In-gel tryptic digestion

Ballihaut, G., Tastet, L., Pecheyran, C., Bouyssiere, B., Donard, O., Grimaud, R., and Lobinski, R., Biosynthesis, purification and analysis of selenomethionyl calmodulin by gel electrophoresis-laser ablation-ICP-MS and capillary HPLC-ICP-MS peptide mapping following in-gel tryptic digestion. Journal of Analytical Atomic Spectrometry 20(6), 493 99, 2005. 

Dissolution in Phosphate buffer, gel electrophoresis, in gel tryptic digestion, petide extraction (CH3CN/H2O/TFA), cleaning on ZipTip CIS Acidification (pH 4.6 with 5% HAc) to precipitate caseins, centrifugation... 

Differential in-gel electrophoresis (DIGE) facilitates protein expression by labeling different populations of proteins with fluorescent dyes. Typically, paired samples from the normal and tumor region are labeled with Cy3 and Cy5. After analysis by differential analysis image software, protein spots that exhibit a significant difference in intensity are excised for in-gel tryptic digestion and MS analysis. 

A significant challenge for neuroscientists in studying the membranous synaptosomal proteome rests squarely in the analysis of its constituent hydrophobic and membrane-bound proteins. These include receptors, transporters, ion channels, and the molecular machinery for synaptic vesicle cycling. Hydrophobic and membrane-bound proteins are poorly resolved by traditional lEF gel technology. Similarly, they tend to resist in-gel tryptic digestion, leading to poor rates of protein identification by PMF (van Montfort et al. 2002). 

HPLC profiles that resulted from the in gel tryptic digest of 34 pmol (2.1 pg) of a 62 kD protein are shown in Fig. 1. At this low level there are at least 5-6 peaks present in the blank chromatogram that are similar in size to peaks... 

Table I. Summary of results from the in gel tryptic digestion of 25 proteins...

Figure 5. Reverse phase HPLC of in gel tryptic digests of 25 pmol transferrin (A) and 10 pmol bovine serum albumin (B) and of the corresponding digests carried out on blank sections of gels (lower profiles shown in above two figures). In each instance 90% of the digest was subjected to HPLC on a 1.0 mm ID Vydac C-18 coluttm eluted at 50 pl/min. The respective full scale deflections were 18.9 mV for panel A and 4.4 mV for panel B with 0.5 volt corresponding to an absorbance of 1.0 at 210 nm.

Koy C, Miklcat S, Raptakis E, Sutton C, Resch M, Tanaka K, Glocker MO. Matrix-assisted laser desorp-tion/ionization-quadrupole ion trap-time of flight mass spectrometry sequencing resolves structures of unidentified peptides obtained by in-gel tryptic digestion of haptoglobin derivatives from human plasma proteomes. Proteomics. 2003 Jun 3(6) 851-8. 

The most common proteomics approach uses two-dimensional gel electrophoresis to separate cellular proteins, followed by in-gel tryptic digestion of the protein spots and identification of the peptide sequences by mass spectrometry. There are s ificant problems with this approach, which so far, have limited its usefulness for drug taiget discovery. A number of protein classes such as int ral membrane proteins, positively chaiged and hydrophobic proteins are difficult to separate. There are often selective losses of individual protdns, for reasons that are not well understood, so the claim for a global picture is not entirefy accurate. The approach is semi-quantitative at best. Importantly, low abundance proteins may not be detected. However, newer approaches are constantly being developei Approaches which... 

Another study on P. aeruginosa (Peng et al. 2005) examined the sarcosine-in-soluble outer membrane fraction upon treatment with ampidllin, kanamycin, and tetracycline to identify proteins related to the respective antibiotic resistances. The authors found 11 difierential proteins, which were excised from the 2D gel and identified by MALDl-TOF MS after in-gel tryptic digestion of the excised spots. Apart from some known antibiotic resistance proteins, Peng et al. discovered some new proteins and thereby novel potential antibiotic targets. 

The same technique of in-gel tryptic digestion and subsequent identification by MALDl-TOF MS was apphed by Dupont and cowoikers in Acinetobacter bau-mannii, an opportunistic bacillus comprising increasing numbers of resistant strains (Dupont et al. 2005). They compared the outer membrane of different strains and found two differentially expressed proteins, one of which was identified as belonging to the OprD family. 

Due to the insolubility of AP-aggregates, fibril formation could not be analyzed directly by mass spectrometry. In order to determine the molecular composition of aggregates, in gel tryptic digestion and mass spectrometric analysis of the gel electrophoresis bands of Ap-oligomers was performed. AP(l-40) was subjected to fibril growth at 1 J,g/ jL (220 pM) in buffer solution, pH 7.5, and incubated for 5 days at... 

S. M. Lubman, D. M. Identification of proteins from two-dimensional gel electrophoresis of human erythroleukemia cells using capillary high performance liquid chromatography/electrospray-ion trap reflectron time-of-flight mass spectrometry with two-dimensional topographic map analysis of in-gel tryptic digest products. Rapid Common. Mass Spectrom. 1999, 73(19), 1907 1916. 

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