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Characterization of Prenylated C-terminal Peptides Using a Thiopropyl-based Capture Technique and LC-MS/MS.

ABSTRACT: Posttranslational modifications play a critical and diverse role in regulating cellular activities. Despite their fundamentally important role in cellular function, there has been no report to date of an effective generalized approach to the targeting, extraction, and characterization of the critical c-terminal regions of natively prenylated proteins. Various chemical modification and metabolic labeling strategies in cell culture have been reported. However, their applicability is limited to cell culture systems and does not allow for analysis of tissue samples. The chemical characteristics (hydrophobicity, low abundance, highly basic charge) of many of the c-terminal regions of prenylated proteins have impaired the use of standard proteomic workflows. In this context, we sought a direct approach to the problem in order to examine these proteins in tissue without the use of labeling. Here we demonstrate that prenylated proteins can be captured on chromatographic resins functionalized with mixed disulfide functions. Protease treatment of resin-bound proteins using chymotryptic digestion revealed peptides from many known prenylated proteins. Exposure of the protease-treated resin to reducing agents and hydro organic mixtures released c-terminal peptides with intact prenyl groups along with other enzymatic modifications expected in this protein family. Database and search parameters were selected to allow for c-terminal modifications unique to these molecules such as CAAX box processing and c-terminal methylation. In summary, we present a direct approach to enrich and obtain information at a molecular level of detail about prenylation of proteins from tissue and cell extracts using high-performance LC-MS without the need for metabolic labeling and derivatization.

SUBMITTER: Wilkins JA

PROVIDER: S-EPMC7261820 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Characterization of Prenylated C-terminal Peptides Using a Thiopropyl-based Capture Technique and LC-MS/MS.

Wilkins James A JA Kaasik Krista K Chalkley Robert J RJ Burlingame Alma L AL

Molecular & cellular proteomics : MCP 20200413 6

Posttranslational modifications play a critical and diverse role in regulating cellular activities. Despite their fundamentally important role in cellular function, there has been no report to date of an effective generalized approach to the targeting, extraction, and characterization of the critical c-terminal regions of natively prenylated proteins. Various chemical modification and metabolic labeling strategies in cell culture have been reported. However, their applicability is limited to cel ...[more]

PMID: 32284353

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Project description:BackgroundThe human brain is the most complex organ in the body, and it is important to have a better understanding of how the protein composition in the brain regions contributes to the pathogenesis of associated neurological disorders.MethodsIn this study, a comparative analysis of the frontal and temporal cortex proteomes was conducted by isobaric tags of relative and absolute quantification (iTRAQ) labeling and two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Brain protein was taken from relatively normal tissue that could not be avoided of damage during emergent surgery of the TBI (traumatic brain injury) patients admitted in Beijing Tiantan Hospital from 2014 to 2017. Eight cases were included. Four frontal lobes and 4 temporal lobes proteome were analyzed and the proteins were quantitated. Gene Ontology (GO), Ingenuity Pathway Analysis (IPA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to analyze the biological function of identified proteins, unchanged proteins, and differentially expressed proteins (DEPs).ResultsA total number of 2127 protein groups were identified in the frontal and temporal lobe proteomes. A total of 1709 proteins could be quantitated in both the frontal and temporal cortex. Among 90 DEPs, 14 proteins were screened highly expressed in the temporal cortex, including MAPT, SNCG, ATP5IF1, GAP43, HSPE1, STMN1, NDUFS6, LDHB, SNCB, NDUFA7, MRPS36, EPDR1, CISD1, and RALA. In addition, compared to proteins expressed in the frontal cortex, 14 proteins including EDC4, NIT2, VWF, ASTN1, TGM2, SSB, CLU, HBA1, STOM, CRP, LRG1, SAA2, S100A4, and VTN were a low expression in the temporal cortex. The biological process enrichment showed that unchanged proteins between the frontal and temporal cortex mainly take part in regulated exocytosis, axon guidance, and vesicle-mediated transport. The KEGG pathway analysis showed that unchanged proteins between the frontal and temporal cortex mainly take part in oxidative phosphorylation, carbon metabolism, Huntington's disease, and Parkinson's disease.ConclusionsThe majority of proteins are unchanged between the frontal and temporal cortex, and unchanged proteins are closely related to its function. Among DEPs, MATP (tau) is upregulated in the temporal cortex, closely related to Alzheimer's disease (AD), and is one of the targets for the treatment of AD. CLU is downregulated in the temporal cortex which functions as an extracellular chaperone that prevents aggregation of non-native proteins. It was suggested that the temporal lobe may not be the "functional dumb area" of the traditional view, but could be involved in important neural metabolic circuits.

Dataset Information

Characterization of Prenylated C-terminal Peptides Using a Thiopropyl-based Capture Technique and LC-MS/MS.

Publications

Characterization of Prenylated C-terminal Peptides Using a Thiopropyl-based Capture Technique and LC-MS/MS.

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