2DE Tandem MS employs several principles of biochemistry to illustrate the properties of gel electrophoresis and peptide fragmentation realistically. Two dimensional electrophoresis begins with IEF (where proteins are separated by isoelectric point) followed by SDS-PAGE (where they are separated by molecular weight). Before the IEF animation of the 2-D electrophoresis simulation, 2DE Tandem MS calculates the pI and MW for each protein and assigns their final positions in the pH range based on their pI value and the min and max pH set by the user (Fig. 1a, Choose pH box). A brief animation runs that visually demonstrates the separation of proteins based on pH, with the bands of protein ending at their intended positions. Before running the SDS-PAGE simulation, the user can select the percent acrylamide using the “Choose Acrylamide %” drop-down menu (Fig. 1a, middle left). At the user's prompting the SDS-PAGE animation begins, the proteins enter the gel, and then migrate toward the bottom of the screen. At this point the percent acrylamide selected by the user determines how far each protein migrates down the gel canvas (Fig. 1a, bottom right).
In an actual experiment, users pick spots from the 2D gels, extract the proteins, digest them and submit them for sequencing by mass spectrometry. This process is replicated in the simulation, with the advantage that the virtual process always works. The protein information box (Fig. 1b) appears when a user clicks on an individual protein spot. Clicking on the “Run Mass Spectrum” button then transfers the protein sequence to the mass spectrometer simulation, which can be accessed by clicking on the Mass Spectrometer tab in the simulation. The tandem mass spectrometry simulation gives the user a choice of four different proteases to digest the proteins. The selection of the protease should be based on the primary sequence of the protein. The simulation uses the standard target sites for trypsin (cuts at the C-terminal side of lys and arg), chymotrypsin (C-term phe, tyr, trp), proteinase K (C-term ala, phe, ile, leu, val, trp, tyr), and thermolysin (N-term ile, leu, met, val) to generate the peptide fragments . The digested fragments undergo ionization and then enter the first mass spectrometer, where they are separated by mass-to-charge ratio. When a peak from the first mass spectrometer (Fig. 2, bottom right) is selected for further fragmentation, the digested peptide enters a collision chamber, where the chemical bond between the carbonyl and amino groups along its backbone are broken, leaving two fragments that each carry a single positive charge: a B fragment, where the carbonyl carbon is triple-bonded to the oxygen, and a Y fragment, where the amino group has two extra hydrogens. There are several other bonds on the peptide molecule that could be broken, but B and Y fragments are the most commonly seen in Tandem MS experiments and the easiest to interpret. The bombardment in tandem mass spectrometry is carefully controlled so that the bulk of the peptides are only broken once, allowing for B and Y fragments of each possible size to be taken from the original peptide . Our simulation replicates this process. Figure 3a indicates the cleavage points on the peptide backbone (ProThrGluGlyCysMetAsnLeu or PTEGCMNL), along with the B fragment (ProThrGluGly or PTEG, Fig. 3b) and Y fragment (CysMetAsnLeu or CMNL, Fig. 3c) that result from cleavage of the peptide bond between glycine and cysteine. The structures of all the B and Y fragments that appear in the tandem MS simulation (Fig. 2, top right) for this peptide can be found in Supporting Information Fig. S1. Because only one CONH bond is broken per peptide fragment and each charged fragment only differs 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 (Fig. 2, upper spectrum) to give the full sequence of the peptide.
Figure 3. Fragmentation of a peptide by tandem mass spectrometry. (a) The complete peptide fragment is shown in a chemical drawing. This is an example of a peptide represented by a selected fragment peak in the lower graph of the tandem MS simulation (bottom of Fig. 2). (b) The B-fragment (B4) of the peptide produced by cleaving the GlyCys bond in the peptide shown in (a). B-fragments have been bombarded with gas molecules so the chemical bond between the carbonyl and amino groups has broken, leaving the carbon triple-bonded to the oxygen, which then carries a positive charge. (c) The Y-fragment (Y4) produced by cleaving the GlyCys bond of the peptide shown in (a). Y-fragments have been bombarded with gas molecules so the chemical bond between the carbonyl and amino groups has broken, leaving the amino group with two extra hydrogens and a positive charge. The chemical drawings were prepared using Marvin (Marvin 5.9.0, 2012, ChemAxon, http://www.chemaxon.com).
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