Integrated analysis of global proteome, phosphoproteome, and glycoproteome enables complementary interpretation of disease-related protein networks

Multi-dimensional proteomic analyses provide different layers of protein information, including protein abundance and post-translational modifications. Here, we report an integrated analysis of protein expression, phosphorylation, and N-glycosylation by serial enrichments of phosphorylation and N-glycosylation (SEPG) from the same tissue samples. On average, the SEPG identified 142,106 unmodified peptides of 8,625 protein groups, 18,846 phosphopeptides (15,647 phosphosites), and 4,019 N-glycopeptides (2,634 N-glycosites) in tumor and adjacent normal tissues from three gastric cancer patients. The combined analysis of these data showed that the integrated analysis additively improved the coverages of gastric cancer-related protein networks; phosphoproteome and N-glycoproteome captured predominantly low abundant signal proteins, and membranous or secreted proteins, respectively, while global proteome provided abundances for general population of the proteome. Therefore, our results demonstrate that the SEPG can serve as an effective approach for multi-dimensional proteome analyses, and the holistic profiles of protein expression and PTMs enabled improved interpretation of disease-related networks by providing complementary information.11103Ysciescopu

Reinspection of a Clinical Proteomics Tumor Analysis Consortium (CPTAC) Dataset with Cloud Computing Reveals Abundant Post-Translational Modifications and Protein Sequence Variants.

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) has provided some of the most in-depth analyses of the phenotypes of human tumors ever constructed. Today, the majority of proteomic data analysis is still performed using software housed on desktop computers which limits the number of sequence variants and post-translational modifications that can be considered. The original CPTAC studies limited the search for PTMs to only samples that were chemically enriched for those modified peptides. Similarly, the only sequence variants considered were those with strong evidence at the exon or transcript level. In this multi-institutional collaborative reanalysis, we utilized unbiased protein databases containing millions of human sequence variants in conjunction with hundreds of common post-translational modifications. Using these tools, we identified tens of thousands of high-confidence PTMs and sequence variants. We identified 4132 phosphorylated peptides in nonenriched samples, 93% of which were confirmed in the samples which were chemically enriched for phosphopeptides. In addition, our results also cover 90% of the high-confidence variants reported by the original proteogenomics study, without the need for sample specific next-generation sequencing. Finally, we report fivefold more somatic and germline variants that have an independent evidence at the peptide level, including mutations in ERRB2 and BCAS1. In this reanalysis of CPTAC proteomic data with cloud computing, we present an openly available and searchable web resource of the highest-coverage proteomic profiling of human tumors described to date

The role of DNA microarrays in Toxoplasma gondii research, the causative agent of ocular toxoplasmosis

Ocular toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, is the leading cause of retinochoroiditis. Toxoplasma is an obligate intracellular pathogen that replicates within a parasitophorous vacuole. Infections are initiated by digestion of parasites deposited in cat feces or in undercooked meat. Parasites then disseminate to target tissues that include the retina where they then develop into long-lived asymptomatic tissue cysts. Occasionally, cysts reactivate and growth of newly emerged parasites must be controlled by the host’s immune system or disease will occur. The mechanisms by which Toxoplasma grows within its host cell, encysts, and interacts with the host’s immune system are important questions. Here, we will discuss how the use of DNA microarrays in transcriptional profiling, genotyping, and epigenetic experiments has impacted our understanding of these processes. Finally, we will discuss how these advances relate to ocular toxoplasmosis and how future research on ocular toxoplasmosis can benefit from DNA microarrays

Compact variant-rich customized sequence database and a fast and sensitive database search for efficient proteogenomic analyses

In proteogenomic analysis, construction of a compact, customized database from mRNA-seq data and a sensitive search of both reference and customized databases are essential to accurately determine protein abundances and structural variations at the protein level. However, these tasks have not been systematically explored, but rather performed in an ad-hoc fashion. Here, we present an effectivemethod for constructing a compact database containing comprehensive sequences of sample-specific variants—single nucleotide variants, insertions/deletions, and stop-codon mutations derived from Exome-seq and RNA-seq data. It, however, occupies less space by storing variant peptides, not variant proteins. We also present an efficient search method for both customized and reference databases. The separate searches of the two databases increase the search time, and a unified search is less sensitive to identify variant peptides due to the smaller size of the customized database, compared to the reference database, in the target-decoy setting. Our method searches the unified database once, but performs targetdecoy validations separately. Experimental results show that our approach is as fast as the unified search and as sensitive as the separate searches. Our customized database includes mutation information in the headers of variant peptides, thereby facilitating the inspection of peptide-spectrum matches.1991sciescopu

Compact variant-rich customized sequence database and a fast and sensitive database search for efficient proteogenomic analyses

In proteogenomic analysis, construction of a compact, customized database from mRNA-seq data and a sensitive search of both reference and customized databases are essential to accurately determine protein abundances and structural variations at the protein level. However, these tasks have not been systematically explored, but rather performed in an ad-hoc fashion. Here, we present an effective method for constructing a compact database containing comprehensive sequences of sample-specific variants-single nucleotide variants, insertions/deletions, and stop-codon mutations derived from Exome-seq and RNA-seq data. It, however, occupies less space by storing variant peptides, not variant proteins. We also present an efficient search method for both customized and reference databases. The separate searches of the two databases increase the search time, and a unified search is less sensitive to identify variant peptides due to the smaller size of the customized database, compared to the reference database, in the target-decoy setting. Our method searches the unified database once, but performs target-decoy validations separately. Experimental results show that our approach is as fast as the unified search and as sensitive as the separate searches. Our customized database includes mutation information in the headers of variant peptides, thereby facilitating the inspection of peptide-spectrum matches. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.