Soybe
an (
Glycine max (L.) Merr. cv J
ac
k) seed development w
as sep
ar
ated into nine defined st
ages (S1 to S9). Test
a (seed co
ats) were removed from developing seeds
at st
ages S2, 4, 6, 8,
and 9,
and subjected to shotgun proteomic profiling. For e
ach st
age ¡°tot
al proteins¡± were isol
ated from 150 mg dry weight of seed co
at using
a phenol-b
ased method, then reduced,
al
kyl
ated,
and digested with trypsin. The tryptic peptides were sep
ar
ated using
a C18-reversed ph
ase m
atrix, then
an
alyzed using
an LTQ Orbitr
ap M
ass Spectrometer. Spectr
a were se
arched
ag
ainst the Phytozome
G. max DB using the Sorcerer 2 IDA Sequest-b
ased se
arch
algorithm. Identities were verified using Sc
affold 3. A tot
al of 306 (S2), 328 (S4), 273 (
S6), 193 (S8),
and 272 (S9) proteins were identified in three out of three biologic
al replic
ates,
and sorted into 11 function
al groups: Prim
ary Met
abolism, Second
ary Met
abolism, Cellul
ar Structure, Stress Responses, Nucleic Acid met
abolism, Protein Synthesis, Protein Folding, Protein T
argeting, Hormones
and Sign
aling, Seed Stor
age Proteins,
and Proteins of Un
known Function. In selected inst
ances, individu
al seed co
at proteins were qu
antified by spectr
al counting. The number of proteins involved in intermedi
ary met
abolism, fl
avonoid biosynthesis, protein folding
and degr
ad
ation
are discussed
as they rel
ate to seed co
at function.
ass=""h4"">Biological significance
Most previous analyses of seed coats have either targeted individual enzymes or used the results from high-throughput transcript profiling to infer biological function. Because there is seldom a linear correlation between transcript and protein levels, we have undertaken a shotgun proteomics-based description of soybean (G. max (L.) Merr. cv Jack) seed coats, as a function of development, in order to bridge this gap and to establish the baseline for a more comprehensive understanding of seed biology.