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Evaluation of Sample Preservation and Storage Methods for Metaproteomics Analysis of Intestinal Microbiomes.

ABSTRACT: A critical step in studies of the intestinal microbiome using meta-omics approaches is the preservation of samples before analysis. Preservation is essential for approaches that measure gene expression, such as metaproteomics, which is used to identify and quantify proteins in microbiomes. Intestinal microbiome samples are typically stored by flash-freezing and storage at -80°C, but some experimental setups do not allow for immediate freezing of samples. In this study, we evaluated methods to preserve fecal microbiome samples for metaproteomics analyses when flash-freezing is not possible. We collected fecal samples from C57BL/6 mice and stored them for 1 and 4 weeks using the following methods: flash-freezing in liquid nitrogen, immersion in RNAlater, immersion in 95% ethanol, immersion in a RNAlater-like buffer, and combinations of these methods. After storage, we extracted protein and prepared peptides for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis to identify and quantify peptides and proteins. All samples produced highly similar metaproteomes, except for ethanol-preserved samples that were distinct from all other samples in terms of protein identifications and protein abundance profiles. Flash-freezing and RNAlater (or RNAlater-like treatments) produced metaproteomes that differed only slightly, with less than 0.7% of identified proteins differing in abundance. In contrast, ethanol preservation resulted in an average of 9.5% of the identified proteins differing in abundance between ethanol and the other treatments. Our results suggest that preservation at room temperature in RNAlater or an RNAlater-like solution performs as well as freezing for the preservation of intestinal microbiome samples before metaproteomics analyses. IMPORTANCE Metaproteomics is a powerful tool to study the intestinal microbiome. By identifying and quantifying a large number of microbial, dietary, and host proteins in microbiome samples, metaproteomics provides direct evidence of the activities and functions of microbial community members. A critical step for metaproteomics workflows is preserving samples before analysis because protein profiles are susceptible to fast changes in response to changes in environmental conditions (air exposure, temperature changes, etc.). This study evaluated the effects of different preservation treatments on the metaproteomes of intestinal microbiome samples. In contrast to prior work on preservation of fecal samples for metaproteomics analyses, we ensured that all steps of sample preservation were identical so that all differences could be attributed to the preservation method.

SUBMITTER: Mordant A

PROVIDER: S-EPMC8672883 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe use of rumen microbial community (RMC) profiles to predict methane emissions has driven interest in ruminal DNA preservation and extraction protocols that can be processed cheaply while also maintaining or improving DNA quality for RMC profiling. Our standard approach for preserving rumen samples, as defined in the Global Rumen Census (GRC), requires time-consuming pre-processing steps of freeze drying and grinding prior to international transportation and DNA extraction. This impedes researchers unable to access sufficient funding or infrastructure. To circumvent these pre-processing steps, we investigated three methods of preserving rumen samples for subsequent DNA extraction, based on existing lysis buffers Tris-NaCl-EDTA-SDS (TNx2) and guanidine hydrochloride (GHx2), or 100% ethanol.ResultsRumen samples were collected via stomach intubation from 151 sheep at two time-points 2 weeks apart. Each sample was separated into four subsamples and preserved using the three preservation methods and the GRC method (n = 4 × 302). DNA was extracted and sequenced using Restriction Enzyme-Reduced Representation Sequencing to generate RMC profiles. Differences in DNA yield, quality and integrity, and sequencing metrics were observed across the methods (p < 0.0001). Ethanol exhibited poorer quality DNA (A260/A230 < 2) and more failed samples compared to the other methods. Samples preserved using the GRC method had smaller relative abundances in gram-negative genera Anaerovibrio, Bacteroides, Prevotella, Selenomonas, and Succiniclasticum, but larger relative abundances in the majority of 56 additional genera compared to TNx2 and GHx2. However, log10 relative abundances across all genera and time-points for TNx2 and GHx2 were on average consistent (R2 > 0.99) but slightly more variable compared to the GRC method. Relative abundances were moderately to highly correlated (0.68 ± 0.13) between methods for samples collected within a time-point, which was greater than the average correlation (0.17 ± 0.11) between time-points within a preservation method.ConclusionsThe two modified lysis buffers solutions (TNx2 and GHx2) proposed in this study were shown to be viable alternatives to the GRC method for RMC profiling in sheep. Use of these preservative solutions reduces cost and improves throughput associated with processing and sequencing ruminal samples. This development could significantly advance implementation of RMC profiles as a tool for breeding ruminant livestock.

Project description:BackgroundSample preparation is a critical process for proteomic studies. Many efficient and reproducible sample preparation methods have been developed for mass spectrometry-based proteomic analysis of human and animal tissues or cells, but no attempt has been made to evaluate these protocols for plants. We here present an LC-MS/MS-based proteomics study of barley leaf aimed at optimization of methods to achieve efficient and unbiased trypsin digestion of proteins prior to LC-MS/MS based sequencing and quantification of peptides. We evaluated two spin filter-aided sample preparation protocols using either sodium dodecyl-sulphate or sodium deoxycholate (SDC), and three in-solution digestion (ISD) protocols using SDC or trichloroacetic acid/acetone precipitation.ResultsThe proteomics workflow identified and quantified up to 1800 barley proteins based on sequencing of up to 6900 peptides per sample. The two spin filter-based protocols provided a 12-38% higher efficiency than the ISD protocols, including more proteins of low abundance. Among the ISD protocols, a simple one-step reduction and S-alkylation method (OP-ISD) was the most efficient for barley leaf sample preparation; it identified and quantified 1500 proteins and displayed higher peptide-to-protein inference ratio and higher average amino acid sequence coverage of proteins. The two spin filter-aided sample preparation protocols are compatible with TMT labelling for quantitative proteomics studies. They exhibited complementary performance as about 30% of the proteins were identified by either one or the other protocol, but also demonstrated a positive bias for membrane proteins when using SDC as detergent.ConclusionsWe provide detailed protocols for efficient plant protein sample preparation for LC-MS/MS-based proteomics studies. Spin filter-based protocols are the most efficient for the preparation of leaf samples for MS-based proteomics. However, a simple protocol provides comparable results although with different peptide digestion profile.

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Evaluation of Sample Preservation and Storage Methods for Metaproteomics Analysis of Intestinal Microbiomes.

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