TCGA Proteomics Services

The Centre for Genomic Application (TCGA) has recently developed an advance mass spectrometry based Proteomics facility. Our facility is equipped with Bruker (Ultraflex) MALDI-TOF/TOF Agilent 2Dnano LC MS systems.Both the instruments have high sensitivity and high resolution. The facility is also equipped with 2D Gel electrophoresis unit for protein separation. Our facility is equipped to analyze by mass spectrometry proteins, peptides, oligonucleotides and small molecules from samples in solution or separated by chromatography or electrophoresis. Activities at the Proteomics facility is taken care and supervised by well-experienced scientists who also advise investigators in sample preparation and analysis. The facility is open to investigators from different institutes in and abroad on a fee-for-service basis

Services Available

The Proteomics facility at TCGA has state-of-the-art equipments for protein identification and sequencing which is available to researchers on a fee-for-service basis. Our facility provides following services

2D Gel electrophoresis of proteins:

TCGA offers high-throughput, two-dimensional gel electrophoresis (Biorad) for a diverse range of sample types. 2DGel electrophoresis is an excellent method for fractionation of complex mixture of proteins into discrete components and for study of differential protein expression. Spots obtained on gels correspond to different proteins and are isolated and subjected to enzymatic digestion for further mass spec analysis. We offer a multitude of gel staining techniques, depending on the nature of the sample being separated. TCGA maintains strict adherence to standardized procedures for 2D gel electrophoresis based protein separation technique.

In gel tryptic digestion
At TCGA we do in-gel tryptic digestion (as well as in-solution digestion for whole proteome) of spots excised from 1D and 2D gels. We use high quality (Promega Trypsin gold Mass Spectroscopy Grade) for digestion.

Protein identification (Peptide mass fingerprinting) and sequencing by MALDI-TOF/TOF
We subject the digested mixture of peptides to MALDI- TOF analysis for Peptide mass fingerprinting and protein sequencing using Bruker Ultraflex MALDI-TOF/TOF. MALDI – is a well established technique for protein identification (Picture1) and sequencing (Picture2). We also use this technique for comparing protein profiles of normal and diseased samples for purposes like biomarker detection (Clinical Proteomics). Serum samples are fractionated and enriched using magnetic beads before MALDI- TOF and specially designed software by Bruker (Clinprot ) is used for data analysis.

Pic.1 MASCOT search result of glycogen phosphorylase Pic.2 MALDI- TOF / TOF profile of peptide Selected selected from beta-amylase PMF

Protein identification and sequencing by 2D Nano LC MS /MS:
Protein identification and sequencing is also carried out at our facility using two dimensional liquid chromatography ESI MS (Agilent 1100 series 2DnanoLC MS). Tryptic digested protein is subjected to column followed by reverse phase separation. Peptides get ionized in the liquid phase in the Electrospray ionizer and enter the ion trap, get fragmented (MS/MS) and detected there. The data is either sent to MASCOT search engine (Pict 3 and Pict 4) or Spectrum Mill software (Agilent) for analysis. MUDPIT (Multidimensional Protein Identification Technology) analysis is also done at TCGA proteomics facility on highly complex proteomic mixtures such as whole proteomes, organelles and protein complexes, provided by customers. This technique helps to alleviate many of the disadvantages associated with two-dimensional gel electrophoresis.

Pic.3 MS/MS data of Oxoacyl ACP reductase
(Plasmodium Falciparum) using LCMS Pic.4 MASCOT score of 1040.Peptides matched 84

*Additional services will be offered as the facility expands.

Technology

The Proteomics facility at The Centre for Genomic Application (TCGA) is well equipped with instruments for protein separation and identification.. Major instrumentation at the core facility includes the following:

Biorad 2D Gel Electrophoresis unit
2D Gel electrophoresis is currently the most widely accepted technique for the isolation/separation/ purification of proteins for further characterization with the mass spectrometry and identification of specific proteins. 2DGE is a method of protein separation, by which proteins in a mixture are separated according to their isoelectric point (pI) in the horizontal direction (isoelectric focusing [IEF]) and molecular weight in the vertical direction [ SDS - PAGE ].The Proteomics facility at TCGA is equipped with Biorad 2D gel electrophoresis unit for separation of protein samples which are further subjected to MALDI and LC MS studies for protein identification

Ultraflex MALDI- TOF / TOF mass spectrometer (Bruker Daltonics):
At TCGA we use Bruker Ultraflex MALDI-TOF/TOF mass spectrometer which is designed for automated high throughput identification and sequencing of proteins. MALDI- TOF stands for Matrix assisted Laser Desorption Ionization –Time of flight ( Hillenkamp F, Karas M.Methods Enzymol. 1990;193:280-95). . The instrument provides very high sensitivity, resolution and high mass accuracy required for protein identification, expression proteomics studies, and Biomarker discovery.

