What kind of samples you accept for Proteomics analysis?

Ans: Most of the samples we accept are derived from model organisms which are generally used for research work in academic labs. These samples may be derived for example from Human/animal cell lines, saccharomyces cerevisiae, saccharomyces pombe and other yeast strains, human/animal tissue samples, biofluids, E.coli bacterial strains, plant samples, Arabidopsis etc. We also accept recombinant expressed proteins for characterization purpose.

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 Orbitrap 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 utilizes 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.


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 0.5-1 µg of peptide mixture for a single run using 10-15cm column and 1-2µg of peptide mixture using 50 cm long column. If the protein is available in sufficient amount, we recommend trypsin digestion for minimum 25- 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 all sample processing and mass spec 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 (Check VPGEL sample processing figure, VPGEL5). 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 in that case solution based methodology provides better results than gel based approaches in terms of number of peptides and protein identification with our top of the line Mass Spectrometry Exploris Machine and standardized protocols, we generally identify 5000-6000 proteins routinely in complex protein lysates e.g. HeLa in a single Mass Spec run.

Some 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 due to invisibility of protein bands in stained gel. In these special cases we recommend to use the VPSOL approach for mass spec analysis which results in identification of most of the proteins (high, medium & low levels) present in the IP complex. Please discuss with us your experimental requirements and we will be happy to provide you right strategy for successful outcome.