1D gel and LC-MS/MS
Bands are excised from the gel. In-gel digestion are performed with-out an reduction-and-alkylation (no cysteines are present in the histones). The proteins bands are digested with a proper cleavage agent (e.g. trypsin). After an over-night digestion, the peptides are extracted and concentrated by lyophilization. Here after, the peptides are analysed by LC-MS/MS [Molina et al., Mol Cell Proteomics, 2005, p637].
The MS/MS data are searched in an yeast database (also containing sequences of possible contaminants e.g. keratins trypsin) using Mascot [Perkins et al., Electrophoresis, 1999, p3551]. Search parameters: Oxidation (M), Phospho (ST), Acetyl (K)*, GlyGly (K)**, Methylation (K), Methylation (R), di-methylation (K), di-methylation (R) and tri-methylation (R) Max number of missed cleavages sites: 4*** The modified peptides are retrieved in the database search, and are manually verified, by reading the amino acide sequence of a peptide and pinpointing the site of the modification - possible. In the tables, it is noted if a modification is pinpointed (Direct evidence) or if a modification have not been directly deduced, but rather concluded from the remaining of a peptide sequence (In-direct evidence).
Fig. 1. Schematics of the experimental flow, for identification of PTM's.
Notes: *) Since the maximum number of variable modification allowed in Mascot is 9 and since this type of analysis requires a maximum number of variable modifications, I have decided to treat an acetylation and a tri-methylation as equeal. This can be done, since the weight of an acetylation and a tri-methylation is both 42Da. In the manual interpetation of an modified peptide, my way of distinguishing acetylations and methylations are 1) the appearance of an, for acetylations, specific marker ion at m/z 126 [Zhang et al., Proteomics, 2004, p1] and 2) a 14Da+14Da+14Da pattern observed in MS. Regarding the "14Da+14Da+14Da" pattern: I have noticed that peptides, differing only in the number of methylations, elute in close proximity of each other. If I observe peptides that can be interpetated as methylated (+14Da) and di-methylated (+28Da) together with a peptide with an +42Da mass difference, and if no marker ion at m/z 126 are observed, the peptide are assigned a tri-methylation status.
**) Ubiqutin units are attached to Lysines via an Gly-Gly-Lys link. When digesting with trypsin, a lysine residue will contain a Gly-Gly attachment to the functional "arm" of lysine.
***) Relevant for trypsin digestions: Most modified lysines and presumably arginies will give rise to a missed cleavage sites. Allowing for 4 missed cleavages sites will allow me to find a peptides with up to 4 modified lysines/arginines.<