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.
Role: Lab Manager
Phone: (617) 496-0460
The first of three Agilent 1100 HPLC is configured for automated Amino Acid Analysis (AAA) for protein quantitation and composition. Controlled by Agilent ChemStation software, these systems provide flexibility and robustness for protein and peptide work. Fee for service instrument.
A second 1100 system can perform Strong Cation Exchange (SCX) and Reverse Phase Liquid Chromatography (RPLC) with automated peak detection and fraction collection for sample prefractionation and purification under various configurations including diode array detection (DAD) and variable wavelength detection (VWD). Controlled by Agilent ChemStation software, these systems provide flexibility and robustness for protein and peptide work. Fee for service instrument.
The third HPLC system is an analytical scale HPLC with a fraction collector which can be used to separate and analyze components within a chemical mixture. The injection volume of the autosampler is 1-100 uL. The pump flow rate is 0.1-2 mL/min. Chemstation software is used for control and data analysis. Fee for service instrument.
Applied Biosystems Vision preparative HPLC A LC system is used for separating and analyzing large scale of chemical mixture. The AFC injection system can inject as much as 1-5 mL samples and the typical flow rate during the run is 10-20 mL/min.
Two of these instruments available.
The Waters Alliance 2695 Separations Module is a high performance liquid chromatographic system with a quaternary, low-pressure mixing pump and inline vacuum degassing. Flow rates from 50 uL/min to 5 mL/min can be generated for use with 2.1 mm ID columns and larger. The autosampler has a maximum capacity of 120 vials (12x32, 2-mL) with programmable temperature control from 4 to 40 degrees C. A heated column compartment provides temperatures from 5 degrees above ambient to 65 degrees C. The detector is a photodiode array (model 2996) with a wavelength range of 190-800 nm and sensitivity settings from 0.0001-2.0000 absorbance units. Fee for service instrument. Instrument availability: 70% of the time.
"The nanoACQUITY UltraPerformance LC® (UPLC® ) System is designed for nano-scale, capillary, and narrow-bore separations to attain the highest chromatographic resolution, sensitivity, and reproducibility.
Direct nano-flow offers significant improvements over conventional nano-flow separations technologies. You’ll see improved peak capacity and peak shape, and increase the number of components that can be detected per separation.
The system’s 10,000 psi operating pressure capability allows for superior high-peak capacity separations by operating longer columns packed with sub-2 micron particles."
The lab contains two of these instruments.
Fee for service instrument. Instrument availability: 70% of the time.
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.
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).
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.
[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.
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 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.
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 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.
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.
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 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.
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.
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 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.
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.
"Agilent ChemStation is the industry leading chromatography data system for Agilent instrumentation, handling the widest variety of separation techniques such as GC, LC, LC/MS, CE and CE/MS. It is a scalable data system ideally suited for applications in all industries ranging from early product development to quality control. Extensive customization capabilities as well as configurable regulatory compliance provide the flexibility to support different workflows. Sophisticated level-5 control and monitoring of LAN-based instruments ensures fast and flexible data acquisition, which is complemented by advanced data analysis and reporting capabilities for highest productivity." Used with Agilent 1100 HPLC.
Used with Agilent 6210 Time-of-Flight LC/MS.
Used with Agilent 6210 Time-of-Flight LC/MS. "The all new Agilent MassHunter Workstation software provides intuitive, yet powerful, instrument control, data acquisition, qualitative and quantitative data analysis, and reporting for Agilent time-of-flight (TOF), quadrupole time-of-flight (Q-TOF), ICP-MS, and triple quadrupole systems. Designed from the ground up to make MS analyses easier—from tuning to final report—MassHunter Workstation software can be complemented by application-specific MassHunter software packages that provide even more power and stream-lined operation for specialized analytical tasks such as expression profiling."
Acquisition software used with Waters Quattro micro GC/MS/MS.
Open access software used with Waters Quattro micro GC/MS/MS.