Read a profile of the Sandler-Moore Mass Spectrometry Core in The Science and Art of Running a Core Facility
The UCSF Sandler-Moore Mass Spectrometry Core Facility is a designated Cancer Center Core that serves as a mass spectrometry resource for the UCSF proteomics research community. The Core’s goals are twofold:
The Core offers a variety of services for protein identification and characterization as well as molecular mass determinations for peptides, intact proteins, protein complexes, and small oligonucleotides (up to 20 bases). A complete list of services and their corresponding recharge rates can be found here. The faculty director and staff strongly encourage all users, particularly first-time clients, to schedule an appointment to discuss study design, proper sample preparation methods, the sample submission process, and data interpretation. Appointments can be made by contacting the faculty director or any staff member by email.
Protein Identification by Tandem Mass Spectrometry (MS/MS)
Tandem mass spectrometry (MS/MS), performed utilizing electrospray ionization (ESI) and a quadrupole quadrupole time-of-flight (QqTOF) mass spectrometer or a MALDI TOF/TOF instrument, are alternative techniques for determining protein identification. Protein identification is accomplished by isolating (in the gas phase) peptide populations consisting of a single mass-to-charge ratio (m/z), within the mass spectrometer, fragmenting this population and then measuring the masses of the peptide fragment ions. The experimentally determined peptide fragment ion masses are used to search a theoretical fragment ion mass database generated by in silico digestion and fragmentation of all proteins in a protein database. Complex mixtures, such as enzymatically produced protein digests, undergo high performance liquid chromatography (HPLC) separation prior to introduction of sample into the mass spectrometer ion source. Choice of ionization mode (MALDI or ESI) and sample introduction (infusion, HPLC, solid phase desorption) will depend on the nature of the sample and experimental information desired.
Confirmation of peptide mass fingerprinting results by tandem mass spectrometry is highly recommended if protein identifications are to be published in peer-reviewed journals. Furthermore, many journals now require that all MS/MS data used to substantiate reported protein identifications be submitted as supplementary material to the main manuscript.
Molecular Weight Determination Service Options
The Core offers two services for molecular weight determination: MALDI TOF and ESI QqTOF mass spectrometry. The size of the molecule in question dictates which type of mass spectrometry to choose for the analysis. The molecular masses of peptides, small proteins (less than 5000 Daltons), and oligonucleotides up to 20 bases are determined on a MALDI TOF mass spectrometer. Intact protein molecular weights (more than 5000 Daltons) are performed on a QqTOF mass spectrometer.
Molecular Weight Determination by MALDI TOF
The molecular weight of synthetic or naturally isolated peptides, small proteins (less than 5000 Da), and oligonucleotides (up to 20 bases) can be measured by MALDI TOF mass spectrometry. The mass accuracy of the molecular weight determination will vary between ~10 ppm to 0.01% depending on the size of the molecule being measured.
Molecular Weight Determination of Intact Proteins by ESI QqTOF
The molecular weight of intact proteins can be measured by ESI QqTOF mass spectrometry within ~0.01-0.02% mass accuracy, provided that the samples are free of salts and detergents.
The success of any mass spectrometry experiment depends on proper sample preparation. There are many salts and detergents that are incompatible with ESI and MALDI that must be avoided from the onset and throughout the entire sample work-up methodology. In addition, for those performing in-gel proteolytic digests, it is imperative that precautions be taken to avoid contamination by human keratin proteins.
A recommended protocol for preparing in-gel protein tryptic digests suitable for mass spectrometry analysis is available for downloading here. First-time users are strongly encouraged to consult with Core staff regarding proper preparation of their samples prior to submission for analysis.
The facility currently has seven mass spectrometers:
The QStar Elite, LTQ XL, and LTQ Orbitrap Velos afford MS and MS/MS capabilities, and are interfaced with Eksigent 2D nanoLC systems for online LC MS/MS operation. The 4000 QTRAP is also interfaced with an Eksigent 2D nanoLC system. The facility also has a ProGest Protein Digestion Station (Genomic Solutions) for automated trypsin digests in a 96-well plate format, and an LC Packings Ultimate 3000 capillary HPLC system interfaced to a Sunchrom spotting robot. The latter provides a direct link between HPLC and MALDI formats of mass spectrometric analysis.
In addition, the Core has two Paradigm MG4 (Michrom BioResources) 2D nano/capillary HPLC systems with HTC PAL liquid handling robots that function as off-line protein/peptide separation and sample preparation workstations. Specifically, these systems are used for fractionating complex protein and peptide mixtures by strong cation exchange or high pH reversed phase chromatography followed by separation of each fraction by reversed phase HPLC.
A searchable database of core facilities at all UCSF campus locations, provided by the Clinical and Translational Science Institute at UCSF, is available here.