260
- Plasman K et al (2013) Contemporary posi-
tional proteomics strategies to study protein
processing. Curr Opin Chem Biol 17:
66–72 - Dix MM et al (2008) Global mapping of the
topography and magnitude of proteolytic
events in apoptosis. Cell 134:679–691 - Mahrus S et al (2008) Global sequencing of
proteolytic cleavage sites in apoptosis by spe-
cifi c labeling of protein N termini. Cell
134:866–876 - Kleifeld O et al (2010) Isotopic labeling of ter-
minal amines in complex samples identifi es
protein N-termini and protease cleavage prod-
ucts. Nat Biotechnol 28:281–288
5. Gevaert K et al (2003) Exploring proteomes
and analyzing protein processing by mass spec-
trometric identifi cation of sorted N-terminal
peptides. Nat Biotechnol 21:566–569
6. Schilling O et al (2010) Proteome-wide analy-
sis of protein carboxy termini: C terminomics.
Nat Methods 7:508–511
7. Van Damme P et al (2010) Complementary
positional proteomics for screening substrates
of endo- and exoproteases. Nat Methods
7:512–515
8. de Poot SA et al (2011) Human and mouse
granzyme M display divergent and species-
specifi c substrate specifi cities. Biochem J 437:
431–442
level of the selected metacaspase. Subsequently, seedlings were
harvested and frozen in liquid nitrogen for protein extraction.- This amount of tissue yields on average about 5 mg of protein
material. - The recommended amount of protein material per sample is
4 mg. - The total amount of labelling reagents added suffi ces to label
the equivalent of up to 4 mg of protein material. - A typical side-reaction of labelling protein primary amino
groups with N -hydroxysuccinimide esters is the acetylation of
hydroxyl groups in proteins (serines, threonine, and tyrosine).
Hydroxylamine is effi cient in reverting this O-acetylation. - At this stage, samples can be subjected to the chromatography
part of the N-terminal COFRADIC procedure as detailed in
ref. 14.
Acknowledgments
L.T. acknowledges support from the VIB International PhD
Program and the Netherlands Proteomics Centre, a program
embedded in The Netherlands Genomics Initiative. P.V.D. is a
Postdoctoral Fellow of the Research Foundation Flanders (FWO-
Vlaanderen) and S.S. is indebted to the Special Research Fund of
Ghent University for a postdoctoral fellowship. F.V.B acknowl-
edges support from grants of the Ghent University Multidisciplinary
Research Partnership “Ghent BioEconomy” 27 (project no.
01MRB510W) and of the Belgian Science Policy Offi ce (project
IAP7/29). F.V.B. and K.G. acknowledge support from the
Research Foundation Flanders (FWO-Vlaanderen), research proj-
ect G.0038.09.References
Liana Tsiatsiani et al.http://www.ebook3000.com