Biomedical subjects

Albert Sickmann

Publications and source records attributed to Albert Sickmann.

43 records · Page 3Linked to original sources

PubMed2003

Mass spectrometry--a key technology in proteome research.

The rapid developments in the field of mass spectrometry have transformed it into a key technology in proteome research. Increased sensitivity in mass spectrometry, as a result of more efficient ionisation techniques and better detectors, has allowed the stepwise reduction of protein quantity for analysis. Protein spots of 2D-PAGE separated samples are now quantitatively sufficient for an unequivocal identification of a protein by mass spectrometry. In addition to protein identification a closer look at posttranslational modifications is now also possible. It is speculated that modifications like phosphorylation or glycosylation exist on every second protein and that they are important for the protein function. This review highlights the different mass spectrometric methods and gives a brief overview of strategies and methods used to identify modifications.

Amino Acid Sequence↗

PubMed2003

Multidimensional nano-HPLC for analysis of protein complexes.

The analysis of macromolecular protein complexes is an important factor in understanding most cellular processes, e.g., protein transport into cell organells, signal transduction via biological membranes, apoptosis, energy metabolism, directed motion of cells, and cell division. These complexes are not only built of various numbers of different proteins but also of prosthetic groups and RNA molecules. To understand the role each protein plays in a complex, a complete analysis of all protein compounds is necessary. Therefore, several separation steps have to be coupled to mass spectrometry to identify the proteins. In this work, we describe the application of multidimensional liquid chromatography, SCX-RP-LC as well as SAX-RP-LC, coupled to electrospray ion trap mass spectrometry. Tryptic digested ribosomes were separated by ion exchange chromatography manually collected and prepared for reversed phase chromatography to analyze the peptides via nano-ESI mass spectrometry. The total numbers of identified proteins are compared in consideration of the separation method (SCX-RP versus SAX-RP).

Amino Acid Sequence↗

PubMed2003

Pex8p: an intraperoxisomal organizer of the peroxisomal import machinery.

Peroxisomes transport folded and oligomeric proteins across their membrane. Two cytosolic import receptors, Pex5p and Pex7p, along with approximately 12 membrane-bound peroxins participate in this process. While interactions among individual peroxins have been described, their organization into functional units has remained elusive. We have purified and defined two core complexes of the peroxisomal import machinery: the docking complex comprising Pex14p and Pex17p, with the loosely associated Pex13p, and the RING finger complex containing Pex2p, Pex10p, and Pex12p. Association of both complexes into a larger import complex requires Pex8p, an intraperoxisomal protein. We conclude that Pex8p organizes the formation of the larger import complex from the trans side of the peroxisomal membrane and thus might enable functional communication between both sides of the membrane.

Carrier Proteins↗

PubMed2002

The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel.

Mitochondrial proteins with N-terminal targeting signals are transported across the inner membrane via the presequence translocase, which consists of membrane-integrated channel proteins and the matrix Hsp70 import motor. It has not been known how preproteins are directed to the import channel. We have identified the essential protein Tim50, which exposes its major domain to the intermembrane space. Tim50 interacts with preproteins in transit and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits the import of preproteins with a classical matrix-targeting signal, while preproteins carrying an additional inner membrane-sorting signal do not strictly depend on Tim50. Thus, Tim50 is crucial for guiding the precursors of matrix proteins to their insertion site in the inner membrane.

Amino Acid Sequence↗

PubMed2002

Towards a high resolution separation of human cerebrospinal fluid.

Human cerebrospinal fluid is an ultrafiltrate of plasma that is largely produced by the choroid plexus. It consists of a mixture of anorganic salts, various sugars, lipids and proteins from the surrounding brain tissues. The predominant proteins in cerebrospinal fluid are isoforms of serum albumin, transferrin and immunoglobulins, representing more than 70% of the total protein amount. A rough overview of the protein compounds of human cerebrospinal fluid including their respective concentrations is given by Blennow et al. [Eur. Neurol. 33 (1993) 129]. In contrast, the aim of this work is to display the detailed protein composition of CSF by two-dimensional gel electrophoresis and to identify both high and low concentrated proteins using different mass spectrometry techniques. This extensive overview of proteins in human cerebrospinal fluid will be highly relevant for clinical research. Furthermore, the comparison of 2D gels will help to analyze the standard protein variability in CSF of healthy persons and detect specific protein variations of patients with various neurological diseases (e.g., Alzheimer's disease, Huntington's chorea). Sample preparation for two-dimensional gel electrophoresis must include concentration and desalting steps such as precipitation and ultrafiltration due to the high amount of salts, sugars and lipids and the low total amount of protein of 0.3-0.7 microg/microl present in human CSF. Up to now we were able to identify more than 480 spots from suchlike generated 2D gels using MALDI- and ESI-mass spectrometry.

Amino Acid Sequence↗

PubMed2002

Identification of modified proteins by mass spectrometry.

Because it is obvious that high-throughput genomics do not lead to a molecular description or even a prediction of protein function, modern techniques for protein analysis become increasingly more important. Sequence analysis of proteins and peptides is not limited to the elucidation of the primary structure of a protein. The analysis of posttranslational modifications is an important task of protein chemistry in proteome research. Increased sensitivity in mass spectrometry as a result of more efficient ionization techniques and better detection systems has allowed the stepwise reduction of protein quantity for analysis. Protein spots of 2D-PAGE separated samples are now sufficient for an unequivocal identification of a protein by mass spectrometry. In addition to protein identification, a closer look at posttranslational modifications is now also possible. It is assumed that modifications such as phosphorylation or glycosylation exist on every second protein and that they are important for the protein function.

Algorithms↗

PubMed2000

Regulation of the glucose-specific phosphotransferase system (PTS) of Staphylococcus carnosus by the antiterminator protein GlcT.

The ptsG operon of Staphylococcus carnosus consists of two adjacent genes, glcA and glcB, encoding glucose- and glucoside-specific enzymes II, respectively, the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The expression of the ptsG operon is glucose-inducible. Putative RAT (ribonucleic antiterminator) and terminator sequences localized in the promoter region of glcA suggest regulation via antitermination. The glcT gene was cloned and the putative antiterminator protein GlcT was purified. Activity of this protein was demonstrated in vivo in Escherichia coli and Bacillus subtilis. In vitro studies led to the assumption that phosphoenolpyruvate-dependent phosphorylation of residue His105 via the general PTS components enzyme I and HPr facilitates dimerization of GlcT and consequently activation. Because of the high similarity of the two ptsG-RAT sequences of B. subtilis and S. carnosus, in vivo studies were performed in B. subtilis. These indicated that GlcT of S. carnosus is able to recognize ptsG-RAT sequences of B. subtilis and to cause antitermination. The specific interaction between B. subtilis ptsG-RAT and S. carnosus GlcT demonstrated by surface plasmon resonance suggests that only the dimer of GlcT binds to the RAT sequence. HPr-dependent phosphorylation of GlcT facilitates dimer formation and may be a control device for the proper function of the general PTS components enzyme I and HPr necessary for glucose uptake and phosphorylation by the corresponding enzyme II.

Bacillus subtilis↗