Mass Spectrometry Core
The aim of the UTMB Mass Spectrometry Core (MSC) is to assemble state-of-the-art instrumentation and highly skilled technical staff to provide experimental capabilities required to support a broad range of molecular-level research. Mass spectrometry (MS) plays an increasingly important role in molecular-level research, and it is central to ‘omics’ research. The MSC is well-equiped to facilitate studies with a suite of LC-MS systems including the ThermoScientific Fusion Orbitrap MS, the Sciex 5600 TripleTOF, 6500 Qtrap, and 5800 MALDI TOF/TOF.
- Molecular weight determination of biomolecules such as lipids, metabolites, peptides and proteins.
- Protein identification from gel plugs or bands and solutions using enzymatic digestion followed by MS or MS/MS using either MALDI or LCMS.
- Proteomics: identification of large numbers of proteins from complex mixtures using LCMS. Can include quantitative measurements.
- Identification of post-translational modifications.
- Differential quantitation by label-free (DIA, DDA, or SWATH), iTRAQ, TMT, or SILAC using the Thermo Orbitrap Fusion or Sciex 5600+ TripleTof.
- Quantitation of molecules using Selected Reaction Monitoring (SRM) or multi-reaction monitoring (MRM) on a 6500 QTrap.
- Advanced informatics from proteomics datasets: Beyond Mascot and Sequest output. See informatics tab:
Scaffold version 4.4.8: Scaffold is used to report results of proteomics experiments in a format that is easy for researchers to understand. In addition, Scaffold facilitates publication of experimental results by providing a description of the data analysis protocol and tools for exporting results in a format that is appropriate for publication.
PEAKS Studio, version 7.5:PEAKS is a tool which provides cutting-edge capabilities for identification and quantification of bottom-up proteomics data. Its search algorithm is complementary to Mascot in that it performs de novo sequencing and uses these results for database searching. In addition, PEAKS has been shown in various ABRF studies to perform very strongly in the identification of single amino acid variants and unexpected post-translational modifications.
Progenesis QI for proteomics, version 2.0: This software is used for label-free quantification based on MS1 peak volumes. LC-MS data files for multiple experimental conditions and analytical replicates are aligned by accurate mass and retention time, and then database searching is performed in order to assign peptide sequences to features identified by the software. The software provides multiple options for using the resulting peptide assignments to infer protein-level quantification. In addition, information for features without spectral identification can be exported and used to drive targeted LC-MS methods in order to gain additional peptide assignments.
Mass Profiler Pro. version 12.6.1: This data analysis software tool provides advanced quantification, statistical analysis and visualization tools for proteomics, transcriptomics, lipidomics and metabolomics experiments.
Skyline: This open source software, from the MacCoss laboratory at the University of Washington, is valuable in the analysis of data from all varieties of quantitative experiments – including MS1-based quantification from DDA experiments, SRM, PRM and DIA.
Ingenuity Pathway Analysis: Our laboratory has a license for this web-based tool, which is a useful hypothesis-generating tool for the analysis of quantitative data. It can also be useful in the integration of data from multi-omics experiments.
Fusion Orbitrap. Ultra-high resolution (214,000 @ 1,000Da) MS coupled to a nano-UPLC. The Fusion is a high performance system that provide high resolution and high mass accuracy for analysis of compounds ranging from small molecules to proteins. They have become particularly popular for proteomics applications, namely the identification of proteins based on analysis of constituent peptides (termed the “bottom-up” method) or analysis of the original intact proteins (termed the “top-down” approach). The fusion offers three fragmentation modes; collision induced dissociation (CID), High energy collision dissociation (HCD), and electron transfer dissociation (ETD). In addition, the Fusion has MSn capabilities combined with the added functionality of electron transfer dissociation are powerful tools needed to resolve the site and type of PTM present.
5600 TripleTOF is typically used for proteomics. It is a hybrid quadrupole TOF platform, accurate mass (<3ppm), high-resolution (30,000) system than can operate by means of information-dependent acquisition (IDA) with speed (up to 50Hz) and sensitivity of a TOF MS and quantification capabilities similar to a triple quadrupole MS. When operated in a data-independent acquisition (DIA) mode, termed SWATH-MS by Sciex, the 5600 offers quantitative accuracy comparable to Selected Reaction Monitoring (SRM), and multiplexing capabilities comparable to DDA proteomics. In SWATH-MS, peptide precursors are sequentially selected in defined precursor ion mass windows for concurrent fragmentation, and the resulting fragment ion spectra are recorded in recursive cycles over the chromatography time.
6500 QTRAP system merges triple quadrupole and linear ion trap capabilities to provide the industry standard in sensitivity and selectivity. With a Dionex 3000 nanoUPLC system at the front end, the QTRAP 6500 UPLC-MS/MS system offers the ultimate sensitivity for selected reaction monitoring for quantitation, complex sample analysis (e.g. amino acids, lipids, metabolites, and peptides).
5800 MALDI TOFTOF is used for obtain masses of lipids, nucleic acids, peptides, proteins, and polymers ranging in molecular mass from 200 Da up to 100 kDa or more. For analysis by MALDI, a sample is mixed or coated with an energy-absorbing matrix and then irradiated with a laser beam (typically a Nd:Yag laser). Singly protonated (positive ionization mode) or deprotonated (negative ionization mode) molecular ions are formed and detected by a time-of-flight mass analyzer. In addition the 5800 has MS/MS capabilities, aiding in the identification of analytes. The 5800 can also be used for molecular imaging of tissues or materials.