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.
"Analyze demanding proteomics, metabolomics, lipidomics and metabolism samples in greater depth using the Thermo Scientific™ Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer. This instrument combines a high-field Thermo Scientific™ Orbitrap™ mass analyzer and Thermo Scientific™ Velos Pro™ ion trap technology to deliver very high resolution, speed, sensitivity and complementary fragmentation information."
Mass Spectrometer used for oligosaccharide structural analyses and MS-MS analysis.
"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.
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).
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.
Starting with a single highly purified protein in an SDS-PAGE gel slice or in a compatible solution, 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. "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.