Instrumentation
ThermoFisher LTQ-Orbitrap
The ThermoFisher LTQ-Orbitrap combines the best of linear ion trap technology in the LTQ, with the novel Orbitrap mass analyzer, which provides high mass accuracy and resolution for both MS and MSn spectra. This instrument is typically run with the Orbitrap acquiring full MS data at low ppm mass accuracy and resolution up to 100,000 HWFM, while the LTQ acquires multiple MSMS scans of peptides for identification and modification site determination. Other operational configurations include SRM/MRM experiments, high energy collisions in the C-trap, and high accuracy MSMS spectra in the Orbitrap. This instrument is essential and continually amazes us with the sensitivity and accuracy we achieve on a daily basis.
Driving the chromatography on the Orbitrap is the Waters nanoACQUITY UPLC system. This system maintains accurate gradient production in a splitless configuration in the sub-μl range at pressures up to 10,000psi. The autosampler allows automated trapping for sample concentration and desalting and provides excellent reproducibility and linearity across the injection volume range of 0.1 to 20μl.
New Objective Inc. provides the nanocapillary chromatography hardware and ESI source used exclusively on the LTQ-Orbitrap. The PicoView source allows easy positioning and visualization of the ESI spray, which is key to operation of the high sensitivity nanocapillary columns with integrated spray tips (Picofrit columns) and trap columns (IntegraFrit columns) provided by this long-time collaborator with our lab. No other chromatographic configuration has demonstrated the versatility, robustness and extremely high sensitivity that these products provide.
Waters Q-TOF Premier
The Waters Q-TOF Premier is a hybrid quadrupole TOF instrument equipped with the Protein Expression System for non-labeled quantitative proteomics analysis. The instrument features Lock Mass spray and MSe, which allows this instrument to skip the isolation step for MSMS, greatly increasing the effective duty cycle when run in this mode. Key to the expression analysis is the stability and reproducibility of the nanoACQUITY UPLC system and the high pressure 1.7μ reversed phase capillary column, which afford the instrument excellent chromatographic properties. This instrument is also capable of intact protein mass determinations by direct ESI infusion and has the full suit of data analysis software from Waters.
Applied Biosystems DE-STR MALDI-TOF Matrix Assisted Laser Desorption Time Of Flight (MALDI-TOF) is s soft ionization technique capable of analyzing proteins, peptides, oligonucleotides and other organic molecules intact up to a mass range of 200 kDa. This instrument provides a complimentary method of screening samples prior to more complicated ESI mass determinations, and can be set up to screen many samples in an automated fashion.
http://www.appliedbiosystems.com/
Applied Biosystems Procise 494-HT
Edman sequencing is a classic protein characterization technique that has been a mainstay in this laboratory for over 20 years. This process requires >1pmol of protein, either in solution of on PVDF blot, and can quantitatively determine the sequence of one or several protein or peptides with a cycle time (one amino acid per cycle) of about 45 minutes. This is not a database dependent technique, so it can be considered the original form of denovo protein sequencing. Typical sequences are 10 amino acids, but extended sequence runs can be preformed for novel proteins or peptides.
Amino Acid Analysis & RPLC/SCX Chromatography on Agilent 1100
One Agilent 1100 HPLC is configured for automated Amino Acid Analysis (AAA) for protein quantitation and composition. A second 1100 system can perform Strong Cation Exchange (SCX) and Reverse Phase Liquid Chromatography (RPLC) with automated peak detection and fraction collection for sample prefractionation and purification under various configurations including diode array detection (DAD) and variable wavelength detection (VWD). Controlled by Agilent ChemStation software, these systems provide flexibility and robustness for protein and peptide work.