The tryptic digested protein is mixed with matrix and subjected to MALDI- TOF . Set of peptides obtained is matched with database using search engine like MASCOT. If the protein is not available in the database (hence not identified), some of the peptides are selected and subjected to further fragmentation and further database search is done to identify the protein. It is also capable of performing peptide de novo sequencing, the analysis of post-translational modifications using the Biotools software.

Agilent 1100 series 2D NanoLC MS

Two dimensional liquid chromatography mass spectrometry is by far the best method to analyze complex mixtures, such as enzymatically digested proteins or whole cells. At TCGA we use 1100 series 2D Nano LCMS which consists of nano LC system coupled to an ESI-MS instrument. In this instrument the digested protein is either directly loaded on reverse phase nano column or allowed to pass through a strong cation exchange column by an injected salt step gradient of increasing salt concentration which is followed by a reversed phase separation. The peptides are then subjected to online MS/MS using ion trap mass spectrometer. The data is analyzed using MASCOT search engine or Spectrum Mill software (Agilent).

The instrument is also widely used for MUDPIT analysis. MUDPIT stands for Multi-Dimensional Protein Identification Technique. It is a technique for the separation and identification of complex protein and peptide mixtures. The complex peptide mixture by digestion of whole cell or organelle is loaded on strong cation exchange columns and peptide fractions are collected using salt gradient. Prior to that, fractions are concentrated using Enrichment columns and then enters C18 reverse phase columns. The MS/MS data of peptide fractions are analyzed by Spectrum Mill software.

Sample Preparation Guideline

Sample Handling
1) Wear powder free nitrile gloves and a lab coat when working with your sample at all the steps ( in pouring the gel, in staining the gel, in cutting out protein from the gel, or in transferring the sample)and work in a dust free environment to avoid keratin contamination. Dust, small hairs and fingerprints are common sources of contamination.

2) Use clean plates and containers for gel casting and staining.

3) To minimize protein losses from adsorption to walls of tubes, use polypropylene tubes siliconized polypropylene tubes. Avoid polystyrene (clear plastic) and glass.

4) While excising the bands be sure to use extremely clean surfaces and new razor blades or use a cleaned scalpel blade every time you cut a gel.Rinse scalpel first with ethanol, then dd H2O after each band that you cut to avoid cross contamination. Ideally this should be done in a laminar flow hood to minimize the possibility of any dust,hair, flakes of skin, or other forms of dust.

5) Cut close to the stained portion of the gel to minimize the excess acryl amide.

6) Clearly label each tube and complete a sample submission form for every sample

7) Use freshly prepared high purity reagents and water. Contaminants from buffers,detergents, urea etc. can prevent good mass spectrometry results.

8) Avoid the use of non-volatile agents like salts(NaCl,CaCl2,KH2PO4), detergents(Tween,Triton,SDS),chaotropic agents(Urea,Guanidium salts) and non volatilesolvents like DMSO,DMF or glycerol .If you can’t avoid these agents, purify.Dialysis, Ziptips and RP-HPLC are good purification methods if you use volatile solvents and buffers (e.g. trifluroacetic acid, NH4HCO3).

9) Suitable Volatile solvents and buffers include water, acetonitrile, trifluroaceticacid, ammonium hydrogen carbonate, ammonium formate, ammonium acetate.

Note: Ziptip purification is done at TCGA facility at extra cost.

How to Submit Your Samples for MALDI-TOF and LC-MS

Samples can be sent as frozen solution, lyophilized solutions, gel plugs or complete gel for spot picking.

In solution, as concentrated as possible, volume 10-20 µl.
Minimum concentration 10 pmol/µl.
Preferred solvent-0.1-1%TFA

Gel plugs for tryptic digestion:

Gel plugs must be in clear polypropylene tubes or plates, clearly labeled, sealed with parafilm and can be sent at room temperature or 40C. For 96-well trypsinization, gel plugs must be in conical, polypropylene 96-well plate Start with well A1, order plugs from well A1-A8, B1-B8, etc. If submitting gel plugs, include a gel plug from your gel without protein to serve as a negative control.

