The Proteomics Center at the Children's Hospital is equipped with state of the art instrumentation, which allows our researchers to push the limits with biological mass spectrometry experiments. These resources provide a unique opportunity for developing methods that allow cutting edge research. Mass spectrometry is a rapidly evolving field and as such has important implications in several areas of research, including protein biomarker discovery and validation/screening. The reagent / instrumentation costs and lack of expertise are a significant barrier to the widespread adoption of mass spectrometry in many laboratories. Boston Children's Hospital has invested in equipment that is accessible to all researchers in the community as a pooled resource.
The Thermo Scientific Q Exactive benchtop LC-MS/MS, is a hybrid mass spectrometer which combines high-performance quadrupole precursor selection with high resolution, accurate-mass Orbitrap detection. The fast Q-Exactive permits the acquisition of 10 MS/MS spectra per second in the Orbitrap, which makes it a perfect platform to analyze complex proteomic samples. In the lab, the Q-Exactive is used for identification and quantification e. g. large scale proteomics with isobaric mass tagging or isotopic labeling methods of peptides, as well as for the mapping and quantification of post translational modifications. The Q-Exactive is also used for a multiple reaction monitoring (MRM)-like scan mode called parallel reaction monitoring (PRM). This scan mode which was introduced on the Q-Exactive enables the accurate and highly reproducible quantification of a list of predefined peptides without a prior choice of the transitions. The high sensitivity and high speed permits deep insights of whole proteomes as well as challenging proteomes such as CSF.
The AB SCIEX TripleTOF 5600 system is a Q-TOF instrument that integrates comprehensive qualitative exploration, rapid profiling, and high-resolution quantitation workflows on a single platform. Because of its high sensitivity and fast scan speed the instrument is used to elucidate complex proteomics samples for peptide identification and quantification via isobaric mass tags or isotopic labels. Besides the Q-Exactive, it is the instrument of choice for deep proteome coverage and precise quantification. The MRMHR workflow is similar to MRM methods on TripleQuad mass spectrometer. This workflow suffers from sensitivity and dynamic range but allows high precision and the highly reproducible quantification of peptides without extensive method development. An addition mode of operation is the MS/MSALL with SWATH acquisition mode., which relies on fragmentation of all precursor ions in a certain mass window and thus permits label free quantification on MS² level without prior knowledge, which is highly applicable to biomarker research.
The Thermo Scientific LTQ-Orbitrap is hybrid instrument that combines a linear quadrupole ion trap with an ion cyclotron resonance mass analyzer for ion detection. Although the LTQ is getting a bit long in the tooth, it is still the work horse in the lab for samples that don’t require high sensitivity and is often used follow improvements in sample preparation optimization experiments. In contrast to the Q-Exactive it has the capability of acquiring MS³ spectra, which can deliver useful information for localization and detection of specific post-translational modifications.
The AB SCIEX QTRAP 5500 LC/MS/MS system combines a standard triple quadrupole with a Linear Accelerator trap, which offers ultra-fast scan speeds, and full MS³ capabilities. The instrument is mainly used for multiple reaction monitoring (MRM) experiments to target proteins in complex sample mixtures and to quantify specific proteins of interest with a high sensitivity and over a wide dynamic range. The workflow is suitable for the analysis of samples where only a small number of proteins are monitored. Applications within the lab typically involve the development of MRM assays, which are subsequently used for the absolute quantification of proteins and determination of the post translational modification states (FLEXIQuant workflow).