As a part of a proteomics facility, our lab has procured a new LC-ESI-MS instrument from Bruker Daltonics. The Esquire 3000 plus ESI-MS can be used for direct mass determination of any compound, be it a peptide, protein or any small molecule. The LC-ESI can not only provide information on the mass of the individual components in a sample, but also aid in the resolution of the different components of a mixture.
The ESI-MS is provided with a spray chamber, an ion focusing and transport region, skimmer, octopoles and lenses. The spray chamber contains a nebulizer, an end plate and drying gas. We use ultra pure grade nitrogen as the drying gas as it is cost effective. The nebulizer needs to be used carefully, as particulate matter can clog the pore, but can be physically removed and cleaned. The ESI-MS also contains a nanospray apparatus. The sample has to be loaded in a thin, yet expensive, capillary (only one per sample). The loading of the sample by this process is very difficult, thus we avoid using this feature.
The HPLC attached to the instrument is required for carrying out LC-ESI-MS, in cases where separation of sample components is desirable or when the sample contains a high amount of salt. It has a built in degassing unit and requires HPLC-grade solvents. We use acetonitrile, water, methanol and 0.01% formic acid in different ratios as the mobile phase. The ESI-MS cannot tolerate any halogenated solutions. An automated sampler can make sample injection easy by handling 96 samples, provided only one protocol is run. Tryptic digests of proteins can be easily resolved and simultaneously fragmented on the LC-ESI. This procedure makes identification of unknown proteins simple and straightforward.
Samples in the nanomolar range are sufficient for LC-ESI-MS. Low molecular weight substances (less than 500Da) can be easily detected as compared to MALDI. For fragmentation purposes, larger proteins give excellent results. We have recorded upto 89kDa on our instrument. A mass width of 4 mass units is generally specified during peak picking. Fragmentation intensity can be varied to get an optimal pattern. The entire process can be completely automated by specifically programming the instrument to fragment every detected peak in an LC-ESI run, to different levels, until the desired fragment sizes are reached. It is advisable not to fragment peptides smaller than 500Da, as results lose accuracy.
The ESI-MS has an ion trap as a detector for accurate mass sampling and determination. It offers the advantage of carrying out (MS)n rounds of fragmentation. Theoretically, upto 11 rounds of fragmentation can be achieved. We find that 5 rounds are sufficient to generate a reliable fingerprint of any protein.
Peak list generation is very easy and each list can be colored differently through the software. Output can be copied directly to Microsoft powerpoint for presentations. The software also provides the facility to analyze data even when it is being acquired. Several packages (for example, MASCOT) are available for database analysis of fragmentation patterns. The machine is also provided with a useful step-by-step guide for assembly, software installation and instrument handling. The software is not difficult to understand, but definitely requires some training.
Vikas Jain
Research Scholar
Indian Institute of Science