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Mass Spectrometry and Proteomics Research Laboratory (FAS)

Location: Mass Spectrometry & Proteomics, 52 Oxford St., Room B247, Cambridge, MA 02138

Core Summary:

The FAS Center for Systems Biology Mass Spectrometry and Proteomics Resource Laboratory provides mass spectrometry and strategic consulting in Proteomics and Small Molecule analysis for Life Science and Chemistry researchers as well as others worldwide. This resource brings together the state-of-the-art expertise and instrumentation of the Microchemistry and Proteomics, CCB Mass Spectrometry, and Bauer Center Core laboratories, leveraging our breadth of experience to provide the best possible support for your research.

Institutions:
  • Harvard Faculty of Arts and Sciences
Personnel/Contact Information:

Member: Budnik, Bogdan
Role: Lab Manager
Phone: (617) 496-0460

Member: General Inquiries
Email

Facilities and Equipment:

Location of Core: Mass Spectrometry & Proteomics, 52 Oxford St., Room B247, Cambridge, MA 02138

Major Equipment:

  • Agilent 1100 HPLC #1
  • Agilent 1100 HPLC #2
  • Agilent 1100 HPLC #3
  • Agilent ChemStation
  • Applied Biosystems Vision Preparative HPLC
  • EasyAccess
  • MassHunter
  • Masslynx
  • OALogin
  • ThermoFisher LTQ-Orbitrap Velos
  • Thermo Fisher Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer
  • Waters 2695 HPLC
  • Waters nanoACQUITY UPLC
  • Waters Q-Tof micro LC/MS/MS
Services:
  • Complex protein mixture analysis service

    Complex mixtures of proteins are identified by a number of single- and multi-dimensional approaches. For example, GeLC, in which an entire lane of an SDS-PAGE gel is excised into sections, affords the user a two dimensional separation of the protein mixture based on protein intact molecular weight (SDS-PAGE) and then individual peptide hydrophobicity by reversed phase chromatography (RPLC). A similar method known as MUDPit (Multidimensional Protein Identification Technology) starts with a solution digestion of the sample, then two dimensional chromatography by strong cation exchange chromatography (SCX) followed by reversed phase chromatography (RPLC).

  • C-terminal sequence analysis service

    In this lab, we use multiple enzymes to obtain redundant peptides which exhaustively define the C-terminal region of a purified protein. Multiple instrument runs are combined with custom bioinformatics tools to provide the final result.

  • Data analysis service

    Data Analysis is an essential part of mass spectrometry, and we encourage our customers to discuss their results with us when this is helpful. The open access instruments are set up to acquire and process data automatically. In most cases, a data report is printed when acquisition has completed. Agilent LC instruments (time of flight and quadrupole) will send a data report via email and the data report can be opened using DataBrowser which can be loaded on your personal computer (Windows platform only). DataBrowser can be obtained from our facility. Fee For Service results are generally delivered in pdf format wherever possible. You are encouraged to contact the facility prior to sample submission, so that we can understand your scientific objectives and design an experiment appropriate to those goals.

  • De novo sequence analysis service

    [Many] mass spectrometry techniques rely on the protein sequence being known and available for comparison of mass spectra to a database. If this is not the case, identical peptides from homologous proteins can often be found, leaving many still unidentified. While software algorithms have advanced, these spectra often require expert manual interpretation. This laboratory has over 40 years of combined experience specializing in de novo interpretation.

  • Exact mass MS & MS/MS electrospray for structural elucidation service

    Fee For Service - allows users to submit samples with a submission form containing sample information to our facility and results are emailed to users once they are obtained. Fees depend upon the nature of the experiments. Please contact our facility for more details.

  • Intact molecular weight determination service

    Intact proteins, oligonulceotides and peptides frequently need an intact mass determination, and MALDI-TOF is the preferred method to obtain this information due to the "soft" ionization technique and low charge states associated with this technique. Samples are applied to a target with an appropriate matrix and allowed to dry fully, concentrating of sample in a crystalized matrix spot. A UV laser imparts energy to the sample through the matrix, causing the sample to ionize (typically a a singly or doubly charged species) and the time it takes to travel along the flight tube is proportional to the mass of the sample molecule. Typically proteins and peptides between 0.5 and 200kDa, and oligonucleotides up to 10kDa can be observed at very high sensitivity. Sample concentration is key to good signal quality, and salts, detergents and other compounds in the sample buffer can reduce the ionization of the molecule significantly.

