M S R e s e a r c h & D e v e l o p m e n t
New Strategies for Phosphoproteomics
Currently, our major effort is focused on method improvement for analysis of protein phosphorylation by nonradioactive methods. For this purpose, we have available in-gel digestion methods with various proteases, nanoESI-MS/MS on a triple-quadrupole system and on a hybrid Q-TOF instrument, capillaryLC-ESI-MS on both instruments, enzymatic and chemical dephosphorylation micromethods, IMAC microenrichment for phosphopeptides, and capillaryLC-element mass spectrometry with phosphorous detection. We are working towards an optimally sensitive combination of these methods to provide complete phosphopeptide coverage and identification of phosphorylation sites for gel-isolated proteins.
Protein Identification
We have established identification of 1D or 2D gel-isolated proteins by a combination of in-gel tryptic digestion, desalting and nanoESI tandem MS. We are using a hybrid Q-TOF instrument with automated MS to MS/MS switching followed by database searching with uninterpreted MS/MS spectra via the Internet search engine Mascot provided by Matrix Science.
Covalent Protein Modification
In addition to the analysis of phosphorylation, we also utilize tandem MS of peptides and MS of intact proteins for identification of other covalent modifications, such as myristoylation, acetylation, gluconoylation, deamidation and isoaspartate formation.
Picomole Quantification of Polar Lipids
NanoESI tandem mass spectrometry was introduced as a new method to quantify the main phospholipid classes in crude lipid extracts of cells and cell organelles. The method has high sensitivity and is capable of quantifying individual molecular species of phospholipids. The method is being applied to new biological questions and to classes of polar lipids that have not been accessible up to now.
Current Research & Thesis Projects
F a u z i S a f i a n, Dipl.-Chem. (PhD, Malaysia State Program)
New quantitative microanalytic methods for determination of organic phosphate bound in phosphoproteins and phospholipids.
Dr. A n n a K o n o p k a, Guest Scientist
Introduction of capLC-element mass spectrometry to absolute phosphopeptide and phosphoprotein quantification. Labeling of peptides and proteins by tags taylored for detection by ICP-MS.
Dr. B e t t i n a H a h n, Postdoc (Systems Biology of Cancer)
Quantitative phosphorylation analysis of signal transduction proteins
Differential and absolute quantification using chemical stable isotope labeling and stable isotope labeled internal standards.
M a r t i n B ö h m, Dipl.-Ing. (FH) Chemie (PhD, LungSys)
Structural and quantitative analysis of erythropoetin receptor (EpoR) by mass spectrometry in cells and tissue related to lung cancer.
S e b a s t i a n T i t t e b r a n d t, Dipl.-Ing. (FH) Biologische Chemie (PhD, AmbiProbe)
Development and application of H/D exchange techniques for probing chemical analyte properties by mass spectrometry.
G e r h a r d E r b e n, Technician
Optimization of analytical services for small molecules employing ESI and MS/MS techniques.
