DISCOVERY & TARGETED PROTEOMICS

Service: VPGEL LC-MS/MS

  • This service is for protein identification from 1D gel bands or 2D gel spots.
  • Single Coomassie brilliant blue protein band/MS compatible silver staining spot
  • Complex mixture analysis of proteins stacked under the border between the stacking and separating gel e.g. pull down complexes, vesicular proteomics, protein fractions etc
  • Phosphorylation and other PTM analysis of gel separated proteins.

Compatible gels: Any protein band which can be visualized by Coomassie brilliant blue staining or silver staining can be identified using this method. We require high resolution pictures of gel (Coomassie/Silver/Sypro Ruby/2D) before and after band excision.

We accept gel bands from

  • Coomassie brilliant blue stained gels 1D gel
  • Silver Stained 1D gel Gels - mass-spec compatible staining methods
  • Sypro Ruby Stained 1D Gels
  • 2D SDS gel electrophoresis-Coomassie/mass-spec compatible silver staining

Service code

VPGEL1

VPGEL2

VPGEL3

VPGEL4

VPGEL5

Sample origin

Coomassie

Silver

Sypro

Coomassie (complex protein mixture, stacked band)

Coomassie /silver /MS compatible stain

Method Specifications

Ultra-high resolution nano-LC MS/MS analysis using 10-15 cm column (1.9µm column resin) Plus Q Exactive series mass spectrometer

diagram1

Organism Database

Customer needs to provide name of organism database for search and analysis.

Sample processing

diagram2

Label free quantification (LFQ) analysis: Clustergram of the ANOVA significant proteins differing among the four strains of Mycobacterium tuberculosis.

We will process the sample (gel bands) using our in-house protocols. The samples will be trypsinized followed by cleaning using C18 columns before analysis on mass spectrometer.

Guidelines for sample preparation

Running protein samples on a gel prior to proteomics analysis has many advantages. Gel removes any foreign particles/substances which are not compatible with downstream analysis using mass spectrometry. The environment we work in is highly polluted with keratin that came from our heads, skin, nails, fingerprints, woolen clothing, and etc. Other rich sources of keratin contamination include paper and cardboard containers. Since mass spectrometers detect proteins at femtomolar levels, the slightest contamination of keratin will result in keratin being identified as the most abundant protein versus being able to identify the proteins that really interest you. Other types of protein contamination such as bovine serum albumin and milk proteins usually come from a staining tray which was previously used to develop a western. Therefore use a brand new staining tray.

SDS PAGE gel: Use high quality reagents for gel preparation, Fresh buffers, and use nitrile gloves for handling the gel, use fresh staining and de-staining solutions. Avoid any dust (Keratin) when gel tray, solutions are exposed to air.

For staining the gels use Coomassie Brilliant Blue or MS-compatible silver staining kits from available vendors.

Non-kit based MS compatible silver staining protocol

  • It is strongly recommended that you use Coomassie brilliant blue stain rather than Silver.  If you cannot visualize bands with Coomassie try to scale up your isolation to increase the amount of protein until you get to Coomassie detectable limits.
  • If you cannot reach Coomassie stainable levels you must use the mass spec compatible Silver Stain protocol below. Only use methanol and acetic acid during the fixing step. Do not use any solutions containing formaldehyde or glutaraldehyde to fix the gel. Refer to Shevchenko et al. (1996) Analytical Chemistry, 68:850-858 for more details.
  • It is better to have all the protein in one band/spot, but if you cannot load more protein in one lane then run several lanes and combine the bands/spots.
  • Use 1.0 to 1.5mm gels.
  • Only stain the gel long enough (usually only a few minutes) to detect the bands of interest.
  • Take a picture of the gel and submit it (photocopy or electronic) along with samples.
  • Excise gel band(s)/spot(s) with as little excess empty gel as possible. store in 1% acetic acid at 4° C.
  • For sample submission, place the gel band(s)/spot(s) into a keratin-free, clean micro centrifuge tube. Ship at ambient temperature.

In case you have used non-compatible silver stain, do let us know to take further steps to get enhance protein recovery.

