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A sample preparation workflow for adipose tissue shotgun proteomics and proteogenomics.

ABSTRACT: Animals with large adipose stores, such as marine mammals, may provide insights into the evolution and function of this multifunctional tissue in health and disease. In the absence of sequenced genomes, molecular information can be rapidly obtained by proteomics and transcriptomics, but their application to adipose tissue is hindered by low nucleic acid and protein yields. We sequenced and compared proteomes isolated from the blubber of four elephant seals using phenol and guanidine thiocyanate (Qiazol) or detergent (sodium deoxycholate) buffer. Qiazol recovered more subcellular proteins such as metabolic enzymes, in addition to extracting RNA, facilitating proteogenomic analyses of small lipid-rich tissue biopsies. We also compared proteomics data analysis platforms and found that de novo peptide sequencing improved protein identification sensitivity compared to database search alone. We report sample preparation and data analysis workflows for proteogenomics and a proteome of elephant seal blubber containing 2678 proteins, including many of interest for further functional studies.This article has an associated First Person interview with the first author of the paper.

SUBMITTER: Khudyakov JI

PROVIDER: S-EPMC6262853 | biostudies-other | 2018 Nov

REPOSITORIES: biostudies-other

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A sample preparation workflow for adipose tissue shotgun proteomics and proteogenomics.

Khudyakov Jane I JI Deyarmin Jared S JS Hekman Ryan M RM Pujade Busqueta Laura L Maan Rasool R Mody Melony J MJ Banerjee Reeti R Crocker Daniel E DE Champagne Cory D CD

Biology open 20181119 11

Animals with large adipose stores, such as marine mammals, may provide insights into the evolution and function of this multifunctional tissue in health and disease. In the absence of sequenced genomes, molecular information can be rapidly obtained by proteomics and transcriptomics, but their application to adipose tissue is hindered by low nucleic acid and protein yields. We sequenced and compared proteomes isolated from the blubber of four elephant seals using phenol and guanidine thiocyanate ...[more]

PMID: 30361411

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Project description:Due to its outstanding throughput and analytical resolution, gel-free LC-based shotgun proteomics represents the gold standard of proteome analysis. Thereby, the efficiency of sample preparation dramatically affects the correctness and reliability of protein quantification. Thus, the steps of protein isolation, solubilization, and proteolysis represent the principal bottleneck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by the application of detergents. However, these compounds ultimately compromise reverse-phase chromatographic separation and disrupt electrospray ionization. Filter-aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols remain to be confirmed. Thereby, the most important gap is the absence of the data on the linearity of underlying protein quantification methods for plant matrices. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (Pisum sativum) seed and Arabidopsis thaliana leaf proteomes by the shotgun approach. For this aim, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Furthermore, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. The spiking experiments revealed high sensitivities (LODs of up to 4 fmol) for spiked BSA and LDRs of at least 0.6 × 102. Thereby, phenol extraction yielded slightly better recoveries, whereas the detergent-based method showed better linearity. Thus, our results indicate the very good applicability of FASP to quantitative plant proteomics with only limited impact of the protein isolation technique on the method's overall performance.

Project description:Background:Proteomic analyses of clinical specimens often rely on human tissues preserved through formalin-fixation and paraffin embedding (FFPE). Minimal sample consumption is the key to preserve the integrity of pathological archives but also to deal with minimal invasive core biopsies. This has been achieved by using the acid-labile surfactant RapiGest in combination with a direct trypsinization (DTR) strategy. A critical comparison of the DTR protocol with the most commonly used filter aided sample preparation (FASP) protocol is lacking. Furthermore, it is unknown how common histological stainings influence the outcome of the DTR protocol. Methods:Four single consecutive murine kidney tissue specimens were prepared with the DTR approach or with the FASP protocol using both 10 and 30 k filter devices and analyzed by label-free, quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). We compared the different protocols in terms of proteome coverage, relative label-free quantitation, missed cleavages, physicochemical properties and gene ontology term annotations of the proteins. Additionally, we probed compatibility of the DTR protocol for the analysis of common used histological stainings, namely hematoxylin & eosin (H&E), hematoxylin and hemalaun. These were proteomically compared to an unstained control by analyzing four human tonsil FFPE tissue specimens per condition. Results:On average, the DTR protocol identified 1841 ± 22 proteins in a single, non-fractionated LC-MS/MS analysis, whereas these numbers were 1857 ± 120 and 1970 ± 28 proteins for the FASP 10 and 30 k protocol. The DTR protocol showed 15% more missed cleavages, which did not adversely affect quantitation and intersample comparability. Hematoxylin or hemalaun staining did not adversely impact the performance of the DTR protocol. A minor perturbation was observed for H&E staining, decreasing overall protein identification by 13%. Conclusions:In essence, the DTR protocol can keep up with the FASP protocol in terms of qualitative and quantitative reproducibility and performed almost as well in terms of proteome coverage and missed cleavages. We highlight the suitability of the DTR protocol as a viable and straightforward alternative to the FASP protocol for proteomics-based clinical research.

Dataset Information

A sample preparation workflow for adipose tissue shotgun proteomics and proteogenomics.

Publications

A sample preparation workflow for adipose tissue shotgun proteomics and proteogenomics.

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