Biomedical subjects

Eugen Damoc

Publications and source records attributed to Eugen Damoc.

9 recordsLinked to original sources

PubMed2006

Detection and purification of tyrosine-sulfated proteins using a novel anti-sulfotyrosine monoclonal antibody.

Protein tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody (called PSG2) that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independently of sequence context. We show that it can detect tyrosine-sulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 showed that certain sperm/epididymal proteins are undersulfated in Tpst2(-/-) mice. This indicates that TPST1 and TPST2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2(-/-) males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms.

Animals↗

PubMed2006

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry.

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.

Animals↗

PubMed2005

Determination of phosphorus-, copper-, and zinc-containing human brain proteins by LA-ICPMS and MALDI-FTICR-MS.

Human brain proteins containing phosphorus, copper, and zinc were detected directly in protein spots in gels of a human brain sample after separation by two-dimensional gel electrophoresis using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). A powerful laser ablation system with cooled laser ablation chamber was coupled to a double-focusing sector field ICPMS. The separated protein spots in 2D gels were fast screened using the optimized microanalytical LA-ICPMS technique measured at medium mass resolution with a focused laser beam (wavelength, 213 nm; diameter of laser crater, 50 mum; and laser power density, 3 x 10(9) W cm(-2)) with respect to selected three essential elements. Of 176 protein spots in 2D gel from a human brain sample, phosphorus, copper, and zinc were detected in 31, 43, and 49 protein spots, respectively. For the first time, uranium as a naturally occurring radioactive element was found in 20 selected protein spots. The detection limits for P, S, Cu, Zn and U were determined in singular protein spots with 0.0013, 1.29, 0.029, 0.063, and 0.000 01 mg g(-1), respectively. A combination of LA-ICPMS with matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) was applied for the identification of selected protein spots from human brain protein separated by 2D gel electrophoresis. Combining MALDI-FTICR-MS for the structure analysis of metal- and phosphorus-containing human brain proteins with LA-ICPMS, the direct analysis of heteroelements on separated proteins in 2D gels can be performed. For quantification of analytical LA-ICPMS data, the number of sulfur atoms per protein (and following the sulfur concentration) determined by MALDI-FTICR-MS was used for internal standardization. From the known sulfur concentration in protein, the concentration of other heteroelements was calculated. In addition, the number of phosphorylation and the phosphorylation sites of phosphorylated proteins in the human brain sample detected by LA-ICPMS were determined by MALDI-FTICR-MS. This technique allows the study of posttranslational modifications in human brain proteins.

Brain↗

PubMed2005

Immunoaffinity reactors for prion protein qualitative analysis.

The cellular prion protein (PrPc) represents the substrate for generation of conformational aberrant PrP isoforms which occur in human and animal prion diseases. The published two-dimensional maps of human PrPc show a vast microheterogeneity of this glycoprotein. The main goal of this project was to develop a highly specific immunoaffinity reactor for qualitative analysis of PrP cellular isoforms isolated from brain homogenate, cerebrospinal fluid and other tissues. New techniques for affinity proteomics, carriers and immobilization chemistry were applied. The choice of matrix (chemical and magnetic properties, particle size and distribution, porosity) was the key factor that influenced the quality of the reactor and the nature of final applications. Mouse anti-prion IgGs directed to N-terminal and C-terminal epitopes (residues 23-40 and 147-165) were grafted in different manners to magnetic micro- and nanoparticles particularly developed for micro-CHIP application. High operational and storage stability of affinity reactors with minimized nonspecific absorption were achieved. The quality of the immunoreactors was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by two-dimensional electrophoresis.

Animals↗

PubMed2004

High resolution mass spectrometric alveolar proteomics: identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients.

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micropreparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products. The high resolution mass spectrometric proteome analysis should facilitate the unequivocal identification of subunits, aggregations, modifications and degradation products of surfactant proteins and hence contribute to the understanding of the mechanistic basis of lung disease pathogenesis.

Bronchoalveolar Lavage Fluid↗

PubMed2003

Identification of N-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry.

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform-ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.

