample preparation (FASP), is making proteomics studies more trustworthy and sensitive [ 61 ].…”

A review of suspension trapping digestion method in bottom‐up proteomics

“…Araújo et al. compared the FASP, S‐Trap, and SP3 methods in proteomic analysis of turbot liver tissues ( Scophthalmus maximus ) and mussel hepatopancreas ( Mytilus galloprovincialis ) [55]. The S‐Trap method resulted in the largest number of peptide and protein identifications for mussel samples, whereas FASP outperformed the other methods for turbot samples.…”

“…SP3 required less time than FASP and S‐Trap. The performance of SP3 for some samples was better than that of S‐Trap [55, 61, 62]. However, other studies have found that S‐Trap performed better in terms of protein and peptide identification, digestion efficiency, and reproducibility.…”

“…In a previous study, the S‐Trap digestion method was used to profile the proteomes of liver tissues of Scophthalmus maximus and hepatopancreas of Mytilus galloprovincialis [55]. The authors identified ∼8000 peptides, ∼1200 proteins, 78 molecular functions, and 215 biological processes in the liver tissues of Scophthalmus maximus .…”