Proteogenomics: concepts, applications and computational strategies

Journal name:
Nature Methods
Volume:
11,
Pages:
1114–1125
Year published:
DOI:
doi:10.1038/nmeth.3144
Received
Accepted
Published online

Abstract

Proteogenomics is an area of research at the interface of proteomics and genomics. In this approach, customized protein sequence databases generated using genomic and transcriptomic information are used to help identify novel peptides (not present in reference protein sequence databases) from mass spectrometry–based proteomic data; in turn, the proteomic data can be used to provide protein-level evidence of gene expression and to help refine gene models. In recent years, owing to the emergence of new sequencing technologies such as RNA-seq and dramatic improvements in the depth and throughput of mass spectrometry–based proteomics, the pace of proteogenomic research has greatly accelerated. Here I review the current state of proteogenomic methods and applications, including computational strategies for building and using customized protein sequence databases. I also draw attention to the challenge of false positive identifications in proteogenomics and provide guidelines for analyzing the data and reporting the results of proteogenomic studies.

At a glance

Figures

  1. Peptide and protein identification in shotgun proteomics.
    Figure 1: Peptide and protein identification in shotgun proteomics.

    (a) Overview of shotgun proteomics. Proteins are digested into peptides, separated using liquid chromatography (LC) coupled online to a mass spectrometer and then analyzed by the mass spectrometer, which generates tandem mass spectra. (b) Peptides are most commonly identified using a sequence database (DB) search approach. Traditionally, experimental MS/MS spectra are matched with theoretical spectra predicted for each peptide contained in a protein sequence database. Sequence tag–assisted database searching starts with extraction of short tags followed by database searching, in which the list of candidate peptides is restricted to only those peptides that contain one of the extracted sequence tags, thereby allowing for mutations in the sequences of candidate database peptides. Peptide sequence can also be extracted directly from the spectrum using de novo sequencing (extracted sequences can then be searched in a protein sequence database to find the exact or a homologous peptide).

  2. The concept of proteogenomics.
    Figure 2: The concept of proteogenomics.

    In a proteogenomic approach, genomic and transcriptomic data are used to generate customized protein sequence databases to help interpret proteomic data. In turn, the proteomic data provide protein-level validation of the gene expression data and help refine gene models. The enhanced gene models can help improve protein sequence databases for traditional proteomic analysis.

  3. Type of peptides identified in proteogenomics.
    Figure 3: Type of peptides identified in proteogenomics.

    Peptides identified by searching customized protein sequence databases (DB) are mapped on the genome. Intergenic peptides map to regions located between annotated gene models, whereas intragenic peptides map to genomic regions contained within or in close proximity to an annotated gene model. Intragenic peptides can be further categorized according to the annotation of the corresponding gene model (for example, “Protein-coding gene,” “Long noncoding RNA (lncRNA) gene” and “Pseudogene”). The majority of peptides map to a protein-coding gene and can be divided into exon and exon-exon junction peptides. Novel peptides include peptides mapping to untranslated regions (3′ or 5′ UTR peptides), intron peptides, peptides spanning the boundary between the coding sequence region and the neighboring UTR or intron region (“Exon boundary”), peptides spanning unannotated (alternative) splice junctions (“Alt junction”), and out-of-frame peptides (“Alt frame”).

  4. Statistical assessment of peptide identifications in proteogenomics.
    Figure 4: Statistical assessment of peptide identifications in proteogenomics.

    MS/MS spectra (Spec) are searched against a customized protein sequence database (DB) that includes target sequences for the organism of interest, i.e., a reference protein database and predicted protein sequences (containing novel peptides). In addition, two 'decoy' databases (for example, containing reversed sequences) of the same sizes as the target reference and predicted databases are appended to the target databases. The best database peptide match for each spectrum is selected for further analysis. Peptide identifications are classified as known or novel (for a decoy peptide, the class—known or novel—is determined by the class of the corresponding target sequence from which the decoy was generated). When simple database search score–based filtering is used, the numbers of target and decoy peptide identifications passing a certain score threshold are counted and used to estimate the FDR corresponding to that threshold. FDR analysis should be done separately for known and novel peptides (class-specific FDR) because of differences in the number of known and novel sequences in the searched customized sequence database and because of the lower likelihood of correctly identifying a novel peptide. For more advanced methods based on computing posterior peptide probabilities, both the database search scores and the peptide class (known or novel) should be taken into consideration.

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Affiliations

  1. Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.

    • Alexey I Nesvizhskii
  2. Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA.

    • Alexey I Nesvizhskii

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