I

MS Detection

Fig. 1.43 Strategies for protein identification. (A) 2D gel electrophoresis approach. (B) 2D liquid chromatography approach. IEF Isoelectric focusing, SCX strong cation exchange column, RP reverse phase column, SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis.

tides present in the digest. The list of peptides is then submitted to a database search to identify the protein. This approach does not work if several proteins are present in the same spot or if the sample is contaminated for example with keratin. The identification of the protein can be improved by sequencing selected peptides either by post source decay (PSD-MALDI) or tandem mass spectrometry (MALDI-TOF/TOF).

High-performance liquid chromatography (HPLC) represents an attractive alternative to two-dimensional electrophoresis for the separation of both proteins and peptides because of its chromatographic resolving power, reproducibility and its compatibility with MS detection. The use of multidimensional chromatogra-phy for the separation of complex protein and peptide mixtures has consequently seen increased use in proteomics studies [87, 88]. A typical approach involves the digestion with trypsin of an extract. Furthermore the preparation and handling of peptides is less tedious than with intact proteins and the whole process can be easily automated. A typical two-dimensional LC experiment (2D-LC) involves the initial separation (first dimension) of the resulting peptide mixture by their electrostatic charge using strong cation exchange (SCX) chromatography. In the second dimension peptides are then separated by their hydrophobicity using reversed phase (RP) chromatography coupled directly to ESI-MS. In a typical analysis of a complex protein mixture from a single sample the procedure is repeated about ten times with increasing salt concentration, resulting in a total analysis time of about 12 h.

As electrospray ionization is concentration-sensitive the last LC dimension uses a nano LC column with an internal diameter of 75 mm to achieve maximum sen-

Fig. 1.44 2D-LC setup. The first ion exchange dimension is performed with a column with an i.d. of 1 mm, at a flow rate of 50 |mL min-1 while the second dimension uses a nanocolumn with an i.d. of 0.75 mm and a flow rate of 300 nL min-1. First dimension ion exchange has ten salt steps: 0, 5, 10, 15, 20, 25, 50, 75, 100, 200 mM KCl. Second dimension is typically an organic gradient: 5% to 80% acetonitrile with 0.1% formic acid in 30 min.

Fig. 1.44 2D-LC setup. The first ion exchange dimension is performed with a column with an i.d. of 1 mm, at a flow rate of 50 |mL min-1 while the second dimension uses a nanocolumn with an i.d. of 0.75 mm and a flow rate of 300 nL min-1. First dimension ion exchange has ten salt steps: 0, 5, 10, 15, 20, 25, 50, 75, 100, 200 mM KCl. Second dimension is typically an organic gradient: 5% to 80% acetonitrile with 0.1% formic acid in 30 min.

sitivity while larger diameters are preferred for the first ion exchange dimension to be able to inject large sample amounts and volumes. A 2D-LC system is depicted in Fig. 1.44. Ion exchange elution can be performed with ammonium acetate buffers which are MS-compatible. More efficient is potassium chloride elu-tion, but the drawback is that it affects the detection of peptides. Therefore it is necessary to implement trapping columns for desalting the fraction before transferring it in the second reversed phase LC dimension. At the end of the analysis all the data are processed together to generate a list of several hundred proteins. For this task efficient bioinformatics tools are essential.

Figure 1.45 illustrates a typical 2D nano LC-MS/MS analysis of a Caenorhabditis elegans extract. For each timepoint a single MS and a product ion spectrum are

Fig. 1.45 Example of a 2D nano LC-MS/MS analysis of a C. elegans extract. (A) Fraction 2, 4 mM KCl salt elution on the strong cation exchange column. (B) Full scan MS spectrum of the peak eluting at RT 26.3 min in (A). (C) product ion spectrum of the doubly charged precursor of (B) at m/z 784.8. Y fragments are typical for C-terminal fragments while b ions are typical for N-terminal fragments.

recorded (Fig. 1.45B, C). With the help of bioinformatic tools the product ion spectrum can be automatically interpreted. The y fragments are typical for C terminal fragments, while the b ions are typical for N-terminal fragments.

Two-dimensional-liquid chromatography (2D-LC) approaches are much easier to automate than 2D-electrophoresis. However 2D electrophoresis has the advantage that separation is performed at the protein and not at the peptide level and that the proteins can be visualized by staining. With 2D-LC one has to wait for bioinformatics treatment to see if the experiment was successful.

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