Animals↗

PubMed2003

"Affinity-proteomics": direct protein identification from biological material using mass spectrometric epitope mapping.

We describe here a new approach for the identification of affinity-bound proteins by proteolytic generation and mass spectrometric analysis of their antibody bound epitope peptides (epitope excision). The cardiac muscle protein troponin T was chosen as a protein antigen because of its diagnostic importance in myocardial infarct, and its previously characterised epitope structure. Two monoclonal antibodies (IgG1-1B10 and IgG1-11.7) raised against intact human troponin T were found to be completely cross reactive with bovine heart troponin T. A combination of immuno-affinity isolation, partial proteolytic degradation (epitope excision), mass spectrometric peptide mapping, and database analysis was used for the direct identification of Tn T from bovine heart cell lysate. Selective binding of the protein was achieved by addition of bovine heart cell lysate to the Sepharose-immobilised monoclonal antibodies, followed by removal of supernatant material containing unbound protein. While still bound to the affinity matrix the protein was partially degraded thereby generating a set of affinity-bound, overlapping peptide fragments comprising the epitope. Following dissociation from the antibody the epitope peptides were analysed by matrix assisted laser desorption-ionisation (MALDI) and electrospray-ionisation (ESI) mass spectrometry. The peptide masses identified by mass spectrometry were used to perform an automated database search, combined with a search for a common "epitope motif". This procedure resulted in the unequivocal identification of the protein from biological material with only a minimum number of peptide masses, and requiring only limited mass-determination accuracy. The dramatic increase of selectivity for identification of the protein by combining the antigen-antibody specificity with the redundancy of peptide sequences renders this "affinity-proteomics" approach a powerful tool for mass spectrometric identification of proteins from biological material.

Amino Acid Sequence↗

PubMed2003

Microfluidic systems in proteomics.

We present the state-of-the-art in miniaturized sample preparation, immunoassays, one-dimensional and multidimensional analyte separations, and coupling of microdevices with electrospray ionization-mass spectrometry. Hyphenation of these different techniques and their relevance to proteomics will be discussed. In particular, we will show that analytical performances of microfluidic analytical systems are already close to fulfill the requirements for proteomics, and that miniaturization results at the same time in a dramatic increase in analysis throughput. Throughout this review, some examples of analytical operations that cannot be achieved without microfluidics will be emphasized. Finally, conditions for the spreading of microanalytical systems in routine proteomic labs will be discussed.

Electrophoresis↗

PubMed2003

High resolution proteome analysis of cryoglobulins using Fourier transform-ion cyclotron resonance mass spectrometry.

Cryoglobulins are cold-precipitable serum immunoglobulins associated with a number of infectious, autoimmune and neoplastic disorders such as hepatitis C, Waldenström's macroglobulinemia, multiple myeloma, chronic lymphocytic leukemia, and rheumatoid arthritis. The mechanism(s) of cryoprecipitation has remained obscure hitherto, which has prompted recent intensive efforts on the identification of cryoglobulin components. In the present study, two-dimensional gel electrophoresis (2-DE) combined with high resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry has been applied as a powerful approach for the analysis of cryoglobulins. While FT-ICR mass spectrometry has been shown to enable the high resolution identification and structure analysis of biopolymers using both electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI), the recently developed MALDI-FT-ICR source is shown here to provide high (sub-ppm) mass determination accuracy and isotopic fine structure as particular advantages in the identification of proteins. The main protein components in a serum cryoprecipitate from a patient with hepatitis C virus (HCV) infection and presenting type II cryogobulinemia are immunoglobulin (Ig)M and IgG which were identified by MALDI-FT-ICR MS analysis after separation by 2-DE as mu- and gamma-heavy chains, kappa- and lambda-light chains, and J-chains. Furthermore, complementarity determining regions CDR1 and CDR2 from monoclonal IgM-RF variable region (V)L were directly identified using accurate mass determinations by FT-ICR-MS. The presence of Spalpha was ascertained as an IgM-associated protein in the serum cryoprecipitate from a patient with HCV infection.

Amino Acid Sequence↗