540-562 First Avenue
New York, NY 10016
The main focus of our lab is the study of proteins and their roles in cellular signaling events. While great strides in understanding intracellular signal transduction have been made in recent years by using molecular biological techniques, we feel that a more complete understanding of the dynamics of intracellular decision-making processes can be gained only by studying the proteins directly. We use mass spectrometry as the main tool for our studies because of the wide variety of information about protein structure it can provide while requiring only small amounts of protein for analysis.
One goal of our research is to identify proteins that interact with each other during specific signaling events. Our lab has a special interest in the study of signal transduction in neurons, especially the ephrin signaling pathway. The basic strategy for these experiments is to isolate protein complexes by immunoprecipitation or affinity chromatography, separate the proteins by SDS-PAGE and then identify proteins of interest from the gel. We do this by excising the protein band from the gel, cleaving the protein with a specific protease such as trypsin, then determining the amino acid sequences of the peptides by Q-TOF tandem mass spectrometry. The masses and tandem mass spectra (containing peptide sequence information) of the peptides are then used to search genome databases to identify the proteins. To get more information about the relative amounts of each protein that participate in signaling complexes in the stimulated and nonstimulated states, we use stable isotope coding methods (for example, stable isotope labeling in cell culture, or SILAC). We also have used mass spectrometry to identify large numbers of proteins present in subcellular organelles. By first separating complex mixtures of peptides by nanoflow HPLC coupled directly to the Q-TOF mass spectrometer, we can identify many dozens of proteins at a time at low femtomolar levels, and many hundreds of proteins in a single experiment. In collaboration with Dr. Ed Ziff''s group, we have identified several hundred proteins in neuronal postsynaptic densities.
Another focus of our work is to identify posttranslational modifications on proteins. An example of a common posttranslational modification with an important role in the regulation of protein activities is phosphorylation on serine, threonine or tyrosine residues. We have established a useful method for identifying potential phosphopeptides present at low levels in complex mixtures of nonphosphorylated peptides by comparison of MALDI-TOF mass spectra taken in both negative and positive ion modes (Figure 1). Another example of a useful method for identification of sites of posttranslational modification is shown in Figure 2. In this case the glycosylated asparagine residue of the urothelial protein uroplakin was partially labeled with 18O by cleaving the sugar residue with PNGase F in the presence of 50% 18O water. After cleaving the uroplakin with trypsin, the peptide containing the labeled asparagine was then identified on the basis of its isotopic mass distribution and sequenced by Q-TOF mass spectrometry.
Subcellular Parkinson's Disease-Specific Alpha-Synuclein Species Show Altered Behavior in Neurodegeneration
Abdullah, Rashed; Patil, Ketan S; Rosen, Benjamin; Pal, Ramavati; Prabhudesai, Shubhangi; Lee, Sungsu; Basak, Indranil; Hoedt, Esthelle; Yang, Peter; Panick, Keith; Ho, Hsin-Pin; Chang, Emmanuel; Tzoulis, Charalampos; Larsen, Jan Petter; Neubert, Thomas A; Alves, Guido; Moller, Simon G. Subcellular Parkinson's Disease-Specific Alpha-Synuclein Species Show Altered Behavior in Neurodegeneration. Molecular neurobiology. 2016 Nov 11;:?-? (2304642)
Cytoplasmic, full length and novel cleaved variant, TBLR1 reduces apoptosis in prostate cancer under androgen deprivation
Daniels, Garrett; Zhang, Xinmin; Zhong, Xuelin; Santiago, Larion; Wang, Ling Hang; Wu, Xinyu; Zhang, Jack Y; Liang, Fengxia; Li, Xin; Neubert, Thomas A; Steinke, Laurey; Shen, Ying; Basch, Ross; Schneider, Robert; Levy, David E; Lee, Peng. Cytoplasmic, full length and novel cleaved variant, TBLR1 reduces apoptosis in prostate cancer under androgen deprivation. Oncotarget. 2016 Jun 28;7(26):39556-39571 (2092672)
Protein Kinase C-theta and vimentin modulate multiple facets of Regulatory T-cell function
McDonald-Hyman, Cameron; Thangavelu, Govindarajan; Saha, Asim; Muller, James; Zhang, Guoan; Kumari, Sudha; Koehn, Brent H; Mitchell, Jason S; Fife, Brian T; Serody, Jonathan S; Osborn, Mark J; Hippen, Keli L; Kelekar, Ameeta; Munn, David H; Altman, Amnon; Neubert, Thomas; Dustin, Michael L; Blazar, Bruce R. Protein Kinase C-theta and vimentin modulate multiple facets of Regulatory T-cell function [Meeting Abstract]. Journal of immunology (1950). 2016 MAY 1;196:?-? (2220252)
In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's Abeta protein
McIntee, Farron L; Giannoni, Patrizia; Blais, Steven; Sommer, George; Neubert, Thomas A; Rostagno, Agueda; Ghiso, Jorge. In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's Abeta protein. Frontiers in aging neuroscience. 2016 ;8:223-223 (2275452)
Ubiquitination role in TCR signaling and costimulation via GITR
Muller, J; Zhang, G; Silva, HM; Neubert, T; Dustin, M. Ubiquitination role in TCR signaling and costimulation via GITR [Meeting Abstract]. European journal of immunology. 2016 AUG;46:1191-1191 (2281752)