Premium
SILIP: a novel stable isotope labeling method for in planta quantitative proteomic analysis
Author(s) -
Schaff Jennifer E.,
Mbeunkui Flaubert,
Blackburn Kevin,
Bird David McK.,
Goshe Michael B.
Publication year - 2008
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2008.03639.x
Subject(s) - monoisotopic mass , stable isotope labeling by amino acids in cell culture , quantitative proteomics , solanum , proteomics , chemistry , isotopes of nitrogen , isotope , mass spectrometry , stable isotope ratio , plant tissue , biology , botany , chromatography , nitrogen , biochemistry , physics , organic chemistry , quantum mechanics , gene
Summary Due to ease of manipulation, metabolic isotope coding of samples for proteomic analysis is typically performed in cell culture, thus preventing an accurate in vivo quantitative analysis, which is only achievable in intact organisms. To address this issue in plant biology, we developed SILIP (stable isotope labeling in planta ) using tomato plants ( Solanum lycopersicum cv. Rutgers) as a method that allows soil‐grown plants to be efficiently labeled using a 14 N/ 15 N isotope coding strategy. After 2 months of growth on 14 N‐ and 15 N‐enriched nitrogen sources, proteins were extracted from four distinct tomato tissues (roots, stems, leaves and flowers), digested, and analyzed by LC/MS/MS (data‐dependent acquisition, DDA) and alternating low‐ and elevated‐energy MS scans (data‐independent acquisition, MS E ). Using a derived relationship to generate a theoretical standard curve, the measured ratio of the M (monoisotopic) and M‐1 isotopologues of 70 identified 15 N‐labeled peptides from 16 different proteins indicated that 15 N incorporation was almost 99%, which is in excellent agreement with the 99.3% 15 N‐enriched nitrate used in the soil‐based medium. Values for the various tissues ranged from 98.2 ± 0.3% 15 N incorporation in leaves to 98.8 ± 0.2% in stems, demonstrating uniform labeling throughout the plant. In addition, SILIP is compatible with root‐knot nematode ( Meloidogyne spp.) development, and thus provides a new quantitative proteomics tool to study both plant and plant–microorganism systems.