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.
- 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
- Fusion Tribrid Mass Spectrometer + Dionex Ultimate 3000
- QE HF mass spectrometer + Eksigent nanoLC-Ultra 2D HPLC system
- UltraflexXtreme MALDI TOF/TOF mass spectrometer
- Proteome Discoverer & MaxQuant (Database search)
- Scaffold (Data processing & visualization)
- Skyline (Targeted proteomics analysis)
- Perseus (Statistical analysis & data visualization)
Crystallography
X-Ray crystallography and cryogenic electron microscopy (CEM) are commonly techniques used to obtain the three-dimensional structure of a macromolecule. 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 structural studies. Once enough pure protein is available, optimum conditions are found to produce either diffraction-quality crystals or high-quality grids for structural determination using an x-ray beam line at the Advanced Photon Source (APS) or National Synchrotron Light Source II (NSLSII) and an electron microscope at New York Structural Biology Center (NYSBC), respectively. Structural features of the protein emerge as the data are processed and the structure is refined.
Users of the Structural Biology Shared Resource will first meet with Dr. Forouhar for a free consultation to discuss the feasibility and strategy for structural studies of their protein of interest.
- Structure Modeling: Computer modeling can be performed using Alpha-Fold2-Multimer and Alpha-Fold3, with manual adjustment of each model, which could be consistent with published biochemical and biophysical data or unpublished one that is provided by the researchers.
- Crystal Screening: Your protein will be screened against 1,536 reagents using a sophisticated robot. We need 450 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.
- Grid Screening: Grids for your protein will be made shortly after your protein being subjected to a gel-filtration column and possibly being concentrated. Grids will be provided either by you or us. In the latter case, you will be charged for each grid and time required for making each. Grids will be screened using the Glacios microscope at the CEM core facility in Hammer building. Each screening session (4 hours), respectively, costs $200 and $340 for any Columbia principal investigator (PI) and any outside PI, in addition to our labor in making grids and screening.
- Crystallization: Once optimum conditions for crystallization are found, they will be reproduced in the lab to form crystals of sufficient size and quantity to yield high quality diffraction data.
- Data Collection for Crystallography: Crystals will be shipped to an American synchrotron (APS or NSLSII) and x-ray datasets will be collected remotely.
- Data Collection and Processing for CEM: Once we obtained promising grids on which well-defined particles could be detected, we will request for a 2-day microscope time at a Krios microscope at NYSBC. The session is free for any Columbia PI but there is a waiting period. We will charge you for setting up the data collection and not for the entire 2-day session. Unlike crystallography, CEM data collection and processing both take much longer time.
- Structure Determination and Refinement for Crystallography: The time required to determine the structure of a protein and to refine that structure depends 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.
- Structure Determination and Refinement for CEM: There is no limit in the size of a protein that is subjected to structure studies using crystallography. In contrast, we prefer to work on a protein or a protein complex that is at least 100 kDa for structural studies using CEM. The required time to determine the structure of a protein and to refine it depends on the size of the protein and the quality of the CEM data. For a CEM map at 3Å resolution, we will use AI-assisted model building, which could greatly facilitate structural determination and refinement. However, due to much more dynamic nature of a protein structure obtained by CEM, the time required to completing a protein structure or a protein complex by CEM is much longer than that obtained by crystallography.
Projected Costs for Crystallography and CEM
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 that 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 no model for the protein of interest in Protein Data Bank (PDB), we will initially use a model generated by Alpha-Fold2-Multimer or Alpha-Fold3 as a template for structural determination. If this method failed, we will use seleno-methionine-labeled protein. In this case, the interested party will provide us with the seleno-methionine (SeMet)-labeled recombinant protein at the start of the service or we could produce the SeMet-labeled protein for an additional charge.
In case of structural studies by CEM, the bulk of cost will be on obtaining good grids from a stable protein or protein complex and data processing and refinement.
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(link sends e-mail)
(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(link sends e-mail)
User Fees and Policies
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 - 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 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 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 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
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 Structural Biology 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.
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
- 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.
- 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.
- 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.
