Dataset Information

Trace sample analysis using Data-Dependent Acquisition Without Dynamic Exclusion

ABSTRACT: In this study, we report the application of turboDDA in improving trace sample analysis. Using different amount of K562 samples, we detected increases in identified protein number of more than 20% compared to all samples analyzed by standard DDA with dynamic exclusion.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ci Wu

LAB HEAD: Shen Zhang

PROVIDER: PXD042385 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

Trace Sample Proteome Quantification by Data-Dependent Acquisition without Dynamic Exclusion.

Wu Ci C Lei Jiao J Meng Fei F Wang Xingyao X Wong Cassandra J CJ Peng Jiaxi J Lin Ge G Gingras Anne-Claude AC Ma Junfeng J Zhang Shen S

Analytical chemistry 20231130 49

Despite continuous technological improvements in sample preparation, mass-spectrometry-based proteomics for trace samples faces the challenges of sensitivity, quantification accuracy, and reproducibility. Herein, we explored the applicability of turboDDA (a method that uses data-dependent acquisition without dynamic exclusion) for quantitative proteomics of trace samples. After systematic optimization of acquisition parameters, we compared the performance of turboDDA with that of data-dependent ...[more]

PMID: 38032138

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Project description:The functional diversity of crustacean hemocyanins is broad, encompassing O2 delivery, innate immune response, metabolite storage, and osmolyte balance, all in a heterogeneous protein structure. As such, the sequence diversity of this class of proteins and its subunit composition are the focus of many studies on crustacean adaptation to environmental challenges. Recent transcriptomic and genomic sequencing on the Pacific whiteleg shrimp Litopenaeus vannamei has identified unique isoforms of hemocyanin including an ancestral β-type subunit thought to be lost in penaeid shrimp. However, it is unknown the degree to which these isoforms are translated as proteins, and whether they differ in function. The present study uses proteomic approaches to characterize the protein-level abundance and organization of these hemocyanin isoforms within their native oligomeric structures. Fractions of each hemocyanin oligomeric form were purified by size-exclusion high performance liquid chromatography for identification of subunit isoforms using tandem mass spectrometry at <1% protein false discovery rate. Relative abundances of hemocyanin oligomers and monomeric subunits from hemolymph and fractions were also quantified by polyacrylamide gel electrophoresis with and without denaturation for comparison of relative abundance. Ten hemocyanin isoforms were identified by tandem mass spectrometry in both hemocyanin oligomer fractions including a single small subunit, eight large subunits, and the first protein-level evidence of a β-type subunit in penaeid shrimp. Hemocyanin subunits were organized primarily as hexamers (95-99% relative abundance) as opposed to dodecamers. Hexamers utilized a significantly higher ratio (2.05:1) of small subunit to large subunit compared to dodecamers (1.04:1), and the relative abundances of the large subunit isoforms was dominated by HcL1 in both fractions. The ability to distinguish and quantify hemocyanin isoforms within oligomeric structures will aid future studies linking hemocyanin genes to function and physiology as well as offer insight into the evolutionary history of crustaceans.

Dataset Information

Trace sample analysis using Data-Dependent Acquisition Without Dynamic Exclusion

Publications

Trace Sample Proteome Quantification by Data-Dependent Acquisition without Dynamic Exclusion.

Similar Datasets

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