Proteogenomic Analysis to Identify Missing Proteins from Haploid Cell Lines

Abstract

Chromosome-centric Human Proteome Project aims at identifying and characterizing protein products encoded from all human protein-coding genes. As of early 2017, 19 837 protein-coding genes have been annotated in the neXtProt database including 2691 missing proteins that have never been identified by mass spectrometry. Missing proteins may be low abundant in many cell types or expressed only in a few cell types in human body such as sperms in testis. In this study, we performed expression proteomics of two near-haploid cell types such as HAP1 and KBM-7 to hunt for missing proteins. Proteomes from the two haploid cell lines were analyzed on an LTQ Orbitrap Velos, producing a total of 200 raw mass spectrometry files. After applying 1% false discovery rates at both levels of peptide-spectrum matches and proteins, more than 10 000 proteins were identified from HAP1 and KBM-7, resulting in the identification of nine missing proteins. Next, unmatched spectra were searched against protein databases translated in three frames from noncoding RNAs derived from RNA-Seq data, resulting in six novel protein-coding regions after careful manual inspection. This study demonstrates that expression proteomics coupled to proteogenomic analysis can be employed to identify many annotated and unannotated missing proteins.

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry

“…279 The results obtained by MD approach can be integrated with the transcriptome and genomic data, not only to identify unannotated missing proteins and/or to conrm annotated coding regions in the genome, but also to correct either the erroneously annotated gene sequences or to correct the proteome sequence databases. [280][281][282] In this connection, there is a huge scope for MD proteomic approach to contribute immensely for the development of this upcoming eld, since investigations thus far involving proteogenomics have mostly followed the BU proteomic approach only. 281,283,284 Additionally, it needs to be noted that there has been no accurate denition for MD approach, which could clearly demarcate the boundary between BU and MD approaches.…”

“…[280][281][282] In this connection, there is a huge scope for MD proteomic approach to contribute immensely for the development of this upcoming eld, since investigations thus far involving proteogenomics have mostly followed the BU proteomic approach only. 281,283,284 Additionally, it needs to be noted that there has been no accurate denition for MD approach, which could clearly demarcate the boundary between BU and MD approaches. For instance, Tsybin and co-workers dened or proposed another approach called 'Extended Bottom-Up', which may be regarded as an offshoot of MD approach.…”

Integration of large-scale multi-omic datasets: A protein-centric view

“…For example, a study in the parasite Blastocystis identified seven new proteins using peptide information at the C-terminal where part of the UAA termination codons are derived by transcript polyadenylation [33]. Other efforts in human cells searched unmatched mass spectra from proteomics experiments against RNA-seq data that was computationally translated into all three possible open reading frames to identify short, missing proteins, identifying a handful of such microproteins [34,35]. Another study used a specific proteomic approach that enriched for the N-terminal ends of proteins that are often missed in both RNA sequencing and proteomics experiments.…”

Dynamics of dual specificity phosphatases and their interplay with protein kinases in immune signaling

“…Systems biology and integrative biology offer several approaches to identify molecular mechanisms operating behind biological processes in unprecedented detail. Integrated approaches such as Proteogenomics can provide macroresolution snapshots to facilitate understanding of intricate molecular mechanisms in cancers [25,26] and infectious diseases [27,28]. Applying integrated approaches in the context of immunology can therefore offer unique insights into mechanisms of innate and adaptive immunity.…”