an-integrated-workflow-identification-quantification-on-fdr-control

An Integrated Workflow of Identification and Quantification on FDR Control-Based Untargeted Metabolome

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an ...

jinan university

In most proteome mass spectrometry experiments, more than half of the mass spectra cannot be identified, mainly because of various modifications. The open search strategy allows for a larger precursor tolerance to utilize more spectra, especially those with post-translational modifications; however, thorough quality control based on independent information is lacking. Here, we used the "Suspicious Discovery Rate (SDR)" based on translatome sequencing (RNC-seq) as an independent source to reference the proteome open search results in steady-state cells. We found that the open search strategy increased the spectra utilization with the cost of increased suspicious identifications that lack translation evidence. We further found that restricting the peptide FDR below 0.1% efficiently controlled the suspicious identifications of open search methods and thus enhanced the confidence of the peptide identification with modifications comparable to the level of the traditional narrow window search. We then demonstrated the successful and validated identification of 27 single amino acid variations from the spectra of two cell lines using the open search strategy without a predefined database. These results validated the proper use of open search methods for higher-quality proteome identifications with information on post-translational modifications and single amino acid polymorphisms.

Optimal Settings of Mass Spectrometry Open Search Strategy for Higher Confidence.

FDR control has been a huge challenge for large-scale metabolome annotation. Although recent research indicated that the target-decoy strategy could be implemented to estimate FDR, it is hard to perform FDR control due to the difficulty of getting a reliable decoy database because of the complex fragmentation mechanism of metabolites and ubiquitous isomers. To tackle this problem, we developed a decoy generation method, which generates forged spectra from the reference target database by preserving the original reference signals to simulate the presence of isomers of metabolites. Benchmarks on GNPS data sets in Passatutto showed that the decoy database generated by our method is closer to the actual FDR than other methods, especially in the low FDR range (0-0.05). Large-scale metabolite annotation on 35 data sets showed that strict FDR reduced the number of annotated metabolites but increased the spectral efficiency, indicating the necessity of quality control. We recommended that the FDR threshold should be set to 0.01 in large-scale metabolite annotation. We implemented decoy generation, database search, and FDR control into a search engine called XY-Meta. It facilitates large-scale metabolome annotation applications.

XY-Meta: A High-Efficiency Search Engine for Large-Scale Metabolome Annotation with Accurate FDR Estimation.

Proficient mismatch repair (pMMR) colorectal adenocarcinoma (CRAC) metastasizes to a greater extent than MMR-deficient CRAC. Prognostic biomarkers are preferred in clinical practice. However, traditional biomarkers screened directly from sequencing are often not robust and thus cannot be confidently utilized.

Robust Glycogene-Based Prognostic Signature for Proficient Mismatch Repair Colorectal Adenocarcinoma.

Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms. Taking the advantage of read length, short-read RNC-seq identified up to 15,289 genes and 15,906 AS isoforms in a single human cell line, much more than the Ribo-seq. The single-molecule long-read RNC-seq supplemented 4,429 annotated AS isoforms that were not identified by short-read datasets, and 4,525 novel AS isoforms that were not included in the public databases. Using such RNC-seq-guided database, we identified 6,766 annotated protein isoforms and 50 novel protein isoforms in mass spectrometry datasets. These results demonstrated the potential of full-length RNC-seq in investigating the proteome of AS isoforms.

Article	Year
Optimal Settings of Mass Spectrometry Open Search Strategy for Higher Confidence. Journal of proteome research\| PubMed ID: 30265008	2018
XY-Meta: A High-Efficiency Search Engine for Large-Scale Metabolome Annotation with Accurate FDR Estimation. Analytical chemistry\| PubMed ID: 32212716	2020
Robust Glycogene-Based Prognostic Signature for Proficient Mismatch Repair Colorectal Adenocarcinoma. Frontiers in oncology\| PubMed ID: 34692502	2021
Efficient Detection of the Alternative Spliced Human Proteome Using Translatome Sequencing. Frontiers in molecular biosciences\| PubMed ID: 35720116	2022

Dehua Li

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