Ans: Most of the samples we accept are derived from model organisms which are generally used for research work in academic labs. We accept both intact cell pellets as well as Mass Spectrometry compatible cell extracts or isolated metabolites. We also arrange dry ice pickup of samples from researcher lab till our Delhi lab for projects having good number of samples. 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.

For our clinical Mass Spectrometry projects, we also accept FFPE tissues, paraffin blocks, Serum/Plasma, Urine, Saliva, CSF etc after detailed project discussions with clinicians and after careful considerations and precautions for handling infectious material. Our advanced histology laboratory can prepare tissue slides as well as high quality proteomics lysates utilizing specially developed protocols. The FFPE slides can also be utilized for spatial proteomics projects for biomarker identification and validation using our laser microdissection methodologies combined with LC-MS/MS analysis.

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 most intense protein in the sliced gel shows highest coverage & identification scores after data analysis. The maximum coverage and number of peptides detected for any protein again depends upon number of protease cleavage sites present in the protein as well as the site location with respect to each other. For Example,very small size (few amino acids) and very big size peptides generally don’t fly well in the mass spec and will not be identified at MS or MS/MS level. Again certain peptides due to their amino acid sequence ionizes and fly very well inside the mass spec and results in very high quality sequencing spectrum and corresponding scores.

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 ionization, fliability and MS1/MS2 level 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 of phospho-protein in your sample have major role to play in successful identification of targeted protein 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.

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 (C-TRAP) before their MS/MS sequencing. In this way not only the coverage of the protein increases but also results 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 in-built automation 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.

Apart from latest machine technology, it is also very important to prepare high quality mass spectrometry sample as well as maintaining the mass spec platform to obtain high quality data.