searches, are required prior to data analysis.
There is no universal proteomics setup and each
of the four steps is crucial in order to obtain a
useful dataset. For plants, protein extraction is
challenging due to interfering compounds such
as polyphenols, terpenes, and organic acids that
are abundant in green tissues and, thus, several
protein extraction protocols have been developed
(Wang et al. 2003 ; Xie et al. 2007 ; Jellouli et al.
2010 ). ForLotustissues, we have successfully
used a SDS/phenol protein extraction followed
by ammonium acetate precipitation and further
removal of interfering compounds by acetone
washes (Dam et al. 2009 ; Nautrup-Pedersen et al.
2010 ; Dam et al. 2014 ).
After extraction, the protein fraction is redis-
solved and proteins are either separated/digested
or digested/separated. For the separation/diges-
tion methodology, proteins are solubilized and
separated using 1D or 2D gels followed by
excision of bands/spots, digestion, and desalting
prior to MS. Alternatively, the peptides can be
further separated using liquid chromatography
(LC) prior to MS which is frequently used for 1D
separation. The 2D gel technology is a two-step
separation of up to 5,000 protein spots (O’Farrell
1975 ) and these can be visualized/quantified
down to 1 ng (Weiss and Görg 2007 ;Görg et al.
2004 ). For the digestion/peptide separation
methodology, the protein fraction is solubilized,
in-solution digested, and peptides are separated
using LC. Prior to LC, peptides can be labeling
with, for example, iTRAQ used for relative
quantification. InLotus, the protein separation/
digestion strategy was used to create proteome
reference maps of seed, pod, nodule, and root
using 2D gels (Nautrup-Pedersen et al. 2010 ;
Dam et al. 2014 ) together with subcellular pro-
tein extraction of seed globulin-, plant cytosolic-,
and symbiosome membrane proteins (Dam et al.
2009 , 2013 , 2014 ; Nautrup-Pedersen et al. 2010 ;
Credali et al. 2013 ; Wienkoop and Saalbach
2003 ) and, furthermore, the digestion/peptide
methodology has been initiated for nodules and
roots. Currently, the majority of proteomics
studies are performed with the digestion/peptide
methodology. However, the 2D gel methology
has the advantage of visualizing PTMs given that
each protein isoform is sufficiently separated in
the 2D gel and, thus, can be analyzed individu-
ally to identify the variety of PTMs for the pro-
tein (Rogowska-Wrzesinska et al. 2013 ).18.2 Mass Spectrometry Techniques
and Data SearchIn the last decade, the MS instruments used for
MS scan and MS/MS analysis have developed
rapidly both in sensitivity and in speed. Mass
spectrometers have three major functional parts,
i.e., ion source, mass analyzer, and detector.
These are developed as independently functional
entities and can be combined differently. For a
more comprehensive description of mass spec-
trometers, see Parker et al. ( 2010 ).
For Lotus proteomics, the matrix-assisted
laser desorption/ionization (MALDI) and elec-
trospray ionization (ESI) were the two ion sour-
ces used. For MALDI, the tryptic desalted
peptides were eluted on a target plate together
with a strong absorption matrix. The mass ana-
lyzer, the time offlight (TOF), detected the mass/
charge (m/z) of peptide ions and TOF/TOF was
used for fragmentation of selected peptide ions.
For the ESI ion source, the desalted peptides
were separated using a C-18 column to reduce
the complexity of peptide ions analyzed. The
peptide ions were sprayed into the mass spec-
trometer and the mass analyzer Q-TOF, ion trap,
and recently orbitrap were used for Lotus
proteomics.
The Mascot software was used for Lotus
protein identifications. The proteomics data was
searched against all predicted Lotus protein
coding genes, and for validation, the data was
analyzed manually using the novel available MS
data miner (MDM) software (Dyrlund et al.
2012 ).
All four major steps in a proteome analysis are
crucial and have to be optimized for each
experimental setup. In the following, proteomics
of differentLotustissues together with some of
the biological interpretations are discussed.202 S. Dam and J. Stougaard