Intact gel:

You can send us the entire gel with well annotated images (PowerPoint) describing which bands to be cut .We can cut the spots for you. Gels can be sent at room temperature in sealed plastic bag containing enough water to maintain moisture so that gel does not get dry. Gels should be packaged in such a way as to avoid tearing of the gel.

Recommended Concentration of samples

Peptides- 5pmol/ µl.

Protein (5-60KD)- 10-20 pmol

Protein (60-100KD)- 20-40pmol

How to Submit Your Samples for 2 D Gel Electrophoresis:

Sample should contain

No salt
Avoid ionic components(like SDS)
Can contain 2-4 % CHAPS, 8 M Urea, and 10-30mM Tris base
Should not contain lipids, nucleic acids, polysaccharide materials, phenolic compounds, and insoluble materials.

Recommended concentrations
Concentration: 5-10 µg/ml
Amount: 100-150µg (up to 1 mg).
If any additional information is required regarding sample preparation & submission and MUDPIT analysis, please mail at somdutta@tcgaresearch.org or call at 91-11-5142220.

FAQs

1.What are the various Proteomics services provided by TCGA?
The proteomics division at TCGA currently provides three major services
1.Separation of proteins by 2D Gel electrophoresis( Biorad)
2.Protein identification by MALDI-TOF/TOF (Bruker Ultraflex)
3.Protein identification by 2D Nano LC MS (Agilent)
4.MUDPIT analysis of whole proteome using 2D Nano LC MS (Agilent)

2.What is the turn around time?
The typical turn around time for MALDI-TOF/TOF is around 7-10 working days. MUDPIT analysis of whole proteome takes around 15 days.

3.How much protein is to be submitted for analysis?
All our instruments have very high sensitivity to detect proteins/peptides. However for better identification and further studies (e.g Post-translational modifications) try to send minimum 5-10pm/ml sample for PMF and MS/MS by MALDI-TOF /TOF and 2DnanoLC- MS. For intact proteins preferred concentration range should be 20-50pmol/m

4.What type of staining is recomended for MALDI and LC MS/MS studies?
We prefer Coomasie or colloidal blue staining. Silver staining is generally not recommended as gels have to be totally destained before enzymatic digestion as trypsin activity is impeded by silver. If it is absolutely essential to use silver staining it is recommended to avoid the use of gluteraldehyde as a fixing agent

5.What kind of buffers/detergents can be tolerated on MALDI?
Triton-X100 should be strictly avoided . Instead use N-octyl-b-glucopyranoside as detergent. Use dialysis or gel filtration techniques for insolution digests.

6.What is the recommended protocol for Tryptic digestion?
For detailed tryptic digestion protocol contact us at the given e-mail address. somdutta@tcgaresearch.org

7.What is MUDPIT analysis and is it possible at TCGA?
Yes we perform MUDPIT analysis quite regularly at TCGA using Agilent 1100series 2D nanoLC MS. It is one of the recommended methods for characterization and identification of all the proteins present in a purified organelle fraction or whole cell. MUDPIT stands for Multi-Dimensional Protein Identification Technique. It is a technique for the separation and identification of complex protein and peptide mixtures. This is a complementary technology to 2D gel based approaches. It is based on two dimensional nanoflow chromatography of peptides produced by tryptic digestion of a complex mixture of proteins which is coupled directly to nanoelectrospray ionization (nano-LC-MS/MS). The whole cell lysate or organelle fraction is subjected to reduction and alkylation steps followed by in-solution digestion. The complex peptide mixture prior to digestion is loaded on strong cation exchange (SCX) columns (3.5um Zorbax ) and peptide fractions are collected using salt gradient. Prior to that fractions are concentrated using Enrichment columns (Zorbax C18,5um ) and then enters C18 reverse phase columns (3.5um Zorbax).The peptides are then subjected to online MS/MS using ion trap mass spectrometer. The data is analyzed using Spectrum Mill software (Agilent) and other search engines.

8.How much is the cost of MALDI-ToF and nanoLC MS services?
Contact somdutta@tcgaresearch.org for details.