Proteomics and Macromolecular Crystallography

  • Rajesh Soni, PhD
    • Manager, Proteomics
  • Farhad Forouhar, PhD
    • Manager, Crystallography

Mass Spectrometry Proteomics

The Proteomics Shared Resource supports both discovery-based and targeted proteomic analysis. The facility provides cancer researchers access to the state-of-the-art mass spectrometers and software for qualitative (e.g. protein identification), quantitative (e.g. relative quantification), and post-translational modification (PTM) analyses. The services include i) mass determination of purified proteins/peptides, ii) identification of purified protein complexes, iii) site-targeted PTM mapping on purified proteins, targeted MRM quantification, iv) global proteome analysis, subproteome isolation and identification, and v) large-scale PTM characterization. The facility director and dedicated technical personnel have extensive expertise in mass spectrometry-based techniques and will provide consultation on experimental design, data analysis, and troubleshooting. We encourage researchers to contact us prior to submission of samples to discuss sample preparation, experimental design, and potential outcomes of research projects.

Specific Services provided:

  • Protein Identification (gel band and section)
  • Quantitative proteomics of affinity enriched protein complexes (IP, BioID, APEX2, etc.)
  • Global quantitative proteomics analysis (Label free (LFQ) & Label (SILAC & TMT)
    • Proteome analysis
    • Post-translational modifications (PTM) analysis (phosphorylation, ubiquitination, etc.)
  • Quantitative and targeted proteomics of cell signaling pathways (Immuno-affinity enrichment)
    • (PI3K/AKT, AMPK, PKC, CDK, MAPK, JNK cascades, T-cell and B-cell receptor signaling, etc.)
  • Exosome proteomics profiling
  • Targeted protein/peptide characterization/ quantification (customized PRM)
  • Custom proteomics for innovative projects

HICCC PSR Mass Spectrometers

  • Fusion Tribrid Mass Spectrometer + Dionex Ultimate 3000
  • QE HF mass spectrometer + Eksigent nanoLC-Ultra 2D HPLC system
  • UltraflexXtreme MALDI TOF/TOF mass spectrometer

Software

  • Proteome Discoverer & MaxQuant (Database search)
  • Scaffold (Data processing & visualization)
  • Skyline (Targeted proteomics analysis)
  • Perseus (Statistical analysis & data visualization)

Crystallography

X-Ray crystallography is the technique used to obtain the three dimensional structure of a macromolecule from a crystal. The macromolecule structure can provide detailed information on the site and specificity of protein-ligand interactions, protein-protein complexes, protein-nucleic-acid complexes, immune complexes, and host-pathogen (i.e. virus, bacterium) interactions. The availability of structural information can bring focus to research in the elucidation of disease mechanisms and in the development of effective drugs. Dr. Forouhar works closely with researchers to develop a project and guide them through the preparation of high quality purified protein samples suitable for crystallization. Once enough pure protein is available, optimum conditions are found to produce diffraction-quality crystals, which are then subjected to an X-ray beam at a synchrotron facility. Structural features of the protein emerge as the data are processed and the structure is refined.

Users of the Crystallography Shared Resource will first meet with Dr. Forouhar for a free consultation to discuss the feasibility and strategy for obtaining a crystal structure from their protein.

Services Include:

  • Structure Modeling: Computer modeling can be performed on a protein which has a structure that is at least 30% identical to that of a protein structure deposited in the Protein Data Bank (PDB).
  • Crystal Screening: Your protein will be screened against 1,536 reagents using a sophisticated robot. We need 460 – 500 µL of the pure protein for the extensive screening. Three types of images, color, UV-TPEF, and SHG will be taken on each crystal hit and analyzed for protein crystal hits versus salt crystals. These three types of images are complementary and confirmatory in distinguishing a real crystal hit (protein crystal) from a false positive hit (salt crystal). At this stage, we can usually tell whether these crystal hits are poor or promising for production of diffraction quality crystals that could lead to structure determination. In case of poor crystal hits, we will take different strategies for improving the hits from poor to diffraction quality crystals. These strategies include 1) surface mutation of charged residues, 2) screening against different protein buffers, and 3) screening against small molecules that could play key roles in improving crystal quality.
  • Crystallization: Once optimum conditions for crystallization are found, those conditions will be reproduced in the lab to form crystals of sufficient size and quantity to yield high quality diffraction data.
  • Data Collection: Crystals will be shipped to an American synchrotron and X-ray data will be collected remotely.
  • Structure Determination and Refinement: The time required to determine the structure of a protein and to refine that structure is highly dependent on the size of the protein and the quality of the diffraction data. An easily completed crystal structure, such as 200-300 amino acids diffracting to 2Å resolution, might take 20 hours. A larger human protein diffracting at 3Å resolution might take many times longer. Throughout the process, we will discuss every step with the researcher.

