Mass spectrometry

The Pandey lab specializes in mass spectrometry based proteomics with technical expertise in liquid chromatography/tandem mass spectrometry. The lab is equipped with various mass spectrometers including LTQ Orbitrap XL mass spectrometer, ETD equipped Agilent ion trap mass spectrometer and quadrupole time-of-flight instruments from applied biosystems (QSTAR) and micromass (Q-TOF). While ETD equipped ion trap mass spectrometer and micromass Q-TOF are exclusively used by members of the Pandey lab, LTQ Orbitrap and QSTAR mass spectrometers are used as a shared resource with other proteomic labs at Johns Hopkins University.

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.

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 nonmodified peptides.

Applied Biosystems QSTAR 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

Fraction collector

Liquid Chromatography

Two CapLC pumps from Agilent and a Micro wellplate auto sampler. The CapLC's are both connected to the mass spectrometers. One set-up is a "high throughput" set-up consisting of a CapLC pump and an auto sampler. The remaning CapLC pump is used together with off-line "bomb" loading.
All separations are performed using homemade fused silica type columns.

Data Databases

Fragmented peptides are searched using a Mascot cluster from Matrixscience.

Schematic of high load flow/low elute flow set-up. Sample is loaded on to the pre column @5-10µL/min with the 6-port valve in "waste" position. When starting to elute, the valve is shifted to "closed" and simultanusely the flow from the pump is lowered to approx. 300nL/min. With the 6-port-valve in "closed" position the flow will forced through the emitter.