Nitroproteins in Human Astrocytomas Discovered by Gel Electrophoresis and Tandem Mass Spectrometry

Abstract

Protein tyrosine nitration is involved in the pathogenesis of highly fatal astrocytomas, a type of brain cancer. To understand the molecular mechanisms of astrocytomas and to discover new biomarkers/therapeutic targets, we sought to identify nitroproteins in human astrocytoma tissue. Anti-nitrotyrosine immunoreaction-positive proteins from a high-grade astrocytoma tissue were detected with two-dimensional gel electrophoresis (2DGE)-based nitrotyrosine immunoblots, and identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifty-seven nitrotyrosine immunopositive protein spots were detected. A total of 870 proteins (nitrated and non-nitrated) in nitrotyrosine-immunopositive 2D gel spots were identified, and 18 nitroproteins and their 20 nitrotyrosine sites were identified with MS/MS analysis. These nitroproteins participate in multiple processes, including drug-resistance, signal transduction, cytoskeleton, transcription and translation, cell proliferation and apoptosis, immune response, phenotypic dedifferentiation, cell migration, and metastasis. Among those nitroproteins that might play a role in astrocytomas was nitro-sorcin, which is involved in drug resistance and metastasis and might play a role in the spread and treatment of an astrocytoma. Semiquantitative immune-based measurements of different sorcin expressions were found among different grades of astrocytomas relative to controls, and a semiquantitative increased nitration level in high-grade astrocytoma relative to control. Nitro-β-tubulin functions in cytoskeleton and cell migration. Semiquantitative immunoreactivity of β-tubulin showed increased expression among different grades of astrocytomas relative to controls and semiquantitatively increased nitration level in high-grade astrocytoma relative to control. Each nitroprotein was rationalized and related to the corresponding functional system to provide new insights into tyrosine nitration and its potential role in the pathogenesis of astrocytoma formation. Graphical Abstract ᅟ.

Prolactin Variants in Human Pituitaries and Pituitary Adenomas Identified With Two-Dimensional Gel Electrophoresis and Mass Spectrometry

“…The silver-stained 2D gel was digitized and analyzed with Discovery Series PDQuest 2D Gel Analysis software (15, 21). …”

Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome

“…2D immunoblotting is thus predestined to be used for targeted PTM screening when specific antibodies are available, as is the case for several PTMs, such as nitrosylation or acetylation [43,44,45]. The major challenge of 2D immunoblot-based screening approaches is the correct matching of the two 2D-gels that are typically run in parallel, one for blotting and immunodetection, and the other for protein staining, excision of spots of interest, and mass spectrometric protein identification.…”

Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects

“…specific nitrated proteins and peptides as biomarkers and predictors of specific disease conditions, their possible participation in signaling cascades, and their role in inflammatory, immune and degenerative diseases (66,70,76). This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.…”

“…antibody specificity and mistake when selecting the superimpose place to cut) that may affect the quality and robustness of the experimental result (76).…”

“…Proteins present in those spots (it is always important to remember that one single, well resolved spot in a 2D gel typically contains more than one protein) should be identified by traditional MS methods (61, 76,114). Once identified all the proteins present in that spot, one should be able to predict in silico (software-assisted) ( This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.…”