Projected Costs
A rough estimate of the total cost for an easy and successful crystal structure of a protein comprising 200-300 amino acids at 2Å resolution is $5,000. However, most human proteins which are implicated in cancer contain flexible regions and are therefore not readily amenable to crystallization and structural studies. In such cases, extensive effort will be spent to obtaining a crystal structure of a human protein. Consequently, the total cost of delivering a protein structure to you will normally be a lot more than $5,000. Since structural studies of human proteins are case by case, we will do our best to realistically estimate the total cost of delivering a protein structure to you after we thoroughly evaluate the biophysical/biochemical properties of your protein of interest.

If there is at least one protein structure in Protein Data Bank (PDB), which shares 40% sequence identity with the protein of interest in its native wild-type form, then we will use the molecular replacement method to determine the structure of the protein of interest using the available protein model in PDB. Again we will charge you for every hour spent on performing structural studies on your protein of interest.

If there is no model for the protein of interest, then the best method for determining its structure is by using a seleno-methionine-labeled protein. In this case, the interested party will provide us with the seleno-methionine-labeled recombinant protein at the start of the service.


Locations and Contacts

The Proteomics facility is located at:
Lasker Biomedical Research Building
3960 Broadway, 3rd Floor, Room 350D (Entrance on 166th St.)

For Proteomics inquiries contact: Rajesh Soni, PhD
E-mail: rs3869@cumc.columbia.edu
(Contact by email is preferred)
Tel: 212-342-4177 (office); 212-342-4179 (lab)

For Crystallography inquiries contact: Farhad Forouhar, PhD
E-mail: ff2106@columbia.edu


User Fees and Policies

TMT-based protein quantification

Global proteome analysis - TMT 6-plex
Cancer Center Member: $3,315
Non-Cancer Center Member: $3,900
Off-campus rate: $6,240

Global proteome analysis - TMT 10-plex
Cancer Center Member: $4,275.50
Non-Cancer Center Member: $5,030
Off-campus rate: $8,048

Global proteome analysis - TMT 11-plex
Cancer Center Member: $4,509.25
Non-Cancer Center Member: $5,305
Off-campus rate: $8,488

Global PTM analysis - TMT 6-plex
Cancer Center Member: $5,440
Non-Cancer Center Member: $6,400
Off-campus rate: $10,240

Global PTM analysis - TMT 10-plex
Cancer Center Member: $6,400.50
Non-Cancer Center Member: $7,530
Off-campus rate: $12,048

Global PTM analysis - TMT 11-plex
Cancer Center Member: $6,634.25
Non-Cancer Center Member: $7,805
Off-campus rate: $12,488

Label-free quantification

Label-free quantification - in-solution digestion
Cancer Center Member: $443.70
Non-Cancer Center Member: $522
Off-campus rate: $835.20

Label-free quantification - in-solution digestion & fractionation
Cancer Center Member: $1,496
Non-Cancer Center Member: $1,760
Off-campus rate: $2,816

Label-free quantification - deep proteome analysis
Cancer Center Member: $5,491
Non-Cancer Center Member: $6,460
Off-campus rate: $10,336

Gel-based protein ID

Gel band protein ID
Cancer Center Member: $229.50
Non-Cancer Center Member: $270
Off-campus rate: $432

Gel section protein ID
Cancer Center Member: $273.70
Non-Cancer Center Member: $322
Off-campus rate: $515.20

Gel based PTM characterization
Cancer Center Member: $409.70
Non-Cancer Center Member: $482
Off-campus rate: $771.20

IP-based protein analysis

IP-based gel band protein ID
Cancer Center Member: $229.50
Non-Cancer Center Member: $270
Off-campus rate: $432

IP-based gel section protein ID
Cancer Center Member: $273.70
Non-Cancer Center Member: $322
Off-campus rate: $515.20

On-bead digestion and protein analysis
Cancer Center Member: $324.70
Non-Cancer Center Member: $382
Off-campus rate: $611.20

Phosphopeptide analysis

Phosphopeptide with single shot enrichment - LFQ/SILAC
Cancer Center Member: $1,283.50
Non-Cancer Center Member: $1,510
Off-campus rate: $2,416

Single shot enrichment with PTMScan
Cancer Center Member: $2,483.70
Non-Cancer Center Member: $2,922
Off-campus rate: $4,675.20

Phosphopeptide with Stage-tip fractionation - LFQ/SILAC
Cancer Center Member: $3,469.70
Non-Cancer Center Member: $4,082
Off-campus rate: $6,531.20

Phosphopeptide with deep proteome analysis - LFQ/SILAC
Cancer Center Member: $5,186.70
Non-Cancer Center Member: $6,102
Off-campus rate: $9,763.20

PTMScan pathway enrichment
Cancer Center Member: $1,710.20
Non-Cancer Center Member: $2,012
Off-campus rate: $3,219.20

Other services

LC-MS/MS per hour (for digested samples only)
Cancer Center Member: $88.40
Non-Cancer Center Member: $104
Off-campus rate: $166.40

