Herbivory leads
to changes in
the alloca
tion of ni
trogen among differen
t pools and
tissues; however, a de
tailed quan
ti
ta
tive analysis of
these changes has been lacking. Here, we demons
tra
te
tha
t a mass spec
trome
tric da
ta-independen
t acquisi
tion approach known as LC鈥揗S
E, combined wi
th a novel algori
thm
to quan
tify heavy a
tom enrichmen
t in pep
tides, is able
to quan
tify elici
ted changes in pro
tein amoun
ts and
15N flux in a high
throughpu
t manner. The reliable iden
tifica
tion/quan
ti
ta
tion of rabbi
t phosphorylase b pro
tein spiked in
to leaf pro
tein ex
trac
t was achieved. The linear dynamic range, reproducibili
ty of
technical and biological replica
tes, and differences be
tween measured and expec
ted
15N-incorpora
tion in
to
the small (SSU) and large (LSU) subuni
ts of ribulose-1,5-bisphospha
te-carboxylase/oxygenase (RuBisCO) and RuBisCO ac
tivase 2 (RCA2) of
Nicotiana attenuata plan
ts grown in hydroponic cul
ture a
t differen
t known concen
tra
tions of
15N-labeled ni
tra
te were used
to fur
ther evalua
te
the procedure. The u
tili
ty of
the me
thod for whole-plan
t s
tudies in ecologically realis
tic con
tex
ts was demons
tra
ted by using
15N-pulse pro
tocols on plan
ts growing in soil under unknown
15N-incorpora
tion levels. Addi
tionally, we quan
tified
the amoun
ts of lipoxygenase 2 (LOX2) pro
tein, an enzyme impor
tan
t in an
tiherbivore defense responses, demons
tra
ting
tha
t the approach allows for in-dep
th quan
ti
ta
tive pro
teomics and
15N flux analyses of
the me
tabolic dynamics elici
ted during plan
t鈥揾erbivore in
terac
tions.
Keywords:
thors" href="http://pubs.acs.org/action/doSearch?action=search&searchText=LC%E2%88%92MSE&qsSearchArea=searchText">LC鈭扢SE; thors" href="http://pubs.acs.org/action/doSearch?action=search&searchText=15N%5C-incorporation&qsSearchArea=searchText">15N-incorporation; thors" href="http://pubs.acs.org/action/doSearch?action=search&searchText=absolute+protein+quantification&qsSearchArea=searchText">absolute protein quantification; thors" href="http://pubs.acs.org/action/doSearch?action=search&searchText=Nicotiana+attenuata&qsSearchArea=searchText">Nicotiana attenuata; thors" href="http://pubs.acs.org/action/doSearch?action=search&searchText=herbivory&qsSearchArea=searchText">herbivory; thors" href="http://pubs.acs.org/action/doSearch?action=search&searchText=soil%5C-grown+plants&qsSearchArea=searchText">soil-grown plants