In-gel trypsin digestion

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

Figure 19-25. Peptide mass mapping of in-gel trypsin digested 31-kDa band by MALDI-MS. (Reprinted from reference 138, with permission of Elsevier Science B. V.)...

A recent study from 2014 exploited the analysis of in-gel trypsin-digested proteins with MALDI-TOF/TOF MS to identify CMY-2-type cephdosporinases inEn-terobacteriaceae (Papagiarmitsis et al. 2014). Apeak uniquely observed in CMY producing isolates was thereby confirmed to represent a C. fremdii-Wke p-lactamase. 

DGE-based Western blotting was used to detect, and LC-MS/MS to characterize, nitroproteins in the human pituitary control tissues [19], Proteins from 2D gel spots that corresponded to the strongly positive anti-nitrotyrosine Western blot spots (Fig. 7) were subjected to in-gel trypsin digestion and LC-MS/MS analysis. MS/MS determined the nitration site of each nitrated peptide. Each amino acid sequence was first determined by the accurate de novo sequence method, and secondly by SEQUEST analysis (Fig. 5). De novo sequencing... 

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... 

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. 

Fig. 1. Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) spectrum of a trypsin-digested one-dimensional gel band. Peaks are labeled with their monoisotopic masses. Note that these are not the masses of the peptides, but of the peptide (pseudo)molecular ions. In MALDI spectra, peptide molecular ions arise predominantly through the addition of a proton to the peptide, giving a mass increase of 1.007 Da. The molecular ions are usually denoted as MH+ or [M+H]+.

Figure 4.4. Protein identification by MALDI-TOF. Steps in protein identification by MALDI-TOF are shown. Prior separation by gel or liquid chromatography to a single protein is needed prior to enzymatic digestion with trypsin. Digested peptides are spotted onto a MALDI target with an appropriate matrix that assists desorption of peptides upon activaton by laser. Peaks from the resulting mass spectrum represent peptide ions that can be searched in a database to match a theoretical tryptic digest from known proteins. The more proteins that are matched, the greater the statistical confidence in assignment of a protein identification.

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