Protocol for Mass Spec compatible Silver staining 1-10ng detection limit

  • Wash your gel with 50%MeOH 10% acetic acid, shaking for 20 minutes.
  • Wash with 20% Ethanol, shaking for 10 minutes.
  • Wash with water for up to 1 hr, frequently changing the water.
  • Rinse with 0.03% sodium thiosulfate 1 min - to sensitize (0.03g/100mL)
  • Rinse in water twice very well.
  • Immerse into 0.1% Ag nitrate at 4° C for 20 minutes ( 0.1g/100mL)
  • Rinse with water twice.
  • Chill developer: add 0.04% formalin (40uL/100mL) (formaldehyde) and 2% sodium carbonate per 100mL.  Shake until dev. yellow, discard, and continue w/ fresh, cold developer.
  • To stop development, add 5% acetic acid to the solution
  • Store in 1% acetic acid at 4° C.

Again, however, we strongly encourage users to use Coomassie brilliant blue instead of silver.

We discourage users from submitting silver stained gels for the following reasons: 

  • Traditional silver stain protocols oxidize amino acid side chains and introduce glutaraldehyde cross-links, interfering with sequence determination.  There are kits may reduce amino acid oxidation, but the other reason is,
  • Silver staining overestimates the amount of protein that's actually present in your sample.  We still need picomoles (or 100s of femtomoles at the very least) to identify your protein.  If you want to identify post-translational modifications (especially phosphorylation) we will need 10X ~ 100X as much material.

Data Analysis

  • Data analysis will be performed using protein identification algorithms using commercial and other software.
  • We generally recommend uniprot organism specific database but the final choice depends upon the customer.
  • The results will be provided in excel table format with details of all the protein and peptide identifications including positions of identified post translational modifications.
  • For advanced users who are interested to further analyze the data, support will be provided based on available time slots.

Frequently Asked Questions

How do I know which stains to use for gel staining and how much protein is required for successful MS analysis?

Ans: For Coomassie brilliant blue staining we recommend to use lab made solutions. Using dust free glassware and fresh solutions are necessary to get good results. Please check the instructions in earlier paragraph for more understanding. You can use commercial available MS compatible stains also for staining the gels. Please excise the gel band of interest as thin as possible with as little excess empty gel as possible. In-principle any Coomassie brilliant blue stained band would be identified by our highly sensitive MS machine. In case of silver staining the detection sensitivity depends on many factors as explained in earlier section. You will provide details regarding your method of protein preparation along with available gel pictures and organism database. We will trypsinize the gel bands ourselves and provide you the best results possible.

I suspect that my sliced gel band may contain many proteins. How do I know which is the right protein for me?

Ans: This is a very common question we face on daily basis. If you have a mixture of protein and many proteins have close molecular mass range as expected they all will be present in the sliced gel piece which you have submitted us for sequencing. In this case all the proteins will be identified by the machine. The relative abundance of proteins in the sliced gel piece will be reflected in the result scores as well as protein coverage. Generally the intense protein in the sliced gel show higher identification scores after data analysis.

I wanted to identify phosphorylation on my protein of interest how much protein is required for successful MS analysis?

Ans: Any Coomassie brilliant blue stained band would be identified by our highly sensitive MS machine at MS and MS/MS level. If a protein sample is in a concentration around 100 fmole/μl, loading 10 μl per lane should result in about 1pmol protein per lane. A protein band of >1 pmol can be visualized by several type of stain (Coomassie colloid, Zn, Cu, etc) and identification of protein in such a gel band is often not very challenging to us. We use state of the art columns, chromatography, mass spec machine and data analysis tools to get best results for you. The maximum coverage of protein depends upon protease digestion efficiency as well as sequencing of peptides inside the mass spec. We use our own Lab controls for checking trypsin digestion efficiency. The stoichiometry of phosphorylated versus non-phosphorylated form in your sample have major role to play in successful identification of phosphorylated peptides. Our machine has sub-femtomole Limit of detection (LOD) and In–principle if we assume that the phosphorylated peptide present in you sample exists in this range and fly well followed by sequencing inside the mass spec machine, we will be able to identify phosphorylation in your sample.

What is the advantage of your nano-ESI MS/MS compared to MALDI-TOF/TOF?

