Application of MALDI-TOF-Mass spectrometry to proteome analysis using stain-free gel electrophoresis
2013, Susnea, Iuliana, Bernevic, Bogdan, Wicke, Michael, Ma, Li, Lui, Shuying, Schellander, Karl, Przybylski, Michael
The combination of MALDI-TOF-mass spectrometry with gel electrophoretic separation using protein visualization by staining procedures involving such as Coomassie Brilliant Blue has been established as a widely used approach in proteomics. Although this approach has been shown to present high detection sensitivity, drawbacks and limitations frequently arise from the significant background in the mass spectrometric analysis. In this chapter we describe an approach for the application of MALDI-MS to the mass spectrometric identification of proteins from one-dimensional (1D) and two-dimensional (2D) gel electrophoretic separation, using stain-free detection and visualization based on native protein fluorescence. Using the native fluorescence of aromatic protein amino acids with UV transmission at 343 nm as a fast gel imaging system, unstained protein spots are localized and, upon excision from gels, can be proteolytically digested and analyzed by MALDI-MS. Following the initial development and testing with standard proteins, applications of the stain-free gel electrophoretic detection approach to mass spectrometric identification of biological proteins from 2D-gel separations clearly show the feasibility and efficiency of this combination, as illustrated by a proteomics study of porcine skeleton muscle proteins. Major advantages of the stain-free gel detection approach with MALDI-MS analysis are (1) rapid analysis of proteins from 1D- and 2D-gel separation without destaining required prior to proteolytic digestion, (2) the low detection limits of proteins attained, and (3) low background in the MALDI-MS analysis.
Site Specific Identification of N-Linked Glycosylation in Proteins by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry
2008, Perdivara, Irina, Iacob, Roxana Elena, Przybylski, Michael, Tomer, Kenneth B.
Recently, we reported the characterization of the glycans attached at the 11 N-glycosylation sites of Hepatitis C virus E2 envelope glycoprotein by tandem mass spectrometry. Infections caused by Hepatitis C virus represent the main cause of liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma. The N-linked sugars consist primarily of high mannose glycans, with structures ranging from the minimal core structure, Man3GlcNAc2 (Man3) up to 12 hexose residues attached to the GlcNAc-ß(l 4)-GlcNAc core (depicted as Hex3Man9GlcNAc2). Furthermore, the site N41 (N423) was observed to contain complex type glycans with the structures Man3-GlcNAc and Man3-GlcNAcFuc, in addition to the high mannose population Man3 through Man6, while the site N48 (N430) was occupied exclusively with complex type glycans (Man3-Fuc, Man3-GlcNAcFuc and Man3-GlcNAc2Fuc). The present contribution summarizes our experimental observations upon the factors which may have an impact on the CID tandem mass spectra of glycopeptides.
Characterization of oligomerization–aggregation products of neurodegenerative target proteins by ion mobility mass spectrometry
2012, Vlad, Camelia, Iurascu, Marius Ionut, Slamnoiu, Stefan, Hengerer, Bastian, Przybylski, Michael
Protein amyloidogenesis is generally considered to be a major cause of two most severe neurodegenerative disorders, Parkinson’s disease (PD) and Alzheimer’s disease (AD). Formation and accumulation of fibrillar aggregates and plaques derived from α-synuclein (α-Syn) and ß-amyloid (Aß) polypeptide in brain have been recognized as characteristics of Parkinson’s disease and Alzheimer’s disease. Oligomeric aggregates of α-Syn and Aß are considered as neurotoxic intermediate products leading to progressive neurodegeneration. However, molecular details of the oligomerization and aggregation pathway(s) and the molecular structure details are still unclear. We describe here the application of ion-mobility mass spectrometry (IMS-MS) to the identification of α-Syn and Aß oligomerization–aggregation products, and to the characterization of different conformational forms. IMS-MS is an analytical technique capable of separating gaseous ions based on their size, shape, and topography. IMS-MS studies of soluble α-Syn and Aß-aggregates prepared by in vitro incubation over several days were performed on a quadrupole time of flight mass spectrometer equipped with a “travelling wave” ion mobility cell, and revealed the presence of different conformational states and, remarkably, truncation and proteolytic products of high aggregating reactivity. These results suggest that different polypeptide sequences may contribute to the formation of oligomeric aggregates of heterogeneous composition and distinct biochemical properties.
Molecular Recognition Specificity of anti-3-nitrotyrosine Antibodies Revealed by Affinity-Mass Spectrometry and Immunoanalytical Methods
2008, Petre, Brînduşa-Alina, Drăguşanu, Mihaela, Przybylski, Michael
Nitration of tyrosine residues in proteins has been mainly characterised by immunoanalytical methods using anti-3-nitrotyrosine antibodies, and nitration sites and sequences have been hitherto identified only in a few cases using mass spectrometric methods. Immuno-analytical methods frequently suffer from low and poorly characterised detection specificity of antinitrotyrosine antibodies, while mass spectrometric methods for identification of Tyrosine nitration may be hampered by low levels of modification, and by possible changes of structure and proteolytic degradation of proteins introduced by the nitration. Moreover, no detailed, molecular characterisation of the specificity of anti-3-nitrotyrosine antibodies has been reported. In this study we describe a molecular study of the recognition specificities and affinities of two commercially available, monoclonal anti-nitrotyrosine antibodies by affinity-mass spectrometry, using different 3-nitrotyrosine containing peptides. Tyrosine-nitrated and non-nitrated substrate peptides of prostacyclin synthase (PCS), an enzyme inactivated by nitration of the active site Tyr-430 residue, were synthesised by solid-phase peptide synthesis (SPPS), purified by reversed phasehigh performance liquid chromatography (RP-HPLC) and characterised by electrospray (ESI) and matrix-assisted laser desorption-ionisation (MALDI) mass spectrometry. Binding affinities and specificities of PCS peptides with different Tyr-nitration sites and sequence mutations adjacent to Tyr-430 were determined by evaluation of anti-nitrotyrosine antibodies using an affinitymass spectrometry approach, compared to immuno-analytical determination using dot-blot and ELISA. The results showed that the antibodies may discriminate in the recognition of peptides with different N-terminal adjacent sequences to the nitrotyrosine residues, depending on the type of immunogen employed. A quantitative ELISA estimation was developed for the determination of antibody binding by Tyrosine-nitrated peptides.
Structural characterization of oligomer-aggregates of ß-amyloid polypeptide using ion mobility mass spectrometry
2010, Iurascu, Marius Ionut, Cozma, Claudia, Langridge, James, Tomczyk, Nick, Desor, Michael, Przybylski, Michael, Iurascu, Marius Ionut