How does Mass Spectrometry Proteomics work?
The most revealing and common form of Mass Spectrometry for Proteomics is HPLC-MS/MS.
- A protein sample is digested by one or more enzymes, this produces peptides. If more than one type of enzyme is used, these fragments will have overlapping sequences to some degree.
- The peptides are separated using high-pressure liquid chromatography (HPLC) and possibly some other methods prior to HPLC. The separation is such that similar or identical peptides arrive at the end of the column at the same time.
- The peptides are ionized
- The mass of the ionized peptides are measured with high accuracy
- The ionized peptides are fragmented
- The mass of the fragments are measured with high accuracy
- Post-analysis of the mass of each peptide and all its fragments can be used to determine the sequence of the peptide. Careful analysis of the peptides can be used to construct the whole protein sequence and/or quantify the proteins in the sample.
What to look for in a Protein Mass Spectrometry Service
Protein Mass Spectrometry Services are available from commercial providers and some core facilities at universities and institutions. Here are some things to consider while choosing a service provider:
- Experience – the techniques are well known, but the problems are complex. Look for technicians and scientists that have been doing the work for years.
- Well developed protocols – does the service own its own mass spectrometers and have they been using the equipment for some time?
- Specialization – the most in depth analysis will come from a service that does hundreds of the same kinds of analysis each year
- Throughput – does the service offer a reasonable turn-around time, high-volume work, and an option for rush service.
- Post analysis – examples of post-analysis reports
Mass Spectrometry Protein Sequencing by LC-MS/MS
One of the most important functions of Mass Spectrometric analysis of proteins is Sequencing. The masses of peptides, and fragments of peptides can be analysed by specialized bioinformatics algorithms such as Novor in order to determine the sequence of each peptide. Further bioinformatic analysis can align the sequences of multiple overlapping peptides (produced by different enzymatic digests) to assemble the sequence of the whole protein. The techniques involved here are well understood. However, the use of specialized workflows, protocols, instrumentation, bioinformatics and expertise are the keys to producing reliable results, quickly and with minimal use of sample.