Description of the Proteomics Core
The Proteomics Core offers superior mass spectrometric analysis using three state-of-the-art mass spectrometers, Qstar XL quadrupole time-of-flight MS, 4800 MALDI-TOF-TOF MS, and 4000 Q-Trap MS. A number of different approaches are used to identify proteins and phosphoproteins by mass spectrometry.
The protein of interest is subjected to gel electrophoresis (2D gel or SDS-PAGE) and protein spots or bands of interest are excised from the gel, destained, digested with trypsin or other chemical or enzymatic cleavage agents as needed, and the resulting peptides extracted. The peptides from each spot are analyzed by the 4800 MALDI-TOF-TOF mass spectrometer. Tryptic peptide masses as well as the tandem MS (MS/MS) spectra of selected peptides are acquired in an automated fashion. Another aliquot is placed in a 96-well autosampler and subjected to LC-MS analysis in an automated mode. Because both 4800 MALDI-TOF-TOF and Qstar XL offer high sensitivity, high resolution, and high quality in MS/MS experiments, a high coverage of sequences can be determined from these peptides. These combined approaches generally permit protein identification without any ambiguity.
The 4800 MALDI-TOF-TOF and 4000 Q-Trap are particularly suitable for protein phosphorylation analysis, The 4800 MALDI-TOF-TOF has high sensitivity and works with the immobilized samples on plate, thus provide better opportunity for analysis compared to LC-MS/MS. The 4000 Q-Trap is valuable because of its multiple reaction monitoring (MRM) capability.
Gel-free Protein Quantification Using Stable Isotope Labeling and iTRAQ Reagents
2D gel electrophoresis is a widely used standard protocol, new technologies using stable isotope labeling and iTRAQ reagents. Stable isotope labeling techniques are well developed as well.
The iTRAQ reagents permit quantitative proteomics analysis using reagents that are isobaric, with a different distribution of isotopes between a reporter and balance groups in tandem MS/MS spectra. Thus labeled peptides are identical in MS mode, but upon fragmentation in MS/MS mode produce strong, diagnostic, low-mass MS/MS signature reporter ions. The peak areas of the reporter ions allows quantification of up to four different samples simultaneously.
Other stable isotope labeling approaches (ICAT and SILAC) only allow binary pair-wise quantification of two samples and thus the iTRAQ approach is preferred and will be used by all biomedical projects and will replace 2D gel electrophoresis wherever appropriate. Both the 4800 MALDI-TOF/TOF and 4000 Q-Trap mass spectrometers are suited for the iTRAQ quantitative analysis.
Mass Spectrometric Analyses of Phosphoproteins and Phosphopeptides
Protein phosphorylation can be studied using two approaches. The first utilizes the ability of 2D gel electrophoresis to separate phosphorylated isoforms due to their different electrofocusing points as described earlier. The second approach is to use immobilized metal affinity chromatograph to enrich the phosphopeptides from tryptic digests of a particular protein or a mixture of proteins from a subcellular compartment. The peptides eluted from the IMAC column are subsequently subjected to online LC-MS/MS analysis using 4000 Q-Trap and/or offline LC-MALDI-TOF-TOF analysis. This approach has proven to be successful to detect phosphoproteins more comprehensively and to identify the phosphorylation sites.
LC-MALDI-MS/MS is available to investigators. This technology marries the power of HPLC separation with the in-depth MALDI-TOF-TOF analysis. A capillary HPLC system is utilized to separate complex tryptic peptides of an isolated subcellular fraction using a C18 RP column. Peptides eluted are collected to a MALDI plate using a micro-fraction collector and subjected to MALDI-TOF-TOF analysis.
The LC-MALDI-MS/MS platform is critical to investigators who will analyze complex samples that require HPLC separation prior to the mass spectrometry analysis.
The Proteomics Component is equipped with the following major items of equipment:
Two-Dimensional Gel Separation Apparatus
The Proteomics Core currently has available a Bio-Rad Protean IPG System for electrofocusing separation of up to 12 samples simultaneously on 7, 11 or 17 cm pre-cast IPG strips coupled with corresponding Protean and Criterion Gel apparatuses for 2nd dimension SDS-PAGE in 7, 11 or 17 cm formats. The latter apparatuses will accommodate up to 4 gels simultaneously.
ABI QStar XL Q-TOF Mass Spectrometer
The QSTAR XL Hybrid LC/MS/MS System is a high-performance, hybrid quadrupole time-of-flight mass spectrometer designed specifically for protein identification and characterization. A dedicated nano-flow HPLC system from Eksigent is configured with the NanoSpray source to allow online LC-MS/MS analysis. Specific scan modes such as precursor ion scanning using patented collision cell technology permits determination of peptide sequence and the type and location of post-translational modifications with outstanding specificity and sensitivity.
ABI 4800 MALDI-TOF-TOF Mass Spectrometer
The core has a matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF-TOF) mass spectrometer obtained through a Shared Instrumentation Grant from NIH/NCRR. The 4800 MALDI-TOF-TOF mass spectrometer offers high sensitivity, efficient fragmentation of singly charged precursor ions generated by MALDI, and superior mass accuracy and resolution in both MS and tandem MS/MS modes. In addition, the Proteomics Core has a separate capillary HPLC system that is configured with a micro-fraction collector. As discussed above, this system provides the new powerful LC-MALDI-MS/MS technology for investigators.
ABI 4000 Q-Trap Mass Spectrometer
The Proteomics Core was also awarded a second Shared Instrumentation Grant from NIH/NCRR to purchase a 4000 Q-Trap linear ion trap mass spectrometer with a Shimadzu UFLC (HPLC). This new LC-MS/MS system offers enhanced sensitivity. Particularly, the Q-Trap mass spectrometer has the multiple reaction monitoring (MRM) capability, is valuable in characterizing phosphorylated peptides.