Dataset Information

Comparative analysis of MS sample preparation methods

ABSTRACT: We compare the most commonly used proteomic sample preparation strategies such as classical in solution digest protocols, SPEED, FASP, STrap, SP3, as well as the commercially available kits iST (PreOmics) and EasyPep (Thermo Scientific) in their efficacy to extract proteomes from HeLa cells. We find a remarkably good performance of the majority of the protocols with high reproducibility, little method dependencies and low levels of artifact formation. Despite these similarities we observe significant differences in the recovery of specific sets of proteins in a k-means cluster analysis.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Wolfgang Reiter

LAB HEAD: Markus Hartl

PROVIDER: PXD030406 | Pride | 2022-09-06

REPOSITORIES: Pride

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Publications

In Search of a Universal Method: A Comparative Survey of Bottom-Up Proteomics Sample Preparation Methods.

Varnavides Gina G Madern Moritz M Anrather Dorothea D Hartl Natascha N Reiter Wolfgang W Hartl Markus M

Journal of proteome research 20220825 10

Robust, efficient, and reproducible protein extraction and sample processing is a key step for bottom-up proteomics analyses. While many sample preparation protocols for mass spectrometry have been described, selecting an appropriate method remains challenging since some protein classes may require specialized solubilization, precipitation, and digestion procedures. Here, we present a comprehensive comparison of the 16 most widely used sample preparation methods, covering in-solution digests, de ...[more]

PMID: 36006919

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Project description:Here we exploit the essential process of X-chromosome dosage compensation to elucidate basic mechanisms that control the assembly, genome-wide binding, and function of gene regulatory complexes that act over large chromosomal territories. We demonstrate that a subunit of C. elegans MLL/COMPASS, a gene-activation complex, acts within the dosage compensation complex (DCC), a condensin complex, to target the DCC to both X chromosomes of hermaphrodites and thereby reduce chromosome-wide gene expression. The DCC binds to two categories of sites on X: rex sites that recruit the DCC in an autonomous, sequence- dependent manner, and dox sites that reside primarily in promoters of expressed genes and bind the DCC robustly only when attached to X. We find that DCC mutants that abolish rex-site binding do not eliminate dox-site binding, but instead reduce it to the level observed at autosomal binding sites in wild-type animals. Changes in DCC binding to these non-rex sites occur throughout development and correlate with transcriptional activity of adjacent genes. Moreover, autosomal DCC binding is enhanced by rex-site binding in cis in X-autosome fusion chromosomes. Thus, dox and autosomal sites exhibit similar binding properties. Our data support a model for DCC binding in which low-level DCC binding at dox and autosomal sites is dictated by intrinsic properties correlated with high transcriptional activity. Sex-specific DCC recruitment to rex sites then greatly elevates DCC binding to dox sites in cis, which lack intrinsically high DCC affinity on their own. We also show here that the C. elegans DCC achieves dosage compensation through its effects on transcription. ChIP-chip experiments using antibodies against DPY-27, SDC-3, DPY-30, DPY-26, MIX-1, SMC-4, ASH-2 in wild-type embryos. ChIP-chip experiments using antibodies against SDC-3, DPY-27, DPY-30, ASH-2, and IgG in different DCC mutants. ChIP-chip experiments using antibodies against RNA Pol II (hypophosphorylated and S2 and S5) in wild type and sdc-2 partial loss of function mutants.

Dataset Information

Comparative analysis of MS sample preparation methods

Publications

In Search of a Universal Method: A Comparative Survey of Bottom-Up Proteomics Sample Preparation Methods.

Similar Datasets

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