Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion.
Peptide mass fingerprinting is a powerful tool for the identification of proteins. Trypsin is the most widely used enzyme for this purpose. Therefore, 104 protein digests from human Jurkat T cells and Mycobacterium were analyzed considering missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation. About 90% of the matched peptides with missed cleavage sites could be classified into three groups: (i) lysine and arginine with a neighbouring proline on the carboxy-terminal side, (ii) neighboring lysines/arginines, and (iii) lysines and arginines with an aspartic acid or glutamic acid residue on either the amino- or carboxy-terminal side. The first group is already accounted for by search programs. The number of missed cleavage sites can be increased without reducing the precision of the database search by taking the other two groups into consideration. Peptides with tryptophan were observed in non, singly (+16 Da) and doubly (+32 Da) oxidized forms. The higher oxidized form was only observed with lower intensity in the presence of the lower oxidized form. Peptides with N-terminal glutamine were found always as pyroglutamate (-17 Da), and in the majority of cases in pairs with unmodified glutamine. These data can be used for the refinement of protein searches by peptide mass fingerprinting.
Thiede
B
B
Max-Planck-Institut für Infektionsbiologie, Monbijoustrasse 2, D-10117 Berlin, Germany. thiede@mpiib-berlin.mpg.de
Lamer
S
S
Mattow
J
J
Siejak
F
F
Dimmler
C
C
Rudel
T
T
Jungblut
P R
PR
D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D019169:Jurkat Cells; D009161:Mycobacterium / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000737:chemistry; D011761:Pyrrolidonecarboxylic Acid / Q000032:analysis*; D012394:Rosaniline Dyes; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry*; D014364:Tryptophan / Q000032:analysis*
D007202:Indicators and Reagents; D010455:Peptides; D011506:Proteins; D012394:Rosaniline Dyes; C048139:Coomassie blue; D014364:Tryptophan; D014357:Trypsin; D011761:Pyrrolidonecarboxylic Acid