The UCSF 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:
- Provide mass spectrometry service through collaborative and fee-for-service venues.
- Provide mass spectrometry education through lectures, training classes, electronic media, seminars, and participation in formal university courses.
The facility's faculty director is Susan Fisher, PhD. Core staff include Steven Hall, PhD and H. Ewa Witkowska, PhD, Co-Directors of Operations; and Eric Johansen, PhD, Core Facility Manager.
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Services
The Core offers a variety of services for protein identification and characterization as well as molecular mass determinations for peptides, intact proteins, 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 Service Options
Protein Identification by Peptide Mass Fingerprinting
Peptide mass fingerprinting allows identification of unknown proteins and identity confirmation of recombinant proteins, present either in solution or isolated from 1- and 2-D gels. Proteins are digested with a proteolytic enzyme, usually trypsin, and a mass spectrum of the digest is acquired without further separation. The experimentally determined peptide masses are used to search a peptide mass database generated by in silico digestion of all protein sequences in a publicly available protein database. The experimentally determined peptide masses are matched with theoretical peptide masses from a protein in the database within a window of mass measurement accuracy. Matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry is the standard method for acquiring PMF data. If samples are properly prepared and relatively pure, then peptide mass fingerprinting can yield high confidence protein identifications. Poor confidence in peptide mass fingerprinting identities can occur when a mixture of proteins or other contaminants is present in the original sample. Generally, it is good strategy to use peptide mass fingerprinting as the first step when attempting to establish protein identity by using mass spectrometry.
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 (<5000 Daltons), and oligonucleotides up to 20 bases are determined on a MALDI TOF mass spectrometer. Intact protein molecular weights (>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 (<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.
Sample Preparation
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.
Equipment
The facility currently has four mass spectrometers: the Voyager DE STR, a research-quality MALDI-TOF instrument; the QStar XL, a QqTOF configuration that can be operated either with electrospray (ESI) or with oMALDI ionization sources; a 4800 Proteomics Analyzer with MS/MS capabilities equipped with MALDI ionization source and TOF/TOF ion optics; and a 4000 ESI QTRAP that can be operated as a standard triple quadrupole instrument or with the third quadrupole as a linear ion trap.
The QStar XL affords MS and MS/MS capabilities in either ionization mode, and it is interfaced with an Eksigent 2D nanoLC system for online LC MS/MS operation. The 4000 QTRAP is also interfaced with an Eksigent 2D nanoLC system. The facility also has an LC Packings Ultimate capillary HPLC system interfaced to a ProBot spotting robot, thus providing a direct link between HPLC and MALDI/oMALDI formats of mass spectrometric analysis.
In addition, the Core has a Paradigm MG4 (Michrom BioResources) 2D nano/capillary HPLC system with an HTC PAL liquid handling robot that functions as an off-line protein/peptide separation and sample preparation workstation. Specifically, this system is used for fractionating complex protein and peptide mixtures by strong cation exchange chromatography followed by separation of each fraction by reversed phase HPLC.
Additional Resources
A searchable database of core facilities at all UCSF campus locations, provided by the Clinical and Translational Science Institute at UCSF, is available here.