Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. by matrix-assisted laser desorption/ionization time of flight mass spectroscopy combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Sieve tubes mediate the long-distance transport of nutrients and signals between source and sink organs of plants. To detect mobile phloem proteins that are differentially distributed in source and sink organs of Cucurbita maxima, we used both one-dimensional gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques revealed that phloem protein patterns depend on the sampling site: whilst several proteins were consistently observed in all phloem samples studied others appeared to occur in a organ-specific manner. For a characterization and identification of distinct phloem polypeptides, two approaches were chosen. First, protein bands resolved by SDS-PAGE were eluted from the polyacrylamide gel and the masses of the proteins were then determined by MALDI-TOF MS. Second, proteins resolved by SDS-PAGE were subjected to proteolytic degradation and the resulting peptides were analyzed by MALDI-TOF MS: the masses of the proteolytic peptides were used for a database search. By the latter approach, three mobile phloem compounds were identified as the phloem-specific protein PP2 (D.E. Bostwick et al., 1992, The Plant Cell 4, 1539-1548) a chymotrypsin and an aspartic proteinase inhibitor. None of the other polypeptides studied corresponded to any of the protein sequences present in the database. Furthermore, MALDI-TOF MS analyses indicated that some of the mobile phloem proteins occur in a covalently modified form and that the extent of the modification depends upon the plant organ.
Kehr
J
J
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Golm, Germany.
Haebel
S
S
Blechschmidt-Schneider
S
S
Willmitzer
L
L
Steup
M
M
Fisahn
J
J
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry; D014675:Vegetables / Q000737:chemistry*
D010940:Plant Proteins; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin