Tryptic protein digest

Another approach to selenized yeast is to characterize Se-containing proteins. The application of polyacrylamide gel electrophoresis (PAGE) has been proposed with the introduction of the protein spot to ICP-MS via LA or ET atomization [62, 93]. The feasibility of matrix-assisted laser desorption ionization (MALDI)D timeDof-Bight mass spectrometry (TOF-MS) prior to ES tandem MS as applied to tryptic protein digests has also been explored [135, 136]. 

FIGURE 8.6 A comparison of estimated and published cross-sections for a set of 32 peptides obtained from four different tryptic protein digests (alcohol dehydrogenase (yeast), aldolase (rabbit), creatine phosphokinase (bovine), and hemoglobin (rabbit)). For each peptide the 2+ charge state was used. The same peptides were analyzed twice with an interval of eight months between experiments. (Reproduced from Thalassinos, K. Grabenauer, M. Slade, S.E. Hilton, G.R. Bowers, M.T. Scrivens, J.H. Ana/. Chem. 2009,81, 248-254. With permission from the American Chemical Society.)... 

Pasihs, S.P, Kertesz, V, Van Berkel, G.J., Schulz, M., Schorcht, S. (2008) Using HPTLC/DESI-MS for peptide identification in ID separations of tryptic protein digests. Analytical and Bioanalytical Chemistry, 391, 317-324. 

Fig. 2 Distributions of OPA-derivatized amino acids and peptides chromatographed by the automatic online OPA/2-mercaptoethanol system. A and B 50 pmol of tryptic peptide digest of proteins M and R, respectively. The peak marked with an asterisk is due to the derivatizing reagents. Column 5-/zm Resolve C, (15 cm X 3.9 mm). Emission at 425 nm and excitation at 338 nm. A comparison between the sequences of peptides M,5 and R,5 is also shown.

FIGURE 1 Example of a gel-free-oriented proteomics nano-LC/MS-MS workflow in which bacterial culture proteins digested to tryptic peptides are separated via LC and peptides subsequently analyzed by mass spectrometry. In the process, the spectrometer rapidly cycles every few seconds and examines a size window in which peptide-derived MSI ions are analyzed to define MS/MS (MS2) spectra. The MS/MS (MS2) spectrum generated for each peptide then enters a bioinformatic pipeline for sequence identification, statistical validation, and quantification. 

The second approach, a shotgun proteomic analysis (LC-MS/MS) of tryptic solution-digests of isolated synaptosomes, identified 209 unique proteins. When the two sets of identified proteins are compared, 46 were found to be common. Accounting for this intersection, the total number of unique proteins identified by the... 

We first evaluated the performance of the device described in Figure 2.5 for the analysis of protein digests. Different standard protein tryptic digests... 

Figure 8.2 NanoLC-chip-MS of replicate injections (n — 5) of an eight-protein digest, 250 fmol each, (a) Overlay of 5 total ion chromatograms for the corresponding analyses, (b) Scatter plot of intensity measurements for 5 replicates and 2230 peptide ion clusters, (c) Distribution of RSD values on retention time measurements, (d) Variation of MS response for different tryptic peptides according to sample amount loaded. Conditions enrichment/trap volume of 40nL LC separation channel of 43 x 0.075 x 0.050mm both packed with Zorbax Ci8 separation media a 5 pL injection of 80 ng tryptic digest of 8 proteins was performed except for (d) where variable amounts (1-1000 ng) of digest were injected.

Figure 4. Tryptic peptide digest spectrum of spot 6 and post source decay spectrum of a tryptic peptide (T8), MYSPTSILDIR, with mass at m/z 1295.94 from the protein at spot 6. (A) MALDI reflector mass spectrum of tryptic peptide mixture derived from spot 6. (B) Mass spectrometric determination of partial peptide sequence of a tryptic peptide (T8). Y" series ions were observed.

---