  • Labeled quantitative proteomics - AQUA service

  • Labeled quantitative proteomics - ICAT service

  • Labeled quantitative proteomics - iTRAQ service

    iTRAQ (Isotope Tags for Relative and Absolute Quantitation) is another popular technique that includes up to 10 isotopic labels for multiplexing experimental variables. The technique is based upon chemically tagging the N-terminus of peptides generated from protein. The labeled samples are then combined (post labeling), fractionated by nano-LC and analyzed by tandem mass spectrometry. Peptides are chromatographically resolved as single peaks with identical full MS masses. Fragmentation of the labeled peptides generates a low molecular mass reporter ion that is unique to the tag used to label each of the samples. Measurement of the intensity of these reporter ions, enables relative quantification of the peptides in each digest and hence the proteins from where they originate. This process has the advantage of no chromatographic interference from the labels but requires a low mass MSMS scan to observe the reporter ions.

  • Labeled quantitative proteomics - SILAC

    Labeled: Quantitative mass spectrometry normally utilizes stable isotope labeling at the whole cell level, intact protein level or even peptide level. There are several well established techniques to do this, and a detailed project consultation prior to beginning an experiment with this goal is mandatory.

    SILAC (stable isotope labeling with amino acids in cell culture)
    ICAT (Isotope Coded Affinity Tags)
    iTRAQ (Isotope Tags for Relative and Absolute Quantitation)
    AQUA method of absolute quantitation.

  • Low resolution GC/MS/MS service

    Open Access Service - offers trained users a walk-up service where users can choose from various pre-programmed experimental methods and obtain results 24 hours, 7 days a week.

  • Low resolution GC/MS service

    Service available at two different levels. Fee For Service allows users to submit samples with a submission form containing sample information to the facility and results are emailed to users once they are obtained. Fees depend upon the nature of the experiments. Open Access Service offers trained users a walk-up service where users can choose from various pre-programmed experimental methods and obtain results 24 hours, 7 days a week.

  • N-terminal Edman Sequence Analysis Service

    N-terminal sequence analysis is a chemical method in which the amino terminal amino acid is labeled with phenylisothiocyanate and specifically cleaved, followed by identification of the released phenylthiohydantoin amino acid by RPLC. This process can sequence upwards of 30 amino acids given sufficient quantities, typically 10pmol or more, of a single protein. Edman sequencing affords the researcher the ability to characterize the N-terminus of a protein directly, including quantitatively differentiating between even single amino acid cleavage sites. This process gives true de-novo sequence information, as it is not a database dependent technique, and has been an established method in protein research for many decades.

    In this lab, we use multiple enzymes to obtain redundant peptides which exhaustivly define the C-terminal region of a purified protein. Multiple instrument runs are combined with custom bioinformatics tools to provide the final result.

  • Posttranslational modification site determination service

    Starting with a single highly purified protein in an SDS-PAGE gel slice, multiple sites of modification, eg. phosphorylation, acetylation and others, can be determined. This process involves a detailed project discussion and careful selection of multiple enzymes to maximize peptide coverage for specific sites of interest.

  • Project consultation service

    One of the hallmarks of this facility is the attention we pay to project discussion, sample preparation, and final data presentation for our clients. John Neveu, and Bogdan Budnik are the scientists who conduct these discussions, ensuring that your experimental design and samples have been prepared in an optimal fashion for their intended analyses.

  • Protein identification service

    Protein identification can be done either by direct N-terminal chemical sequencing or by digestion and LC/MS/MSMS analysis. After an in-depth project discussion, the sample is prepared by the user following simple protocols, and submitted to the facility for analysis. Samples are enzymatically digested, run on nano-capillary HPLC/MSMS, and the MSMS spectra are correlated against a specific database for peptide identification. When applicable, N-terminal Edman sequencing is available.

  • Quantitative proteomics service

    One of the major challenges in modern proteomics is characterizing the differences in protein expression between two or more samples in a statistically relevant method. For instance, these methods could show differences in protein expression between treated and non-treated cell lines, healthy and sick animals, or between knockout and wild type organisms.


Departmental Web Link(s):

Last updated: 2015-12-01T15:32:37.610-06:00