Plant Proteomics Methods and Protocols - Plant Proteomics: Methods and Protocols - page 9

quantifi cation of proteins based on stable isotope-labeled integral
standard peptides and liquid chromatography coupled to selective
reaction monitoring has been reported ( see Chapter 15 ) .
Finally, the techniques used can be grouped according to the
MS equipment, which results in different combinations of ionizer
and analyzers, with MALDI sources usually coupled to TOF ana-
lyzers and ESI to quadrupoles, although lately a number of hybrids
have been commercialized ( see Chapter 6 ) .
The most appropriate workfl ow and protocols to be used
depend on the biological system and must be optimized for them
(e.g., species, organ, tissue, cell), as well as they depend on the
objectives of the research (descriptive, comparative, PTMs, inter-
actions, targeted proteomics) ( see Chapters 23 , 34 , 38 , 39 ) .
Alternative workfl ows, strategies and protocols are complementary
to each other; all of them together contributing to a deeper pro-
teome coverage and biological knowledge ( see Chapters 8 , 10 , 34 ) .
No specifi c technique can be considered better than another.
Despite there is no single mass analyzer capable of performing all
applications required in proteomic research, knowledge of the ana-
lytical capabilities and performances of the different mass analyzer
confi gurations available in the market, proteomic services, or in the
laboratories of colleagues, is a key element to take full advantage of
this powerful tool for biological research ( see Chapter 6 ) .
I would advise, when approaching proteomics for the fi rst
time, to start with the simplest alternative, which tends to be 1-D
electrophoresis (proven to be very valuable in the analysis of simple
proteomes, see Chapters 28 and 29 ) , and then moving to the most
complicated, including 2-DE and gel-free. 1-D has provided good
results in Holm oak proteotyping and variability studies by analyz-
ing the profi le of acorn fl our ( see Chapter 49 ) . In our group, we
apply the “funnel strategy,” which means starting with simple tech-
niques for high number of samples, and depending on the results
moving to more complicated strategies with reduced number of
samples. Opinions and views of classical techniques, like 2-DE,
determined as being outdated and poorly effi cient, must be
discarded ( see Chapters 4 and 11 ) . In the case of plant proteomics,
most of the papers published belong to the descriptive, comparative,
or subcellular proteomics categories, using 2-DE-based strategies
(Chapter 11 ) . For some purposes, like the analysis of protein com-
plexes, electrophoretic-based techniques, such as the Blue Native
PAGE or the Clear Native PAGE, have provided excellent results
( see Chapter 46 ) . Even though LC-based separation techniques of
peptides (bottom-up, MudPIT) or proteins (top-down, see Chapter
10 ) , second- and third-generation techniques for quantitative pro-
teomics, including both label (DIGE, ICAT, iTRAQ, SILAC) and
label-free protocols, are still the exception rather than the rule, this
tendency is being changed since the last 2 years ( see Chapters 12 ,
13 , 20 , 22 , 32 , 34 ).