MALDI-MS per hour (1/2 hour minimum)
Cancer Center Member: $189.55
Non-Cancer Center Member: $223
Off-campus rate: $356.80

Structure Modeling
Cancer Center Member: $90
Non-Cancer Center Member: $100
Off-campus rate: $160

Crystallization
Cancer Center Member: $105
Non-Cancer Center Member: $115
Off-campus rate: $184

Data Collection
Cancer Center Member: $92
Non-Cancer Center Member: $102
Off-campus rate: $163.20

Structure determination & refinement
Cancer Center Member: $90
Non-Cancer Center Member: $100
Off-campus rate: $160

Screening
Cancer Center Member: $400
Non-Cancer Center Member: $400
Off-campus rate: $640


Online Reservations

The Proteomics and Macromolecular Crystallography Shared Resource uses the iLab Core Management System for service requests and billing. Before you can request services, you must log into iLab and be approved as a lab member.

Glossary of Terms

Electrospray Ionization (ESI): Ionization by applying high voltage to an analyte solution, often the eluate of an HPLC column, flowing through a nano-bore spray capillary. The high potential causes the liquid to spray out of the tip as a butt of tiny, highly charged droplets. The solvent molecules evaporate and the remaining charged analyte molecule, such as a peptide or protein, enters the mass analyzer. This method of ionization is frequently coupled with HPLC (LC-MS).

Mass Spectrometer: An instrument that ionizes sample molecules and separates the ions according to their mass-to-charge (m/z) ratio. A mass spectrum is a graphical plot of measured ion abundance vs. m/z.

MS/MS: Also called tandem mass spectrometry. A technique which utilizes two stages of mass analysis. In the analysis of a peptide digest, mass of the peptide ion formed in the ion source is first measured by the mass analyzer. Then the selected ion is activated in a collision cell with a neutral gas such as Ar, and the resulting collision-induced fragment ions are analyzed by a second mass analyzer.

MS/MS spectrum: A plot of the abundance of fragment ions produced in the collision cell of the mass spectrometer vs. m/z. Masses of fragment ions can be predicted from a known sequence and are used to identify proteins with the help of bioinformatic tools. Sequence information can be obtained from the MS/MS spectrum manually if the protein is not in the searchable database (de novo sequencing).

Matrix-assisted Laser Desorption Ionization (MALDI or MALDI-TOF): Ionization technique in which the sample is co-crystallized with a matrix of small organic molecules then irradiated with a laser which causes the analyte to vaporize, passing into the mass analyzer as charged ions.

Time-of-Flight (TOF): Method of mass analysis in which ions formed are accelerated by a pulsed potential down an evacuated tube. The time of flight down the tube correlates to mass, with lighter molecules taking less time to arrive at the detector than heavier ones.


Sample Preparation Recommendations

Recommendations for gel band-based protein identification

  • Use commercially available SDS-PAGE instead of home-made SDS-PAGE.
  • Coomassie staining instead of silver staining if possible (contact the director if coomassie staining is not possible).
  • Stain the gel in a closed container all the time to minimize contamination and destain the gel with milliQ water,
  • Wear gloves the entire time of handling the samples.
  • Take a digital image of the gel indicating the band(s) to cut and send the digital file to the director via email.
  • Make an appointment to bring the gel over in a covered container with milliQ H2O.

Recommendations for in-solution (IP) based protein identification

  • If possible, conjugate the primary antibody to solid support (preferably magnetic beads).
  • Start with as much starting material as possible.
  • Optimize antibody to antigen/bait protein ratio. Only use as much antibody as to be able to pull down >90% antigen/bait.
  • Do not use a wash buffer containing carrier protein (e.g. BSA, milk).
  • Verify the IP by Western Blot and submit the beads after the last wash without any liquid.
  • If non-MS compatible detergents are used in your buffers, wash IP with buffer not containing detergent and switch to new tubes a few times during washing.
  • Store the beads at -20oC prior to submitting the sample.

Recommendations for in-solution (IP) based protein identification

  • For adherent cell keep the confluency 80-90%, after trypsinization, collect the cells by low speed centrifuge and wash the cells twice with ice cold phosphate-buffered saline (PBS), transfer into 1.5-ml tubes and centrifuge to remove supernatant completely. Bring the cell pellets on the dry ice or store the cell pellet at -800C until lysis.
  • Suspension cell harvest: collect cells in 15-50 ml tubes, wash the cells twice with ice cold phosphate-buffered saline (PBS), transfer into 1.5-ml tubes and centrifuge to remove supernatant completely. Bring the cell pellets on the dry ice or store the cell pellet at -800C until lysis.
  • Tissue collection: collect tissues in 1.5 ml tubes, and keep tissues in liquid nitrogen immediately after dissection and store at −80°C. For possible blood contamination, the samples should be rinsed twice with ice cold PBS.