Tandem Mass Spectrometer - RITJBF/JBioFramework GitHub Wiki

The Tandem Mass Spectrometry simulation can accept manual input, fasta file input, and input from the 2D Electrophoresis simulation. The simulation runs a mass spectrum of the inputted protein sequence and can run a second mass spectrum of individual peptide fragments.

#Entering a Peptide Sequence

There are three ways to enter a peptide sequence for analysis by tandem mass spectroscopy.

1. Type the sequence in the input protein sequence box:

2. Load a sequence from a fasta file by clicking the ‘Load Sequence From File’ button and navigating to the file through the directory.

3. After running the SDS-Page animation in the Electrophoresis 2D simulation, click on a protein dot that represents the protein to be analyzed, and in the Protein Information Frame, click on the ‘Run Mass Spectrum’ button. The peptide sequence for the protein will be put in the input protein sequence box in the Mass Spectrometer simulation.

#Selecting a Protease

There are four proteases available to use to digest the peptide sequence entered in the protein sequence box.

Proteases are enzymes that cut apart a peptide sequence at specific points by recognizing a pattern of residues. Each protease recognizes a different pattern. After the sequence has been cut apart, or digested, by the protease it exists as numerous peptide fragments that the mass spectrometer will analyze the size and charge of.

The proteases to choose from are:

Trypsin: will cut a peptide sequence after (C-terminal side) an Arginine(R) or Lysine(K), except when either is followed by a Proline(P).

Chymotrypsin: cuts a peptide sequence after Tyrosin(Y), Tryptophan(W), or Phenylalanine(F).

Proteinase K: cuts a peptide sequence after Alanine(A), Phenylalanine(F), Isoleucine(I), Leucine(L), Valine(V), Tryptophan(W), or Tyrosine(Y).

Thermolysin: cuts before (N-terminal side) Isoleucine(I), Leucine(L), Methionine(M), or Valine(V).

#Entering a Mass Charge Ratio

The mass charge range input boxes provide a way to control how expansive the output graph’s and tandem output graph’s m/e scales are. m/e stands for mass divided by charge, and arranges the peptide fragments digested by the selected protease by their weight over charge ratios. Because all fragments have a charge of +1 from the amino-terminal end, this is essentially a mass scale.

This range can be changed by selecting one of the two pre-made options or by selecting 'Enter a Range' and typing your own in manually as shown below.

Peptide fragments possessing a mass/charge ratio outside of the range selected by the user will not have their peaks displayed on either graph. The m/e scale will change to reflect the user’s choice of range.

If a range possessing a lower limit higher than the upper limit or a negative number for either limit is entered the offending limit will be set to its default value; 0 for the lower limit, and 3000 for the upper limit. Non-number input will be corrected in the same way.

Output Graph:

Tandem Output Graph:

#Running a Tandem Mass Spectrum

To analyze a peptide fragment of a protein and experimentally determine its residue sequence using the Mass Spectrometer simulation, follow these steps:

1. Enter a peptide sequence into the input protein sequence box.

2. Select a protease for digesting the peptide chain.

3. Enter the desired mass/charge ratio range to adjust the two graph’s x-axis scales.

4. Click the 'Run Spectrum' Button

4.5) (optional) Adjust the mass/charge ratio range to reflect the x-axis scale desired for the tandem mass spectrum output.

5. Click on one of the peaks (lines) on the output graph (the graph on the lower half of the screen) to sequence the fragment that peak represents.

6. Toggle the b-fragments and y-fragments check boxes to facilitate reading the peptide sequence off of the tandem output graph (the graph on the upper half of the screen).

#Finding the Mass of Peptide Fragment

To determine the exact mass of a peptide fragment after running a mass spectrum, click on the line, or peak, on the output graph (the graph on the lower half of the screen) to select the fragment that peak represents. A tandem mass spectrum will be run on the fragment, but the mass label on the lower left-hand side of the screen will also change to reflect the exact mass of the peptide fragment.

#B Fragments and Y Fragments

After performing a tandem mass spectrometry the graph on the upper half of the screen will display at least two colored lines, or peaks. The blue peak(s) represent the b-fragments and the red peak(s) represent the y-fragments.

When a large number of a specific type of protein is digested by a protease, several types of smaller peptide fragments are left over. It is these smaller peptide fragments that show up as lines, or peaks, on the graph on the lower half of the screen. The ‘tandem’ in tandem mass spectrometry comes in to play here; by clicking on one of the peaks on the lower graph, the type of peptide fragments it represents is broken down at specific points along the peptide backbone. For each CO-NH bond along the peptide backbone, two fragments (one b and one y) will be created by a single such bond being broken. If the resulting positive charge ends up on the CO side of the bond, the new fragment is called a b-fragment. If the resulting positive charge ends up on the NH side of the bond, the new fragment is called a y-fragment. The side of the bond that does not receive the positive charge remains uncharged, and so does not show up on the tandem output graph.

Because only one CO-NH bond is broken per fragment in the large amount of fragments the peak on the output graph represented, and the charged fragments will only differ in size by the mass of one residue compared to its neighbors, it is possible to use the mass difference between successive b-fragment peaks or y-fragment peaks to ‘read’ the residue sequence of the selected output graph peptide fragment. The b-fragments are read left to right, and the y-fragments are read right to left.

Source: ‘An Introduction to Mass Spectrometry’ by Dr. Alison E. Ashcroft at the University of Leeds. http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm

To change the tandem output graph’s display so only one type of fragment, b or y, is shown, toggle the B Fragment and Y Fragment check boxes.

#Intensity

The y-axis of the output graph (the graph on the lower half of the screen) and the tandem output graph (the graph on the upper half of the screen) represents the intensity of the peptide fragments.

Intensity is calculated relative to the most abundant peak. If there is a specific pattern of residues in the analyzed protein’s peptide sequence that occurs more than once, the fragment(s) that pattern of residues creates will be more abundant than fragments from patterns that occur only once.

The most abundant type of peptide fragment is given an intensity of 100. If a different type of peptide fragment occurs three-quarters as often, it is given an intensity of 75. If a still different type of peptide fragment occurs half as often as the most abundant type, it is given an intensity of 50, and so on and so forth.