scholarly journals Metabolic Labeling of Prenylated Proteins Using Alkyne-Modified Isoprenoid Analogues

2018 ◽  
Vol 10 (3) ◽  
pp. e46 ◽  
Author(s):  
Kiall F. Suazo ◽  
Alexander K. Hurben ◽  
Kevin Liu ◽  
Feng Xu ◽  
Pa Thao ◽  
...  
Keyword(s):  
Metabolic Labeling ◽  
Prenylated Proteins

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

2020 ◽  
Vol 19 (6) ◽  
pp. 1005-1016 ◽  
Author(s):  
James A. Wilkins ◽  
Krista Kaasik ◽  
Robert J. Chalkley ◽  
Alma L. Burlingame
Keyword(s):  
Cell Culture ◽  
Posttranslational Modifications ◽  
High Performance ◽  
Direct Approach ◽  
Metabolic Labeling ◽  
Tissue Samples ◽  
Mixed Disulfide ◽  
Cell Extracts ◽  
Prenylated Proteins

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.

scholarly journals Characterization of Prenylated C-terminal Peptides Using a Novel Capture Technique Coupled with LCMS

2020 ◽  
Author(s):  
James A. Wilkins ◽  
Krista Kaasik ◽  
Robert J. Chalkley ◽  
Alma Burlingame
Keyword(s):  
Cell Culture ◽  
High Performance ◽  
Direct Approach ◽  
Metabolic Labeling ◽  
Tissue Samples ◽  
Mixed Disulfide ◽  
Cell Extracts ◽  
Protein Family Database ◽  
Prenylated Proteins

Post-translational 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 labelling 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 labelling. 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 LCMS without the need for metabolic labeling and derivatization.

scholarly journals A combination of metabolic labeling and 2D-DIGE analysis in response to a farnesyltransferase inhibitor facilitates the discovery of new prenylated proteins

2014 ◽  
Vol 10 (5) ◽  
pp. 1094-1103 ◽  
Author(s):  
Charuta C. Palsuledesai ◽  
Joshua D. Ochocki ◽  
Todd W. Markowski ◽  
Mark D. Distefano
Keyword(s):  
Metabolic Labeling ◽  
Farnesyltransferase Inhibitor ◽  
2D Dige ◽  
Prenylated Proteins ◽  
Inhibitor Treatment ◽  
Dige Analysis

Prenylated proteins sensitive to farnesyltransferase inhibitor treatment were identified by combining metabolic labeling with 2D-DIGE. Using this method, GNAI-1 and GNAI-2 were identified as potential novel prenylated proteins.

scholarly journals Metabolic labeling with an alkyne probe reveals similarities and differences in the prenylomes of several brain-derived cell lines and primary cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kiall F. Suazo ◽  
Angela Jeong ◽  
Mina Ahmadi ◽  
Caroline Brown ◽  
Wenhui Qu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Metabolic Labeling ◽  
Primary Cells ◽  
Protein Prenylation ◽  
Single Experiment ◽  
Related Cell ◽  
Proteomic Approach ◽  
C Terminus ◽  
Similarities And Differences ◽  
Prenylated Proteins

AbstractProtein prenylation involves the attachment of one or two isoprenoid group(s) onto cysteine residues positioned near the C-terminus. This modification is essential for many signal transduction processes. In this work, the use of the probe C15AlkOPP for metabolic labeling and identification of prenylated proteins in a variety of cell lines and primary cells is explored. Using a single isoprenoid analogue, 78 prenylated protein groups from the three classes of prenylation substrates were identified including three novel prenylation substrates in a single experiment. Applying this method to three brain-related cell lines including neurons, microglia, and astrocytes showed substantial overlap (25%) in the prenylated proteins identified. In addition, some unique prenylated proteins were identified in each type. Eight proteins were observed exclusively in neurons, five were observed exclusively in astrocytes and three were observed exclusively in microglia, suggesting their unique roles in these cells. Furthermore, inhibition of farnesylation in primary astrocytes revealed the differential responses of farnesylated proteins to an FTI. Importantly, these results provide a list of 19 prenylated proteins common to all the cell lines studied here that can be monitored using the C15AlkOPP probe as well as a number of proteins that were observed in only certain cell lines. Taken together, these results suggest that this chemical proteomic approach should be useful in monitoring the levels and exploring the underlying role(s) of prenylated proteins in various diseases.

Stable isotope metabolic labeling of a mouse model reveals synaptic biomarkers for anxiety disorders

2009 ◽  
Vol 42 (05) ◽  
Author(s):  
MD Filiou ◽  
YY Zhang ◽  
B Bisle ◽  
E Frank ◽  
MS Kessler ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Mouse Model ◽  
Anxiety Disorders ◽  
Metabolic Labeling

scholarly journals High Glycogen Levels in Brains of Rats with Minimal Environmental Stimuli: Implications for Metabolic Contributions of Working Astrocytes

2002 ◽  
Vol 22 (12) ◽  
pp. 1476-1489 ◽  
Author(s):  
Nancy F. Cruz ◽  
Gerald A. Dienel
Keyword(s):  
Rat Brain ◽  
Sensory Stimulation ◽  
Enzyme Inactivation ◽  
Biologically Active ◽  
Metabolic Labeling ◽  
Tissue Sampling ◽  
Normal Rat ◽  
Sampling Procedures ◽  
Very High

The concentration of glycogen, the major brain energy reserve localized mainly in astrocytes, is generally reported as about 2 or 3 μmol/g, but sometimes as high as 3.9 to 8 μmol/g, in normal rat brain. The authors found high but very different glycogen levels in two recent studies in which glycogen was determined by the routine amyloglucosidase procedure in 0.03N HCl digests either of frozen powders (4.8 to 6 μmol/g) or of ethanol-insoluble fractions (8 to 12 μmol/g). To evaluate the basis for these discrepant results, glycogen was assayed in parallel extracts of the same samples. Glycogen levels in ethanol extracts were twice those in 0.03N HCl digests, suggesting incomplete enzyme inactivation even with very careful thawing. The very high glycogen levels were biologically active and responsive to physiologic and pharmacological challenge. Glycogen levels fell after brief sensory stimulation, and metabolic labeling indicated its turnover under resting conditions. About 95% of the glycogen was degraded under in vitro ischemic conditions, and its “carbon equivalents” recovered mainly as glc, glc-P, and lactate. Resting glycogen stores were reduced by about 50% by chronic inhibition of nitric oxide synthase. Because neurotransmitters are known to stimulate glycogenolysis, stress or sensory activation due to animal handling and tissue-sampling procedures may stimulate glycogenolysis during an experiment, and glycogen lability during tissue sampling and extraction can further reduce glycogen levels. The very high glycogen levels in normal rat brain suggest an unrecognized role for astrocytic energy metabolism during brain activation.

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