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.<