Can mass spectrometry help in protein analysis and identification?
Mass spectrometry (also known as 'mass spec' or MS) is one of the most important tools used in the study of proteins. By employing a variety of mass spectrometry techniques, researchers can accurately identify and quantitate proteins in a given solution, identify amino acid sequences, and determine the overall structure of your protein of interest.
Mass Spectrometry: A Quick Overview
Mass spectrometry is basically an analytic technique that determines the relative masses of molecular ions and fragments. Using this process, the gas phase molecules are ionized to determine their mass-to-charge ratio. Since lighter ions will travel faster and be detected first when an electric field is applied, the relative mass can be accurately measured and the composition of the molecule can then be identified. In addition, the sequence of component amino acids can also be identified using the same procedure.
How Does Mass Spectrometry Work?
In analyzing proteins using mass spectrometry, the proteins are first broken down into their component peptides. Trypsin is usually the protease most researchers use in digesting proteins due to a number of reasons. First, it cleaves proteins into its component peptides with an average size of 700 to 1500 Daltons (the ideal size for mass spec). Second, it specifically cleaves the protein at the carboxyl side of arginine and lysine residues. The C-terminals of these peptides are charged and are therefore easily detectable by mass spectrometry. Third, trypsin is highly active and can tolerate a number of additives. And lastly, trypsin can be modified by the methylation of lysines to prevent self-digestion at these sites.
After the proteins have been digested into peptides, they are then separated using reverse phase column with acetonitrile gradient. Note: Acetic acid should be used with the solvents since trifluoroacetic usually interferes with the ionization process.
The ionized peptides are then made to pass through the column eluate which contains peptides and solvent. After the solvent evaporates, their charged surfaces move the ionized peptides into the mass spectrometer. This method, which uses chromatography to introduce molecules into a mass spectrometer, is called high performance liquid chromatography or HPLC.
After this step, the mass of peptides and the mass of peptide fragments produced through collision-induced dissociation (CIS) or collisionally-activated dissociation (CAD) using tandem mass spectrometry or MS/MS can now be measured.
Multiple stages of mass analysis separation can be accomplished with (1) individual mass spectrometer elements separated in space or (2) a single mass spectrometer with the MS steps separated in time. Data analysis is mostly done with the help of a computer program such as SEQUEST but for new sequences and the confirmation of new sequences, most researchers prefer to do it by hand.
Please take note that you may not get a complete data sequence from your protein samples every time due one or a combination of the following reasons:
The protein was not fully digested.
The peptides are too large or too small to be analyzed.
The peptides are either hydrophilic or hydrophobic.
However, if you can show 70% of the protein sequence (70% coverage), your analysis is considered highly successful.
On the other hand, if you want a less complex method for identifying proteins, you can use a MALDI instrument to measure the masses of intact peptides. However, this only works if the sequence of your protein of interest is available and/or if your sample only contains one or two proteins. Remember, if you want to get accurate results from your research, you should be able to produce clean, reproducible peptide samples for your mass spectrometry experiments. One great way to make sure you do is by using high quality, MS grade trypsin in digesting your protein samples. If you are not sure if your current product has what it takes to give you the desired results then take our Trypsin for Mass Spectrometry Challenge to see how your product compares with ours.