Global Mass Spectrometry and Transcriptomics Array Based Drug Profiling Provides Novel Insight into Glucosamine Induced Endoplasmic Reticulum Stress

Abstract

We investigated the molecular effects of glucosamine supplements, a popular and safe alternative to nonsteroidal anti-inflammatory drugs, for decreasing pain, inflammation, and maintaining healthy joints. Numerous studies have reported an array of molecular effects after glucosamine treatment. We questioned whether the differences in the effects observed in previous studies were associated with the focus on a specific subproteome or with the use of specific cell lines or tissues. To address this question, global mass spectrometry-and transcription arraybased glucosamine drug profiling was performed on malignant cell lines from different stages of lymphocyte development. We combined global label-free MS-based protein quantitation with an open search for modifications to obtain the best possible proteome coverage. Our data were largely consistent with previous studies in a variety of cellular models. We mainly observed glucosamine induced O-GlcNAcylation/O-GalNAcylation (O-HexNAcylation); however, we also observed global and local changes in acetylation, methylation, and phosphorylation. (4,5). The clinical relevance of GlcN, a dietary supplement used in osteoarthritis patients, is unclear (6). Previous investigations of long-term GlcN administration did not reveal risks or concerns (7), which is also consistent with recent studies (8). However, in recent clinical studies, GlcN oral administration (8) did not demonstrate any benefits, and the concentration used in vitro was not comparable with the levels observed in the plasma in vivo after the oral administration of GlcN, which raised skepticism (8, 9). Therefore,

New insights into functional regulation in MS-based drug profiling

“…The subcellular fractions are designated MCCT/MCCTT, sN/sNT, MT/MTT, MC/MCT and iN/iNT, where the absence or presence of ending “T” indicate control or treated, respectively. The fractions MCCT, MT and sN were previously validated by Western blot which confirmed that the expected proteins were indeed enriched 5 . The fractions MCCT, MT and sN were also found, based on the identified proteins, to mainly contain cytosolic, mitochondrial and nuclear proteins, respectively.…”

“…Currently employed approaches for global drug profiling include methods based on epigenomics by next generation sequencing 1 , transcriptomics using either microarrays or next generation sequencing 2 and mass spectrometry for profiling proteins 3 and metabolites 4 . Global proteome MS-based drug profiling was originally grounded on 2D gel electrophoresis for separation and quantitation followed by mass spectrometry based identification 5 . With the latest generation of sensitive and high resolution accurate mass spectrometers, new methods are emerging which can be divided into two main methodologies: (1) pre-fractionation of peptides and/or (2) pre-fractionation of proteins previous to LC-MS. Multi-dimensional liquid chromatography 6 7 and isoelectric focusing 8 are examples of peptide pre-fractionation methods.…”

“…With the latest generation of sensitive and high resolution accurate mass spectrometers, new methods are emerging which can be divided into two main methodologies: (1) pre-fractionation of peptides and/or (2) pre-fractionation of proteins previous to LC-MS. Multi-dimensional liquid chromatography 6 7 and isoelectric focusing 8 are examples of peptide pre-fractionation methods. One-dimensional SDS-polyacrylamide gel electrophoresis 9 10 , size exclusion chromatography 11 and to a less extent subcellular fractionation 5 10 have been used to resolve protein mixtures prior to LC-MS analysis.…”

Overview of proteomics studies in obstructive sleep apnea

“…3. This was accomplished by using state-of-the-art mass spectrometers and by using an open search for modifications when matching MS/MS spectra against sequence databases [46–48]. …”

Post-translational Modifications Regulate Class IIa Histone Deacetylase (HDAC) Function in Health and Disease

“…In recent years, the class IIa HDACs have been established as critical regulators of numerous cellular processes (14,15), with misregulation being associated with a wide range of human diseases. Roles for class IIa HDACs have been reported in the development of cardiovascular disease (16), cancer (17), immune response to viral infection (18), epigenetic response to drug stimulus (19), diabetes (20), and neurodegenerative diseases, such as Huntington's disease (21).…”