title: Performance of isobaric and isotopic labeling in quantitative plant proteomics.
abstract: 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.
authors.affiliation: Proteomic Unit, Institute of Chemistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Fábio C S
authors.initials: FC
authors.lastname: Nogueira
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Palmisano
authors.forename: Veit
authors.initials: V
authors.lastname: Schwämmle
authors.forename: Francisco A P
authors.initials: FA
authors.lastname: Campos
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Gilberto B
authors.initials: GB
authors.lastname: Domont
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D056625:Endosperm / Q000737:chemistry / Q000254:growth & development; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry*; D059748:Proteolysis; D020543:Proteome / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012278:Ricinus / Q000737:chemistry* / Q000254:growth & development; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
chemical_list: D010455:Peptides; D010940:Plant Proteins; D020543:Proteome; D012996:Solutions; D014357:Trypsin