Ans: We utilize chromatography gradients to separate the peptide mixture based on their hydrophobicity. This decreases the complexity of the peptide mixture and thousands of MS and MS/MS scans are performed during the machine run. The machine gets sufficient time to enrich peptides in ion trap before their MS/MS sequencing. In this way not only the coverage of the protein increases but also result in better quality data than MALDI-TOF/TOF. In MALDI-TOF/TOF analysis all the peptides present in the mixture fly simultaneously during laser excitation and operator has to manually decide which MS/MS needs to be performed on the sample based on PMF (MS1) spectrum. In our method the machine utilize very smart automatic technologies like Automatic gain control (AGC) and data dependent acquisition (DDA) to sequence as many peptides as possible in the sample. We provide more coverage, better quality of spectrum and sensitivity compared to any MALDI-TOF/TOF technology available. Our technology very confidently identifies proteins, protein complexes, PTM modifications much better than MALDI-TOF/TOF.

Service: VPSOL LC-MS/MS

  • Service code
  • VPSOL1

  • VPSOL2

  • VPSOL3

  • type
  • Global proteome analysis1

    Sample origin: Pull down, Affinity purified, Tag based complexes or characterization of individual proteins, phosphorylation analysis on Affinity purified proteins.

  • Global proteome analysis2

    Sample origin: vesicular proteomics, Sub-cellular fractions membrane proteomics, cellular compartment analysis, phosphorylation analysis of isolated proteins, Phosphoproteome analysis after enrichment

  • Global proteome analysis3

    Sample origin: complex cellular lysates e.g. eukaryotic organisms, Phosphoproteome analysis after enrichment

  • Method Specifications
  • UPLC-MS/MS, 15 cm column, medium gradient, QExactive

  • UPLC-MS/MS, 50 cm column, Long gradient, QExactive

  • UPLC-MS/MS, 50 cm column, extended
    gradient, QExactive

Data Analysis

  • Data analysis will be performed using protein identification algorithms using commercial and other software.
  • We generally recommend uniprot organism specific database but the final choice depends upon the customer.
  • The results will be provided in excel table format with details of all the protein and peptide identifications including positions of identified post translational modifications.
  • For advanced users who are interested to further analyze the data, support will be provided based on available time slots.
service-graph

Typical total ion current chromatogram of a 3 hour LC-MS/MS run using 50cm long column attached with proxeon nano-LC1000 and Q Exactive mass spectrometer.

Sample E. histolytica whole cell proteome-3000 Proteins/20000 peptides identified

Frequently Asked Questions

My samples are in liquid form, how much protein is required for successful MS analysis?

Ans:This actually depends upon the type of experiment. Generally we load 1-2 µg of peptide mixture for a single run using 10-15cm column and 2-4 µg of peptide mixture using 50 cm long column. If the protein is available in sufficient amount, we recommend trypsin digestion for minimum 50 µg of protein. Please discuss with us your sample preparation protocol to avoid contaminants which may interfere with protein digestion and sample analysis on Mass spec machine. After discussion of the suitable protocol we will get protein samples from you and perform trypsin digestion and further process in our lab.

What is the advantage of solution based proteome analysis as compared to gel based technique?

Ans: If your samples are having low level of complexity, we recommend running them on SDS-PAGE gel which removes contaminants and MS interfering substances. Just run for short time to allow the mixture of proteins to enter the stacking gel and concentrate as a coarse band. But not all the proteins are resolved well in SDS-PAGE gel especially membrane proteins. If you are preparing complex cell lysates using a proteomics compatible lysis buffer than solution based samples provide better results than gel based approaches in terms of number of peptides and protein identification. Many times experiments e.g. Pull down analysis, Protein-protein interaction, where starting material and resulting eluate protein concentration is very limiting gel based analysis may not be possible. In these special cases we recommend to use the trypsinized eluates directly for mass spec analysis. Please discuss with us your experimental requirements and we will be happy to provide you right strategy for successful outcome.

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VProteomics

K-37A, Ground Floor
Green Park Main,
New Delhi 110016
india

Phone: +91-11-4050 2621

Email: info@vproteomics.com

Website: www.vproteomics.com

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