While enzymes, such as trypsin, can be used to cleave proteins and peptides at specific amino acid linkages, we can also fragment peptides inside of a mass spectrometer to obtain additional information. These types of experiments are called tandem MS or MS-MS experiments. These experiments are particularly helpful for “shotgun” proteomics or bottom-up proteomics. In these experiments, protein mixtures are first digested with enzymes (such as trypsin), then separated by one or more chromatography steps, and then electrosprayed into a mass spectrometer. A mass analyzer is then used to select a precursor ion with a specific m/z value for fragmentation. Fragmentation requires that some energy be added to the system. The most common method of fragmentation in MS-MS experiments is collision-induced dissociation (CID). In CID, the precursor ion is accelerated into an interaction cell that contains a collision gas, such as helium or nitrogen. When the precursor ion collides with the collision gas, the ion can fragment into two fragments, an ion and a neutral. The fragment ions are then analyzed to produce the MS/MS spectrum.
To better understand the operation of a commonly used triple quadruple or “triple quad” MS/MS, watch an animation, then answer the questions below.
1. What is the purpose of the skimmer in the instrument?
2. The animation uses color to indicate difference m/z value ions. Which “color” of ion is selected as the precursor ion?
3. Although this style of instrument is commonly called a “triple quad,” the collision cell is not actually a quadrupole. What is it?
4. The last quadrupole selected fragment ions to be sent to the detector. Neutrals also pass through this quadrupole. Why don’t they produce a signal at the detector?
MS/MS experiments are useful because the fragment m/z values give information about the analyte’s molecular structure. Low energy CID often produces small neutral losses, such as H2O, CH3OH, CO, CO2, NH3, and CN. Higher energy collisions can lead to retrosynthetic reactions, in which characteristic bonds in the precursor ion are broken. For example, CID of peptides often results in cleavage of peptide bonds along the backbone.
1. Complete the table below to summarize the expected mass differences from common neutral losses. Round expected masses to the nearest amu.
Table 1. Common neutral losses produced by CID of peptide ions.
|Neutral Loss||Mass (amu)|
The identity of the precursor ion can be scanned through the mass range so that a mass spectrum of each precursor ion’s corresponding fragments is obtained. These experiments produced large quantities of data extremely rapidly. Interpreting these large data sets usually involves specialized software programs that identify peptides from their fragments and then identify proteins from their peptides; however, for simple mixtures, the data may be interpreted manually since peptide fragmentation in tandem MS experiments is well-characterized.