The Art of Validating Quantitative Proteomics Data

Handler, David C. L.; Pascovici, Dana; Mirzaei, Mehdi; Gupta, Vivek; Salekdeh, Ghasem Hosseini; Haynes, Paul A.

doi:10.1002/pmic.201800222

Cited by 27 publications

(22 citation statements)

References 22 publications

(31 reference statements)

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“…To validate the MS/MS proteomic results, the expression levels of CYR61, laminin subunit β-2 and superoxide dismutase were assessed by immunofluorescence and immunoblotting in both culture groups. The rationale behind selecting targets to validate by Western blot was to verify relevant biological aspects of the proteomic dataset, as recommended elsewhere [ 22 ], instead of just validating the most abundant proteins. Because the proteomic dataset indicated that proteins related to inflammation, differentiation, cell adhesion and superoxide dismutase were upregulated in the sericin-treated group, targets to be validated by immunoblotting were chosen to represent some of these biological functions.…”

Section: Resultsmentioning

confidence: 99%

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins

et al. 2020

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We previously demonstrated that the silk protein sericin promotes pigmentation of retinal pigment epithelium (RPE) by activating the NF-κB pathway. Among numerous agents, NF-κB can be activated by hydrogen peroxide. In the present study, we explored possible associations between reactive oxygen species and sericin-induced melanogenesis in RPE. The proteome of human fetal RPE cultured for seven days with or without 1% sericin was analyzed using ingenuity pathway analysis (IPA). The proteomic data was verified by immunofluorescence and immunoblotting. Light microscopy and scanning electron microscopy were used to assess morphology. Dihydroethidium (DHE) and dihydrorhodamine (DHR) assays were used to measure superoxide and hydrogen peroxide species. Expression levels of proteins related to inflammation, differentiation, cell survival and cell adhesion were higher in cells cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 1% sericin, whereas cells cultured in DMEM alone showed higher expression levels of proteins associated with Bruch’s membrane and cytoskeleton. Despite upregulation of inflammatory proteins, sericin co-cultured RPE yielded significantly higher cell viability compared to cells cultured without sericin. Addition of sericin to culture media significantly increased hydrogen peroxide-levels without significantly affecting superoxide-levels. We suggest that sericin-induced melanogenesis in cultured RPE is associated with elevated levels of superoxide dismutase, hydrogen peroxide and inflammatory proteins.

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Section: Resultsmentioning

confidence: 99%

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins

et al. 2020

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“…If proteomics is considered with equal latitude, this will only result in the identification of new unique variants. If some of these variants prove to be biologically relevant and/or differentially expressed, then manual or secondary verification would be performed as a matter of course [36].…”

Section: Discussionmentioning

confidence: 99%

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

Prakash¹,

Taylor

Varkey³

et al. 2021

Cancers

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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.

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“…Currently, the validation of proteomic results by WB is a popular matter of debate. Although it is robust to affirm that alterations detected by this technique clearly reflect changes in the proteomic profile, it has some major limitations including: (1) the user has to select the proteins with high abundance in the proteomic analysis to increase the probability of validation by WB, which has a lower sensitivity; and (2) the use of housekeeping proteins as internal standard for WB analysis, because their expression may be different in health or disease conditions [ 42 ]. We tried to overcome the above limitations by applying the following criteria: (1) selection of proteins with very low, low, medium, and high abundance; and (2) use of total protein staining as internal standard for WB analysis rather than the expression of a housekeeping protein.…”

Section: Discussionmentioning

confidence: 99%

Distinct Proteomic Profile of Spermatozoa from Men with Seminomatous and Non-Seminomatous Testicular Germ Cell Tumors

Selvam

Alves

Dias

et al. 2020

IJMS

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Testicular germ cell tumors (TGCTs) are predominant in young males (15–44 years). Seminomatous and non-seminomatous TGCTs account for about 98% of all TGCTs cases. In this study, we aimed to compare the sperm proteome of patients with seminomatous and non-seminomatous TGCTs to identify possible protein biomarkers that could help distinguish between them in a non-invasive manner. We analyzed semen samples from patients with seminomatous or non-seminomatous TGCTs (n = 15/group) that were cryopreserved before the start of cancer treatment. Quantitative proteomic analysis was conducted on pooled samples (n = 3/group) and a total of 258 differentially expressed proteins (DEPs) were identified. The overexpression of acrosin precursor (ACR) and chaperonin containing TCP1 subunit 6B (CCT6B) as well as the underexpression of S100 calcium-binding protein A9 (S100A9) in the spermatozoa of patients with non-seminomatous TGCTs were validated by western blotting conducted on individual samples (n = 6 for seminomatous group and n = 6 for non-seminomatous group). Our overall results suggest an association between the higher and faster invasiveness of non-seminomatous TGCTs and the altered protein expressions, providing important information for future studies.

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The Art of Validating Quantitative Proteomics Data

Cited by 27 publications

References 22 publications

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins

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

Distinct Proteomic Profile of Spermatozoa from Men with Seminomatous and Non-Seminomatous Testicular Germ Cell Tumors

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