Performance of Isobaric and Isotopic Labeling in Quantitative Plant Proteomics

Mass spectrometry has become indispensable for peptide
and protein
quantification in proteomics studies. When proteomics technologies
are applied to understand the biology of plants, two-dimensional gel
electrophoresis is still the prevalent method for protein fractionation,
identification, and quantitation. In the present work, we have used
LC–MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical
labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at
three developmental stages (IV, VI, and X). Endosperm proteins of
each stage were trypsin-digested in-solution, and the same amount
of peptides was labeled with ICPL and iTRAQ tags in two orders (forward
and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap
high-resolution mass spectrometer. Comparing labeling performance,
iTRAQ was able to label 99.8% of all identified unique peptides, while
94.1% were labeled by ICPL. After statistical analysis, it was possible
to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are
specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies.
We noted that the iTRAQ quantification could be influenced by the
tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification
depends on several parameters, both labeling methods were able to
successfully quantify proteins present in the endosperm of castor
bean during seed development and, when combined, increase the number
of quantified proteins.