In-gel digests

Several protocols including in-gel digestion, preparing gel slices, and some others for MS analysis. 

Mass spectrometry provides a more direct and precise technique to study histone modifications. As with the other methods discussed above, mass spectrometry also has several pitfalls that should be taken into account when analyzing histone modifications. First of all histones and especially the core histones H3 and H4 are rich in lysine residues. Consequently, trypsin as an enzyme that is routinely used for the identification of proteins via peptide mass fingerprints cannot be used for regular in gel digestion of histones. Other enzymes that have a different specificity (such as Asp-N or Arg-C) are more frequently used in the analysis of histones [25]. A drawback... 

Pre-concentration by vacuum centrifugation is a common technique used in many versions of current in-gel digestion methods and pHPLC. Throughout the process, the sample is exposed to many surfaces, which may contain a minimal number of active sites where losses can occur. The amount of sample loss may be negligible for concentrated samples, but loses at lower peptide concentrations, such as the femtomole (fm=10 M) level (see Table 3.1), increase markedly. ... 

Timperman, A. T. and Aebersold, R., Peptide electroextraction for direct coupling of in-gel digests with capillary LC-MS/MS for protein identification and sequencing. Analytical Chemistry 72( 17), 4115-4121, 2000. 

Speicher, K. D., Kolbas, O., Harper, S., and Speicher, D. W., Systematic analysis of peptide recoveries from in-gel digestions for protein identification in proteome studies.. Journal of Biomolecular Techniques 11(2), 74-86, 2000. 

Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P. (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203, 173-9. 

On-blot digestion is another method used for proteins blotted onto nitrocellulose or PVDF membranes. Procedure is similar to in-gel digestion with the exception of elution from the membrane surface. 

Trypsin is the most frequently used serine protease. Generally, porcine or bovine pancrease is used as the source of pure trypsin. Trypsin usually digests proteins at their lysine and arginine residues. The superiority of trypsin is that it displays good activity both in solution and in-gel digestion protocols. 

Keywords Proteomics MALDI-TOF mass spectrometry SDS-PAGE 2D-gel in-gel digestion peptide mass fingerprint protein identification database searching. 

The identification of proteins by MALDI-MS following in-gel digestion in a low-salt, nonvolatile buffer of the proteins was simplified by collecting the resulting peptides in a small volume under sonication for 5 min this facilitated automation of the overall process [77]. 

In Gel Digestion of SDS PAGE-Separated Proteins Observations from Internal Sequencing... 

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