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Development of a maleimide-based chemical proteomic method for quantitative analysis of cysteine reactivity

ABSTRACT: Cysteine is the most intrinsically nucleophilic residue in proteins and serves as a sentinel against increasing reactive oxygen species (ROS) via reversible thiol oxidation. Despite the importance of Cys oxidation in understanding biological stress response, it remains largely unknown and a major analytical challenge to determine which protein Cys-sites are the most reactive toward ROS. Herein, we describe initial coverage and sensitivity of a chemical proteomic method to quantify site-specific Cys reactivity using a maleimide-activated, thiol-specific probe (N-propargylmaleimide, NPM).

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Chlamydomonas Reinhardtii

TISSUE(S): Photosynthetic Cell

SUBMITTER: Evan McConnell

LAB HEAD: Leslie M. Hicks

PROVIDER: PXD015409 | Pride | 2020-10-20

REPOSITORIES: Pride

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			Action	DRS
	20190425_EWM_NPM9_250_ELU_ABC_2h_5-35_5uL.mgf	Mgf
	20190425_EWM_NPM9_250_ELU_ABC_2h_5-35_5uL.mzid.gz	Mzid
	20190425_EWM_NPM9_250_ELU_ABC_2h_5-35_5uL.pride.mgf.gz	Mgf
	20190425_EWM_NPM9_250_ELU_ABC_2h_5-35_5uL.pride.mztab.gz	Mztab
	20190425_EWM_NPM9_250_ELU_ABC_2h_5-35_5uL.wiff	Wiff

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Project description:BACKGROUND: Microarray comparative genomic hybridization (CGH) is currently one of the most powerful techniques to measure DNA copy number in large genomes. In humans, microarray CGH is widely used to assess copy number variants in healthy individuals and copy number aberrations associated with various diseases, syndromes and disease susceptibility. In model organisms such as Caenorhabditis elegans (C. elegans) the technique has been applied to detect mutations, primarily deletions, in strains of interest. Although various constraints on oligonucleotide properties have been suggested to minimize non-specific hybridization and improve the data quality, there have been few experimental validations for CGH experiments. For genomic regions where strict design filters would limit the coverage it would also be useful to quantify the expected loss in data quality associated with relaxed design criteria. RESULTS: We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10 C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data quality when varying the oligonucleotide length between 50 and 70, and even when using an isothermal design strategy. CONCLUSIONS: We have determined experimentally the effects of varying several key oligonucleotide microarray design criteria for detection of deletions in C. elegans and humans with NimbleGen's CGH technology. Our oligonucleotide design recommendations should be applicable for CGH analysis in most species. One-copy deletions were studied in both the C. elegans (2 samples) and human (1 sample) genomes. There were no replicate or dye-swap hybridizations.

Project description:Numerous reagents have been developed to enable chemical proteomic analysis of small molecule-protein interactomes. However, the performance of these reagents has not been systematically evaluated and compared. Herein, we report our efforts to conduct a parallel assessment of two widely-used chemically-cleavable linkers equipped with dialkoxydiphenylsilane (DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular cysteinome using iodoacetamide alkyne probe demonstrated a significant discrepancy between the experimental results obtained through the application of each of the reagents. To better understand the source of observed discrepancy, a mass tolerant database search strategy using MSFragger software was performed. This resulted in identifying a previously unreported artifactual modification on the residual mass of the azobenzene linker. Furthermore, we conducted a comparative analysis of enrichment modes using both cleavable linkers. This effort determined that enrichment of proteolytic digests yielded a far greater number of identified cysteine residues than the enrichment conducted prior to protein digest. Inspired by recent studies where multiplexed quantitative labeling strategies were applied to cleavable biotin linkers, we combined this further optimized protocol using the DADPS cleavable linker with tandem mass tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase inhibitor dacomitinib against the cysteinome of an epidermoid cancer cell line. Our analysis resulted in the detection and quantification of over 10,000 unique cysteine residues, a nearly 3-fold increase over previous studies that used cleavable biotin linkers for enrichment. Critically, cysteine residues corresponding to proteins directly as well as indirectly modulated by dacomitinib treatment were identified. Overall, our study suggests that the dialkoxydiphenylsilane linker could be broadly applied wherever chemically cleavable linkers are required for chemical proteomic characterization of cellular proteomes.

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Development of a maleimide-based chemical proteomic method for quantitative analysis of cysteine reactivity

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