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School of Medicine
Akhilesh Pandey, M.D., Ph.D.
Our laboratory comprises an interdisciplinary group of molecular biologists, mass spectrometrists and computational biologists who are interested in systems biology. As a participating laboratory in the Technology Center for Networks and Pathways of Lysine Modification, our laboratory will develop methods for quantitative and qualitative analysis of lysine modifications.
Specifically, we are developing enrichment methods and applying stable isotope labeling with amino acids in cell culture (SILAC) method for mass spectrometry-based quantitation. These methods will allow for a systematic, global and quantitative profiling of lysine modifications to obtain a dynamic picture of signaling in cells.
Below are some of the analytical devices at our disposal and an example of how multiplex SILAC analysis can inform a biological process.
- Figure 1. Thermo LTQ Orbitrap XL mass spectrometer
- Figure 2. ETD equipped Agilent 6340 ion trap mass spectrometer
- Figure 3. Micromass Q-TOF mass spectrometer
- Figure 4. Application of SILAC to study temporal dynamics of adipocyte differentiation
Figure 1. Thermo LTQ Orbitrap XL mass spectrometer.
Thermo LTQ Orbitrap XL mass spectrometer is a hybrid Fourier Transform mass spectrometer which combines features of linear ion trap and orbitrap mass analyzer. This combination of fast scanning linear ion trap and high resolving power and mass accuracy of orbitrap mass analyzer makes it one of the best mass spectrometers for proteomic research. The instrument also features an additional collision cell for higher energy collisional dissociation (HCD) which is particularly useful for analyzing ions in the low-mass region of the spectrum. Culminating almost all the capabilities of previous mass spectrometers while providing higher sensitivity, resolution and mass accuracy makes it suitable for a variety of proteomics studies. We use LTQ Orbitrap for both qualitative and quantitative proteomics studies including identification of biomarkers of GI cancers and characterization of post-translational modifications like phosphorylation and glycosylation.
Figure 2. ETD equipped Agilent 6340 ion trap mass spectrometer.
The Agilent 6340 ion-trap LC/MS system provides flexibility to choose between collision induced dissociation (CID) and electron transfer dissociation (ETD) methods. Although CID has been traditionally used as a preferred dissociation method for most proteomic studies involving protein identification, its utility has been limited while analyzing labile post-translational modifications. However, recently introduced electron transfer dissociation (ETD) method is particularly suitable for studying labile post-translational modifications such as phosphorylation and glycosylation of serine and threonine residues. By carrying out alternating fragmentation of a peptide by both CID and ETD, it is now possible to get better sequence coverage of a peptide resulting in confident protein identification. Our lab uses this instrument to study phosphorylation and O-GlcNAcylation of serine/threonine residues and utility of alternating CID/ETD fragmentation for the analysis of non-modified peptides.
Figure 3. Micromass Q-TOF mass spectrometer.
Micromass Q-TOF Ultima API-US (quadrupole time of flight mass spectrometer) equipped with an Agilent Capillary LC pump and a cooled Micro-well plate autosampler.
Figure 4. Application of SILAC to study temporal dynamics of adipocyte differentiation.
A schematic of the development and application of a five-plex SILAC experiment to study temporal dynamic proteome during the course of adipocyte differentiation. A: Differentiation of adipocytes was confirmed by Oil red O staining. B: Using five different stable isotopic forms of arginine, a five-plex SILAC experiment was performed to study the nuclear proteome and the secretome during the course of adipocyte differentiation. C: A representative MS spectrum from a 5-state labeled peptide is shown. D: A representative elution profile from a 5-state labeled peptide is shown. E: The heat map shows the temporal profile of each protein quantitated by 5-plex SILAC based MS analysis.
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